Anti-APOE antibodies: compositions and methods of making and using the same

Abstract

Compositions and methods of use for modulating APOE mediated signaling are provided. For example, immunomodulatory agents are provided that reduce APOE expression, ligand binding, crosslinking, APOE mediated signaling, or a combination thereof. In another embodiment, immunomodulatory agents are provided that enhance or promote APOE expression, ligand binding, crosslinking, APOE mediated signaling, or a combination thereof. Such agents can be used to modulate an immune response in a subject in need thereof, wherein the subject has a neurodegenerative disease, such as Alzheimer's disease or cerebral amyloid angiopathy.

Description

ANTI-APOE ANTIBODIES: COMPOSITIONS AND METHODS OF MAKING AND USING THE SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 649,509 filed on May 20, 2025, and is incorporated by reference in its entirety.FIELD

[0002] The invention generally relates to compositions and methods for treating APOE-mediated diseases and conditions, and more particularly to compositions and methods for modulating immune responses in a subject with neurodegenerative and / or neuroinflammatory diseases and conditions, such as Alzheimer’s Disease.BACKGROUND

[0003] Amyloidosis broadly encompasses a variety of diseases that are characterized by the extracellular or intracellular deposition of amyloid proteins in tissues and / or organs. Amyloids are insoluble fibrous protein / peptide aggregates and their deposition may occur in localized sites or systemically. The fibrillar composition of these deposits is an identifying characteristic for the various forms of amyloid disease. In some cases the amyloid protein / peptide accumulates intracellullary, resulting in cell dysfunction and ultimately cell death. Examples of intracellular amyloid proteins include a-synuclein, forming Lewy bodies in Parkinson's disease, and forming neuronal inclusions in Huntington disease. The pathogenesis of Alzheimer's disease (AD), the most common of the amyloid related neurodegenerative disorders, is linked to the cleavage of the amyloid precursor protein generating the amyloid-P (AP) peptide which undergoes a shape change into a pathological conformer having a high P-sheet content. Intracerebral and cerebrovascular deposits composed primarily of fibrils of the pathological Ap peptide are characteristic of both familial and sporadic forms of AD. In addition to Ap, abnormally phosphorylated tau protein forms toxic oligomeric structures and neurofibrillary tangles in AD. Similar to AD, prion-associated diseases, such as Creutzfeld-Jacob disease, have also been characterized as amyloid diseases. The pathogenesis of prion disease is linked to a change of the cellular prion protein (PrPc) into the disease associated PrPSc(Sc for scrapie).

[0004] An active area of translational research and current clinical trials for amyloid disease has focused on immunotherapy, using both passive and active immunization against amyloid proteins, particularly Ap in AD (Wisniewski et al., “Amyloid-P Immunization for Alzheimer's Disease,” Lancet Neurol 7: 805-811 (2008)). Although immunotherapy holds great promise as a means of reducing amyloid deposition, it, unfortunately, has been accompanied by major obstacles. Specific problems associated with immunotherapy that were identified in a clinical trial for AD include the potential of toxicity from encephalitis (related to excessive cell mediated immunity), the immunological targeting of both the normal and abnormal Ap peptide, the failure to address tau related pathology, and the apparent poor efficacy. Moreover, although autopsy data from this early immunotherapy vaccine trial suggested that many patients had a significant reduction in amyloid burden, these patients exhibited only minor cognitive benefits (Wisniewski et al., “Amyloid-P Immunization for Alzheimer's Disease,” Lancet Neurol 7:805-811 (2008) and Holmes et al., “Long Term Effects of AP42 Immunization in Alzheimer's Disease: Immune Response, Plaque Removal and Clinical Function,” Lancet 372:216-223 (2008)). Thus, an immunotherapeutic approach that can effectively reduce amyloid burden and overcome the aforementioned problems is warranted.

[0005] Apolipoprotein E (APOE) s4 has been shown to increase risk for Alzheimer's disease and is also associated with an earlier age of disease onset. Studies have shown that having one or two APOE s4 alleles increases the risk of developing Alzheimer's. Approximately 25 percent of people carry at least one copy of APOE e4, and 2 to 3 percent carry two copies.

[0006] Therefore, it is an object of the invention to provide compositions that modulate APOE mediated signal transduction thereby promoting a suppressive immunological response. Such compositions are useful for the treatment of inflammatory diseases and disorders and autoimmune diseases. It is also an object of the invention to provide compositions that modulate APOE mediated signal transduction to thereby enhance or promote an activating immunological response. Such compositions are useful for the treatment of cancer and infectious diseases.SUMMARY

[0007] Compositions and methods of their use for modulating APOE-mediated signal transduction are provided. One aspect of the invention provides an apolipoprotein E(APOE) immunomodulatory agent that modulates APOE expression, ligand binding, crosslinking, APOE-mediated signaling, or a combination thereof, comprising an anti- APOE antibody and antigen-binding fragments thereof, an APOE fusion protein and binding fragments thereof, or an APOE antibody-drug conjugate, wherein the immunomodulatory agent immunospecifically binds to APOE having at least 99% sequence identity to SEQ ID NO: 51, 52, 53, 54, or 55. In one embodiment the anti-APOE antibody and antigen-binding fragments thereof comprises a variable light chain domain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and a variable heavy chain domain having at least 99% sequence identity to selected from the group consisting of SEQ ID NOs: 21, 25, 29 and 33. In another embodiment provides the anti-APOE antibody and antigenbinding fragments thereof has the variable light chain domain of the anti-APOE antibody and antigen-binding fragments thereof comprising complementarity-determining regions (CDRs) selected from CDRs consisting of at least 99% sequence identity to SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, or combinations thereof and the variable heavy chain domain of the anti-APOE antibody and antigen-binding fragments thereof comprise complementaritydetermining regions (CDRs) selected from CDRs consisting of at least 99% sequence identity to SEQ ID NO: 48, 49, 50, or combinations thereof.

[0008] In another embodiment the APOE fusion protein and binding fragments thereof comprise a light chain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, and a heavy chain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID Nos: 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, and 36.

[0009] In one another embodiment the APOE antibody and binding fragments thereof have the light chain and heavy chain selected from a group of sequences having: : a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; avariable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; or a variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: SEQ ID NO:21, 25, 29, or 33.

[0010] In yet another embodiment provides the APOE fusion protein and binding fragments thereof have the light chain and heavy chain selected from a group of sequences having: a light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:4 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 6 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 8 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100%sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 10 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 12 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 14 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 16 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 18 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; or a light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:20 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36.

[0011] In a particular embodiment the APOE antibody and binding fragments thereof have a light chain with at least 99% sequence identity to SEQ ID NO: 15 and heavy chain domains with at least 99% sequence identity to SEQ ID NO: 33.

[0012] Another aspect of the invention provides an APOE antibody-drug conjugate comprising an APOE antibody or fragment thereof as disclosed herein, a linker, a payload, and an optional moiety, wherein the antibody binds to APOE, and wherein the optional moiety is selected from the group consisting of transferrin receptor, CD98hc, basigin, Glutl extracellular domain (ECD) 1, ECD2, ECD3, ECD4, ECD5, and ECD6, IGF-1R, INSR ECD1, and INSR ECD2.

[0013] In one embodiment, the linker is selected from an extracellular protease cleavable linker, a cathepsin cleavable linker, or a non-cleavable linker. Another embodiment provides that the payload is selected from a pro-synaptic agent, a neurostimulatory agent, or a pro-inflammatory agent. In another embodiment, the pro- synaptic agent is selected from EphB2 small molecule agonists, ephrin-B, IGF1, 5-HT or5-HT2A agonists, Ngrl inhibitors, rapamycin, or TrkB agonists. In yet another embodiment, the neurostimulatory agent is selected from a sodium channel agonist, a calcium channel agonist, memantine, or donazepil. In one other embodiment, the payload is selected from an anti-inflammatory agent, selected from an IL-lb inhibitor, an IL-6 inhibitor, a TNF alpha inhibitor, a type 1 IFN inhibitor, or a corticosteroid. In an additional embodiment, the optional moiety is selected from a transferrin receptor, CD98hc, basigin, or Glutl.

[0014] Yet another aspect of the invention provides a pharmaceutical composition for treating a neurodegenerative disease in a subject in need thereof, comprising the APOE immunomodulatory agents disclosed herein. In one embodiment, the APOE immunomodulatory agent is selected from a therapeutically effective amount of the APOE antibody or antigen binding fragments thereof; a therapeutically effective amount of the APOE fusion protein and binding fragments thereof; or a therapeutically effective amount of the antibody-drug conjugate of claim 5; wherein the pharmaceutical composition is administered to the subject to treat or prevent a neurodegenerative disease. In another embodiment, the neurodegenerative disease is selected from Alzheimer’s disease, cerebral amyloid angiopathy, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington’s disease. In yet another embodiment, the APOE immunomodulatory agent binds to amyloid-associated APOE aggregates contained within amyloid plaques in a subject’s brain parenchyma and brain vasculature. In an additional embodiment, the binding of amyloid-associated APOE aggregates contained within amyloid plaques reduces APOE4 levels and lowers the brain amyloid protein levels in the subject’s brain. In another embodiment, the pharmaceutical composition is administered parenterally, orally, or topically.

[0015] An additional aspect of the invention provides a method of treating a neurodegenerative disease comprising administering to a subject in need thereof the pharmaceutical composition of claim 16, wherein the pharmaceutical composition binds to amyloid-associated APOE aggregates contained within amyloid plaques in the subject’s brain parenchyma and brain vasculature to reduce APOE4 levels and lower the brain amyloid protein levelsBRIEF DESCRIPTIONS OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate (one) several embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

[0017] Figure 1 is an illustration showing a model of APOE antibody activity.

[0018] Figures 2A-2B show functional characterization of the ApoE4 antibody.

[0019] FIG.2A is a bar graph showing the binding of APOE mAb to native and heat- induced APOE4 aggregates. FIG. 2B is a binding curve showing APOE mAb binding to APOE4 was enhanced following APOE4 heat treatment.

[0020] Figures 3A-3B are binding curves showing that the APOE mAb binding to APOE4 is enhanced by heat-treatment, to a point (55°C). FIG. 3 A shows APOE mAb binds APOE4 with higher affinities as APOE is pre-treated at progressively higher temperatures. FIGs. 3 A and 3B show that beyond 55°C of heat treatment, APOE mAb binding to heat- treated APOE4 is lost.

[0021] Figures 4A-4C show the characterization of antibody variant expression. FIG. 4A is a bar graph showing the quantification of IgGl concentration by Octet. FIGs. 4B-4C are SDS-PAGE Coomassie stains showing the evaluation of protein expression.

[0022] Figures 5A-5F show that humanized variants and the parent molecule bind comparably to ApoE4. FIGs. 5A-5E are representative biding data for variants 2, 6, 12, 16 and the parent molecule. FIG. 5F shows the calculated ECso for variants 2, 6, 12, 16 and the parent molecule.

[0023] Figures 6A-6I show comparable staining of plaque-associated ApoE. FIGs. 6A-6I show the APOE mAb variant 16 binds similarly to APOE4 co-deposited with parenchymal and vascular amyloid (CAA). APOE- / - tissues were included as native controls.

[0024] Figure 7 is a binding curve showing the antibody binding signal for APOE mAb Variant 16 binding to 6xHis-fusions of native human APOE4 (•) or cyno APOE (■).

[0025] Figures 8A-8C show that ApoE mAb does not bind to plasma ApoE. FIGs. 8A-8B show ELISA signals from a pan-APOE antibody was used to establish equivalent coating concentrations between plasma APOE3 (FIG. 8A) and recombinant APOE3 (FIG.8B). FIG. 8C shows APOE mAb variant 16 binding data compared at the plasma and recombinant protein dilutions selected in top panel.

[0026] Figures 9A-9B show that a CDR mutation may disrupt binding to an ApoE4 intermediate. Modeled dose-response curves for binding to native or heat-denatured APOE4 were calculated for APOE mAb parent (FIG. 9A) and a point mutant (FIG. 9B).

[0027] Figures 10 A- 10C show APOE mAb variant 16 binding affinity with(squares) and without (circles) heating samples to ~40 C for A) full-length APOE4, B) APOE4 fragment amino acids 150-160, and C) APOE4 fragment amino acids 140-160. APOE4 mAb variant 16 binds more strongly to APOE4 fragment 140-160 than to fragment 150-160 or full-length.DETAILED DESCRIPTION

[0028] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.I. Definitions

[0029] To facilitate an understanding of the principles and features of the various embodiments of the disclosure, various illustrative embodiments are explained herein. Although exemplary embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the description or examples. The disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0030] In describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named.

[0031] Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value.

[0032] As used herein, the terms “immunomodulatory agent” and “binding moiety” are used interchangeably.

[0033] As used herein, the term “APOE immunomodulatory agent” refers to apolipoprotein E (APOE) binding moieties including, but not limited to antibodies and antigen binding fragments thereof, and APOE fusion proteins and binding fragments thereof. In one embodiment, APOE has an amino acid sequence according to UniProtKB - P02649 (APOE HUMAN) which is incorporated by reference in its entirety.

[0034] As used herein, a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecifically binding to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. In some embodiments, however, antibodies (and their antigen binding fragments) will not cross-react with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

[0035] As used herein, a molecule is said to “physiospecifically bind” a second molecule if such binding exhibits the specificity and affinity of a receptor to its cognate binding ligand. A molecule can be capable of physiospecifically binding to more than one other molecule.

[0036] As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from a “Complementarity Determining Region” or “CDR” (z.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26- 32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies (mAb), multi-specific antibodies, human antibodies, variant antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1 :253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231 :25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Patent No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti -Id and anti-anti -Id antibodies to antibodies). In particular, such antibodies include immunoglobulin molecules of any type e.g., IgG, IgE, IgM, IgD, IgA and IgY), class e.g., IgGi, IgG?, IgGs, IgG4, IgAi and IgA2) or subclass.

[0037] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody’s “variable region” antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins including the antibody’s “variable region” antigen recognition site and a heterologous protein e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etcf

[0038] As used herein, the term “fragment” refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0039] As used herein the term “modulate” relates to a capacity to alter an effect, result, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (z.e., attenuating, but not abolishing) or it can completely abolish such activity e.g., neutralizing). Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In a still further embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction. For example, the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments, such modulation will provide at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100- fold change in such activity.

[0040] The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is able to substantially block an activity of a ligand or receptor if the extent of blockage is physiologically or therapeutically relevant (for example if such extent is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).

[0041] As used herein, the “activating” or “stimulatory” signals encompass signals that result in enhancing an activity or enhancing signal transduction.

[0042] As used herein, “suppressive” signals refer to signals that suppress immune activity.

[0043] The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of a parent or reference antibody but which differs in amino acid sequence from the parent or reference antibody or antigen binding fragment thereof by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or antigen binding fragment thereof. In some embodiments, such derivatives will have substantially the same immunospecificity and / or characteristics, or the same immunospecificity and characteristics as the parent or reference antibody or antigen binding fragment thereof. The amino acid substitutions or additions of such derivatives can include naturally occurring (z.e., DNA-encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, chimeric variants, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

[0044] As used herein, a “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0045] As used herein, the term “variant antibody” refers to an immunoglobulin including a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85- 99%, or about 95% or more identical. Hence, all parts of a variant immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A variant antibody is an antibody including a variant light chain and a variant heavy chain immunoglobulin. For example, a variant antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human.

[0046] The term “endogenous concentration” refers to the level at which a molecule is natively expressed (i.e., in the absence of expression vectors or recombinant promoters) by a cell (which cell can be a normal cell, a cancer cell or an infected cell).

[0047] As used herein, the terms “treat,” “treating,” “treatment” and “therapeutic use” refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder. As used herein, a “therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0048] As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease prior to the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0049] As used herein, an “immune cell” refers to any cell from the hemopoietic origin including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0050] As used herein, “inflammatory molecules” refer to molecules that result in inflammatory responses including, but not limited to, cytokines and metalloproteases such as including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

[0051] As used herein, “valency” refers to the number of binding sites available per molecule.

[0052] As used herein, the terms “immunologic,” “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and / or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced byadministration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+T helper cells and / or CD8+cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

[0053] An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

[0054] As used herein, 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.

[0055] As used herein, the term “Ap amyloidosis” is clinically defined as evidence of Ap 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 overthe expected risk given the subject's age, family history, genetic status and other known risk factors.

[0056] As used herein, the term “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 W E et al. Ann Neurol 55(3) 2004, and Fagan A M 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 Ser. 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.

[0057] As used herein, 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, andorganization. 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.

[0058] As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.

[0059] As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. The polypeptides can be “exogenous,” meaning that they are “heterologous,” z.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0060] As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0061] Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0062] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0063] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can besubstituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0064] Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that one amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0065] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Vai: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

[0066] The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0067] For purposes herein, the % sequence identity of a given nucleotides or amino acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:100 times the fraction W / Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

[0068] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.II. Compositions

[0069] One embodiment of the present invention provides compositions that specifically bind to APOE and modulate signal transduction through APOE, for example to reduce, inhibit, or block APOE mediated signal transduction in immune cells. Representative immune cells include, but are not limited to, microglia. Another embodiment provides compositions that inhibit, reduce, or block the interaction with APOE with one or more ligands of APOE, and thereby reduce, inhibit, or block APOE mediated signal transduction in immune cells. The binding moieties can bind directly to APOE and inhibit, reduce or block the interaction of APOE with one or more of its ligands. In still another embodiment, the binding moieties specifically bind or to a complex of APOE with one or more ligands.

[0070] Another embodiment provides immunomodulatory compositions that specifically bind to APOE and enhance or promote APOE mediated signal transduction. Inone embodiment, immunomodulatory compositions enhance APOE mediated signal transduction and thereby induce or enhance an immune response in a subject. In another embodiment the immunomodulatory agent binds to APOE, an extracellular domain of APOE, or to a ligand of APOE and promotes signal transduction though APOE to induce, enhance, or promote and immune response in a subject in need thereof.A. APOE Polypeptides

[0071] APOE polypeptides are disclosed as well as binding moieties that specifically bind to APOE, for example bind immunospecifically to APOE, and modulate APOE mediated signal transduction. Modulating APOE mediated signal transduction includes agonizing or antagonizing APOE mediated signal transduction.

[0072] Inhibiting, reducing, or blocking APOE signal transduction in immune cells can occur as a result of the binding moiety binding directly to APOE. In some embodiments the APOE binding moiety binds to APOE and inhibits, reduces or blocks the interaction or association of APOE and one or more of ligands of APOE. APOE binding moieties can include APOE polypeptides including the amino acid sequence of full-length APOE, or a fragment or variant thereof, or a fusion protein thereof.

[0073] In other embodiments, enhancing or promoting APOE signal transduction in immune cells can occur as a result of the binding moiety binding directly to APOE.

[0074] The APOE gene encodes a protein associating with lipid particles that mainly functions in lipoprotein-mediated lipid transport between organs via the plasma and interstitial fluids. APOE is a core component of plasma lipoproteins and is involved in their production, conversion, and clearance. A specific gene that directly causes late-onset Alzheimer's disease has not been identified to date. However, having a genetic variant of the APOE gene on chromosome 19 has been shown to increase a person's risk. The APOE gene is involved in making a protein that helps carry cholesterol and other types of fat in the bloodstream.

[0075] APOE comes in several different forms, or alleles. Each person inherits two APOE alleles, one from each biological parent. APOE s2 is relatively rare and may provide some protection against the disease. If a person with this allele develops Alzheimer's disease, it usually develops later in life than it would in someone with the APOE s4 gene. APOE s3, the most common allele, is believed to play a neutral role in the disease — neither decreasing nor increasing risk. APOE s4 has been shown to increase riskfor Alzheimer's disease and is also associated with an earlier age of disease onset. About25 percent of people carry one copy of APOE e4, and 2 to 3 percent carry two copies.1. Human APOE

[0076] Sequences for human APOE are known in the art. For example, the nucleic acid sequence for human APOE is as follows:

[0077] TTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTTAAGTGATTCGCCCACTGTGGCCTCCCAAAGTGCTG GGATTACAGGCGTGAGCTACCGCCCCCAGCCCCTCCCATCCCACTTCTGTCCAGCCCCCTAGCCCTACTTTCTTTCTGGGATCCAGGAGTCCAGATCCCCAGCCCCCTCTCCAGATTACATTCATCCAGGCACAGGAAAGGACAGGGTCAGGAAAGGAGGACTCTGGGCGGCAGCCTCCACATTCCCCTTCCACGCTTGGCCCCCAGAATG GAGGAGGGTGTCTGTATTACTGGGCGAGGTGTCCTCCCTTCCTGGGGACTGTG GGGGGTGGTCAAAAGACCTCTATGCCCCACCTCCTTCCTCCCTCTGCCCTGCTGTGCCTGGGGCAGGGGGAGAACAGCCCACCTCGTGACTGGGGGCTGGCCCAG CCCGCCCTATCCCTGGGGGAGGGGGCGGGACAGGGGGAGCCCTATAATTGGACAAGTCTGGGATCCTTGAGTCCTACTCAGCCCCAGCGGAGGTGAAGGACGTCCTTCCCCAGGAGCCGGTGAGAAGCGCAGTCGGGGGCACGGGGATGAGCTCAGGGGCCTCTAGAAAGAGCTGGGACCCTGGGAACCCCTGGCCTCCAGGTAGTCTCAGGAGAGCTACTCGGGGTCGGGCTTGGGGAGAGGAGGAGCGGGGGTGAGG CAAGCAGCAGGGGACTGGACCTGGGAAGGGCTGGGCAGCAGAGACGACCCGACCCGCTAGAAGGTGGGGTGGGGAGAGCAGCTGGACTGGGATGTAAGCCATAGCAGGACTCCACGAGTTGTCACTATCATTTATCGAGCACCTACTGGGTGTCC CCAGTGTCCTCAGATCTCCATAACTGGGGAGCCAGGGGCAGCGACACGGTAGCTAGCCGTCGATTGGAGAACTTTAAAATGAGGACTGAATTAGCTCATAAATGGAACACGGCGCTTAACTGTGAGGTTGGAGCTTAGAATGTGAAGGGAGAATGA GGAATGCGAGACTGGGACTGAGATGGAACCGGCGGTGGGGAGGGGGTGGGGGGATGGAATTTGAACCCCGGGAGAGGAAGATGGAATTTTCTATGGAGGCCGA CCTGGGGATGGGGAGATAAGAGAAGACCAGGAGGGAGTTAAATAGGGAATGGGTTGGGGGCGGCTTGGTAAATGTGCTGGGATTAGGCTGTTGCAGATAATGCAACAAGGCTTGGAAGGCTAACCTGGGGTGAGGCCGGGTTGGGGCCGGGCTGG GGGTGGGAGGAGTCCTCACTGGCGGTTGATTGACAGTTTCTCCTTCCCCAGACTGGCCAATCACAGGCAGGAAGATGAAGGTTCTGTGGGCTGCGTTGCTGGTCA CATTCCTGGCAGGTATGGGGGCGGGGCTTGCTCGGTTCCCCCCGCTCCTCCCCCTCTCATCCTCACCTCAACCTCCTGGCCCCATTCAGGCAGACCCTGGGCCCCCTCTTCTGAGGCTTCTGTGCTGCTTCCTGGCTCTGAACAGCGATTTGACGCTCTCTGGGCCTCGGTTTCCCCCATCCTTGAGATAGGAGTTAGAAGTTGTTTTGTTGTTGTTGTTTGTTGTTGTTGTTTTGTTTTTTTGAGATGAAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGGGATCTCGGCTCACTGCAAGCTCCGCCTCCCAGGTCCACGCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACATGCCACCACACCCGACTAACTTTTTTGTATTTTCAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTGGAACTCCTGACCTCAGGTGATCTGCCCGTTTCGATCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCACCTGGCTGGGAGTTAGAGGTTTCTAATGCATTGCAGGCAGATAGTGAATACCAGACACGGGGCAGCTGTGATCTTTATTCTCCATCACCCCCACACAGCCCTGCCTGGGGCACACAAGGACACTCAATACATGCTTTTCCGCTGGGCGCGGTGGCTCACCCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGAGGATCACTTGAGCCCAGGAGTTCAACACCAGCCTGGGCAACATAGTGAGACCCTGTCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGCCACACACCTGTGCTCTCAGCTACTCAGGAGGCTGAGGCAGGAGGATCGCTTGAGCCCAGAAGGTCAAGGTTGCAGTGAACCATGTTCAGGCCGCTGCACTCCAGCCTGGGTGACAGAGCAAGACCCTGTTTATAAATACATAATGCTTTCCAAGTGATTAAACCGACTCCCCCCTCACCCTGCCCACCATGGCTCCAAAGAAGCATTTGTGGAGCACCTTCTGTGTGCCCCTAGGTACTAGATGCCTGGACGGGGTCAGAAGGACCCTGACCCACCTTGAACTTGTTCCACACAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGTGAGTGTCCCCATCCTGGCCCTTGACCCTCCTGGTGGGCGGCTATACCTCCCCAGGTCCAGGTTTCATTCTGCCCCTGTCGCTAAGTCTTGGGGGGCCTGGGTCTCTGCTGGTTCTAGCTTCCTCTTCCCATTTCTGACTCCTGGCTTTAGCTCTCTGGAATTCTCTCTCTCAGCTTTGTCTCTCTCTCTTCCCTTCTGACTCAGTCTCTCACACTCGTCCTGGCTCTGTCTCTGTCCTTCCCTAGCTCTTTTATATAGAGACAGAGAGATGGGGTCTCACTGTGTTGCCCAGGCTGGTCTTGAACTTCTGGGCTCAAGCGATCCTCCCGCCTCGGCCTCCCAAAGTGCTGGGATTAGAGGCATGAGCCACCTTGCCCGGCCTCCTAGCTCCTTCTTCGTCTCTGCCTCTGCCCTCTGCATCTGCTCTCTGCATCTGTCTCTGTCTCCTTCTCTCGGCCTCTGCCCCGTTCCTTCTCTCCCTCTTGGGTCTCTCTGGCTCATCCCCATCTCGCCCGCCCCATCCCAGCCCTTCTCCCCGCCTCCCACTGTGCGACACCCTCCCGCCCTCTCGGCCGCAGGGCGCTGATGGACGAGACCATGAAGGAG TTGAAGGCCTACAAATCGGAACTGGAGGAACAACTGACCCCGGTGGCGGAGG AGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGG GCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCC CACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGA AGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGG GCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGG CCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGC TGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCG CCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGC CCAGCGACAATCACTGAACGCCGAAGCCTGCAGCCATGCGACCCCACGCCAC CCCGTGCCTCCTGCCTCCGCGCAGCCTGCAGCGGGAGACCCTGTCCCCGCCCCAGCCGTCCTCCTGGGGTGGACCCTAGTTTAATAAAGATTCACCAAGTTTCACG CATCTGCTGGCCTCCCCCTGTGATTTCCTCTAAGCCCCAGCCTCAGTTTCTCTT TCTGCCCACATACTGGCCACACAATTCTCAGCCCCCTCCTCTCCATCTGTGTCTGTGTGTATCTTTCTCTCTGCCCTTTTTTTTTTTTTTAGACGGAGTCTGGCTCTGT CACCCAGGCTAGAGTGCAGTGGCACGATCTTGGCTCACTGCAACCTCTGCCTC TTGGGTTCAAGCGATTCTGCTGCCTCAGTAGCTGGGATTACAGGCTCACACCACCACACCCGGCTAATTTTTGTATTTTTAGTAGAGACGAGCTTTCACCATGTTG GCCAGGCAGGTCTCAAACTCCTGACCAAGTGATCCACCCGCCGGCCTCCCAA AGTGCTGAGATTACAGGCCTGAGCCACCATGCCCGGCCTCTGCCCCTCTTTCTTTTTTAGGGGGCAGGGAAAGGTCTCACCCTGTCACCCGCCATCACAGCTCACT GCAGCCTCCACCTCCTGGACTCAAGTGATAAGTGATCCTCCCGCCTCAGCCTT TCCAGTAG (SEQ ID N0:51, NCBI Reference Sequence: NG_007084.2 which is incorporated by reference in its entirety).

[0078] The consensus amino acid sequence for APOE is:MKVLWAALLVTFLAGCOAKVEOAVETEPEPELROQTEWOSGORWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAAT VGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQ AQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH (SEQ ID NO:52 NP 000032.1, UniProtKB Identifier P02649 which is incorporated by reference in its entirety).

[0079] One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO: 52 or a functional fragment thereof and modulates APOE mediated signal transduction.

[0080] The underlined amino acids 1-18 of SEQ ID NO:52 represent the signal sequence of human APOE. The unmarked amino acids 19-317 of SEQ ID NO: 52 represent the polypeptide chain in the mature human APOE protein following processing or proteolytic cleavage and has the following sequence:KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLS SQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGAD MEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRI<LRI<RLLRDADDLQI<RLA VYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGER LRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFE PLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH (SEQ ID NO: 53).

[0081] One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:53, or a functional fragment thereof, and modulates APOE mediated signal transduction.2. Murine APOE

[0082] The amino acid sequence for murine APOE is:MKALWAVLLVTLLTGCLAEGEPEVTDQLEWQSNQPWEQALNRFWDYLRWVQT LSDQVQEELQSSQVTQELTALMEDTMTEVKAYKKELEEQLGPVAEETRARLGKE VQAAQARLGADMEDLRNRLGQYRNEVHTMLGQSTEEIRARLSTHLRKMRKRL MRDAEDLQKRLAVYKAGAREGAERGVSAIRERLGPLVEQGRQRTANLGAGAAQ PLRDRAQAFGDRIRGRLEEVGNQARDRLEEVREHMEEVRSKMEEQTQQIRLQAE IFQARLKGWFEPIVEDMHRQWANLMEKIQASVATNPIITPVAQENQ (SEQ ID NO:54) Uniprot accession number P08226 which is incorporated by reference in its entirety.

[0083] The underlined amino acids 1-18 of SEQ ID NO:54 represent the signal sequence of mouse APOE. The unmarked amino acids 19-311 of SEQ ID NO: 54 represent the polypeptide chain in the mature human APOE protein following processing or proteolytic cleavage and has the following sequence:EGEPEVTDQLEWQSNQPWEQALNRFWDYLRWVQTLSDQVQEELQSSQVTQELT ALMEDTMTEVKAYKKELEEQLGPVAEETRARLGKEVQAAQARLGADMEDLRN RLGQYRNEVHTMLGQSTEEIRARLSTHLRI<MRI<RLMRDAEDLQI<RLAVYI<AG AREGAERGVSAIRERLGPLVEQGRQRTANLGAGAAQPLRDRAQAFGDRIRGRLE EVGNQARDRLEEVREHMEEVRSKMEEQTQQIRLQAEIFQARLKGWFEPIVEDMH RQWANLMEKIQASVATNPIITPVAQENQ (SEQ ID NO:55).

[0084] One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:55, or a functional fragment thereof, and modulates APOE mediated signal transduction.B. Immunomodulatory Agents or Binding Moieties

[0085] One embodiment provides immunomodulatory agents that specifically bind to a ligand of APOE and modulates APOE mediated signal transduction

[0086] Immunomodulatory agents or binding moieties, including agonists and antagonists of APOE, are provided. An agonist of APOE typically induces, promotes, or enhances APOE mediated signaling. An antagonist of APOE typically inhibits, reduces, or blocks APOE mediated signaling. The disclosed compositions and methods can be used to modulate APOE and / or counter-receptor signaling on, for example, immune cells including but not limited to monocytes, Tregs, Myeloid Derived Suppressor Cells (MDSC), T cells, Th2 cells, myeloid cells including antigen-presenting cells (e.g., monocyte, macrophage, or dendritic cell), Natural Killer (NK) cells, or a combination thereof. In some embodiments, the compositions are specifically targeted to one or more cells types. In some embodiments, the disclosed compositions can be used on tumor cells.

[0087] In some embodiments, the anti-APOE agonists induce, promote, or enhance APOE mediated signaling through a known ligand or unknown counter-receptor through APOE interaction with said known or unknown counter-receptor. For example, in some embodiments, the APOE agonist binds to, induces, promotes or creates a conformations change, or otherwise promotes APOE mediated signal transduction.

[0088] In some embodiments, the anti-APOE antagonists inhibit, reduce, block, or otherwise disrupt signaling through a known or unknown counter-receptor through blockade of APOE interaction with said known or unknown counter-receptor. For example, in some embodiments, the APOE antagonist binds to, inhibits, blocks, creates a conformations change, or otherwise interferes with APOE mediated signal transduction.1. Antibodies

[0089] In one embodiment the immunomodulatory agent or binding moiety is an antibody. Suitable antibodies can be prepared using similar methods familiar to those with skill in the art (for example, see Almagro, et al. Front. Biosci. 2008, 13(5): 1619-33).. Nucleic acid and polypeptide sequences for APOE are known in the art and exemplary sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare an antibody or antigen binding fragment thereof specific for APOE. The antibody or antigen binding fragment therefore, can be an agonist or antagonist of APOE mediated signaling.

[0090] The activity of an antibody or antigen binding fragment thereof that is specific for APOE can be determined using functional assays that are known in the art, and include the assays discussed below. Typically, the assays include determining if the antibody or antigen binding fragment thereof increases (z.e., agonist) or decreases (z.e., antagonist) signaling through APOE.

[0091] In some embodiments, the disclosed antibodies and antigen binding fragments thereof immunospecifically bind to human or mouse APOE. In some embodiments, the antibody binds to an extracellular domain of human or mouse APOE.

[0092] For example, molecules are provided that can immunospecifically bind to APOE:(I) arrayed on the surface of a cell (especially a live cell);(II) arrayed on the surface of a cell (especially a live cell) at an endogenous concentration;(III) arrayed on the surface of a live cell, and modulates binding between APOE and a ligand thereof;(IV) arrayed on the surface of a live cell, and reduces or inhibits immune response by APOE;(V) arrayed on the surface of a live cell, wherein the cell is a tumor cell;(VI) combinations of I-IV and V;(VII) combinations of I-III and V; and

[0093] To prepare an antibody or antigen binding fragment thereof that specifically binds to APOE purified proteins, polypeptides, fragments, fusions, or epitopes to APOE or polypeptides expressed from nucleic acid sequences thereof can be used. The antibodies or antigen-binding fragments thereof can be prepared using any suitable methods known in the art, such as those discussed in more detail below. a. APOE Antibodies

[0094] The sequences of light and heavy chain variable regions for monoclonal APOE antibodies are provided below. CDRs are underlined and bolded in the context of the light and heavy chain sequences. i. Variable Light Chain aa. APOE mAb VL-1

[0095] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFLQRPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEI K* (SEQ ID NO: 1).

[0096] The light chain CDRs of APOE mAb VL-1 are bolded and are as follows:

[0097] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0098] CDR2 KVSNRFS SEQ ID NO: 42

[0099] CDR3 FQGSHVPYT SEQ ID NO: 43 bb. APOE mAb VL-1 Light chain (IGKC)

[0100] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFLQRPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQ ID NO: 2).

[0101] The light chain CDRs of APOE mAh VL-1 Light chain (kappa constant domain (IGKC)) are bolded and are as follows:

[0102] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0103] CDR2 KVSNRFS SEQ ID NO: 42

[0104] CDR3 FQGSHVPYT SEQ ID NO: 43

[0105] The human kappa light chain is underlined and has at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 37). cc. APOE mAb VL-2

[0106] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKL EIK* SEQ ID NO:3).

[0107] The light chain CDRs of APOE mAb VL-2 are bolded and are as follows:

[0108] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0109] CDR2 KVSNRFS SEQ ID NO: 42

[0110] CDR3 FQGSHVPYT SEQ ID NO: 43 dd. APOE mAb VL-2 Light chain (IGKC)

[0111] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO:4).

[0112] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-2 Light chain (IGKC) are bolded and are as follows:

[0113] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0114] CDR2 KVSNRFS SEQ ID NO: 42

[0115] CDR3 FQGSHVPYT SEQ ID NO: 43 ee. APOE mAb VL-3

[0116] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFLQKPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEI K* (SEQ ID NO:5).

[0117] The light chain CDRs of APOE mAb VL-3 are bolded and are as follows:

[0118] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0119] CDR2 KVSNRFS SEQ ID NO: 42

[0120] CDR3 FQGSHVPYT SEQ ID NO: 43 ff. APOE mAb VL-3 Light chain (IGKC)

[0121] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFLQKPGQSPELLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEI KRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSO ESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 6).

[0122] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-3 Light chain (IGKC) are bolded and are as follows:

[0123] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0124] CDR2 KVSNRFS SEQ ID NO: 42

[0125] CDR3 FQGSHVPYT SEQ ID NO: 43 gg. APOE mAb VL-4

[0126] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKL EIK* (SEQ ID NO: 7).

[0127] The light chain CDRs of APOE mAb VL-4 are bolded and are as follows:

[0128] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0129] CDR2 KVSNRFS SEQ ID NO: 42

[0130] CDR3 FQGSHVPYT SEQ ID NO: 43 hh. APOE mAb VL-4 Light chain (IGKC)

[0131] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGNTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO:8).

[0132] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-4 Light chain (IGKC) are bolded and are as follows:

[0133] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0134] CDR2 KVSNRFS SEQ ID NO: 42

[0135] CDR3 FQGSHVPYT SEQ ID NO: 43ii. APOE mAb VL-5

[0136] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGNTYLEWFLQKPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEI K* (SEQ ID NO:9).

[0137] The light chain CDRs of APOE mAb VL-5 are bolded and are as follows:

[0138] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0139] CDR2 KVSNRFS SEQ ID NO: 42

[0140] CDR3 FQGSHVPYT SEQ ID NO: 43 jj. APOE mAb VL-5 Light chain (IGKC)

[0141] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGNTYLEWFLQKPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSO ESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 10).

[0142] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-5 Light chain (IGKC) are bolded and are as follows:

[0143] CDR1 RSSQNIIHSNGNTYLE SEQ ID NO: 41

[0144] CDR2 KVSNRFS SEQ ID NO: 42

[0145] CDR3 FQGSHVPYT SEQ ID NO: 43 kk. APOE mAb VL-6

[0146] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGNTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKL EIK* SEQ ID NO: 11).

[0147] The light chain CDRs of APOE mAh VL-6 are bolded and are as follows:

[0148] CDR1 RSSQNIIHSQGNTYLE SEQ ID NO: 44

[0149] CDR2 KVSNRFS SEQ ID NO: 42

[0150] CDR3 FQGSHVPYT SEQ ID NO: 4311. APOE mAb VL-6 Light chain (IGKC)

[0151] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

[0152] DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGNTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYT FGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC* (SEQ ID NO: 12).

[0153] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-6 Light chain (IGKC) are bolded and are as follows:

[0154] CDR1 RSSQNIIHSQGNTYLE SEQ ID NO: 44

[0155] CDR2 KVSNRFS SEQ ID NO: 42

[0156] CDR3 FQGSHVPYT SEQ ID NO: 43 mm. APOE mAb VL-7

[0157] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGQTYLEWFLQKPGQSPELLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEI K* (SEQ ID NO: 13).

[0158] The light chain CDRs of APOE mAb VL-7 are bolded and are as follows:

[0159] CDR1 RSSQNIIHSNGQTYLE SEQ ID NO: 45

[0160] CDR2 KVSNRFS SEQ ID NO: 42

[0161] CDR3 FQGSHVPYT SEQ ID NO: 43 nn. APOE mAb VL-7 Light chain (IGKC)

[0162] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGQTYLEWFLQKPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSO ESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 14).

[0163] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-7 Light chain (IGKC) are bolded and are as follows:

[0164] CDR1 RSSQNIIHSNGQTYLE SEQ ID NO: 45

[0165] CDR2 KVSNRFS SEQ ID NO: 42

[0166] CDR3 FQGSHVPYT SEQ ID NO: 43 oo. APOE mAb VL-8

[0167] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGQTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKL EIK* (SEQ ID NO: 15).

[0168] The light chain CDRs of APOE mAb VL-8 are bolded and are as follows:

[0169] CDR1 RSSQNIIHSNGQTYLE SEQ ID NO: 45

[0170] CDR2 KVSNRFS SEQ ID NO: 42

[0171] CDR3 FQGSHVPYT SEQ ID NO: 43pp. APOE mAb VL-8 Light chain (IGKC)

[0172] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSNGQTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 16).

[0173] The human kappa light chain is underlined and the light chain CDRs of APOE mAh VL-8 Light chain (IGKC) are bolded and are as follows:

[0174] CDR1 RSSQNIIHSNGQTYLE SEQ ID NO: 45

[0175] CDR2 KVSNRFS SEQ ID NO: 42

[0176] CDR3 FQGSHVPYT SEQ ID NO: 43 qq. APOE mAb VL-9

[0177] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

[0178] DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGQTYLEWFLQKPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYT FGGGTKLEIK* SEQ ID NO: 17).

[0179] The light chain CDRs of APOE mAb VL-9 are bolded and are as follows:

[0180] CDR1 RSSQNIIHSQGQTYLE SEQ ID NO: 46

[0181] CDR2 KVSNRFS SEQ ID NO: 42

[0182] CDR3 FQGSHVPYT SEQ ID NO: 43 rr. APOE mAb VL-9 Light chain (IGKC)

[0183] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

[0184] DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGQTYLEWFLQKPGQSPELLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYT FGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC* (SEQ ID NO: 18).

[0185] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-9 Light chain (IGKC) are bolded and are as follows:

[0186] CDR1 RSSQNIIHSQGQTYLE SEQ ID NO: 47

[0187] CDR2 KVSNRFS SEQ ID NO: 42

[0188] CDR3 FQGSHVPYT SEQ ID NO: 43 ss. APOE mAb VL-10

[0189] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

[0190] DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGQTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYT FGGGTKLEIK* (SEQ ID NO: 19).

[0191] The light chain CDRs of APOE mAb VL-10 are bolded and are as follows:

[0192] CDR1 RSSQNIIHSQGQTYLE SEQ ID NO: 47

[0193] CDR2 KVSNRFS SEQ ID NO: 42

[0194] CDR3 FQGSHVPYT SEQ ID NO: 43 tt. APOE mAb VL-10 Light chain (IGKC)

[0195] One embodiment provides a monoclonal antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

[0196] DVVMTQSPLSLPVTLGQPASISCRSSQNIIHSQGQTYLEWFQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCFQGSHVPYT FGGGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC* (SEQ ID NO:20).

[0197] The human kappa light chain is underlined and the light chain CDRs of APOE mAb VL-10 Light chain (IGKC) are bolded and are as follows:

[0198] CDR1 RSSQNIIHSQGQTYLE SEQ ID NO: 47

[0199] CDR2 KVSNRFS SEQ ID NO: 42

[0200] CDR3 FQGSHVPYT SEQ ID NO: 43 ii. Variable Heavy Chain aa. APOE mAb VH-1

[0201] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYFCAR YYSSSPFAY WGQGTLVTVSS* (SEQ ID NO:21).

[0202] The heavy chain CDRs of APOE mAb VH-1 are bolded and are as follows:

[0203] CDR1 GYIFTDY SEQ ID NO: 48

[0204] CDR2 FPGSGVSY SEQ ID NO: 49

[0205] CDR3 YYSSSPFAY SEQ ID NO: 50 bb. APOE mAb VH-1 Heavy Chain (IgGl constant domain)

[0206] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFPGSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYFCAR YYSSSPFAYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO:22).

[0207] The heavy chain CDRs of APOE mAb VH-1 Heavy Chain (IgGl constant domain) are bolded and are as follows:

[0208] CDR1 GYIFTDY SEQ ID NO: 48

[0209] CDR2 FPGSGVSY SEQ ID NO: 49

[0210] CDR3 YYSSSPFAY SEQ ID NO: 50

[0211] The human IgGl Fc is underlined and has at least 60, 65, 70, 7595, 99, or 100% sequence identity with the following:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G* (SEQ ID NO: 38) cc. APOE mAb VH-1 Heavy Chain (IgGl constant domain, G1FES)

[0212] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYFCAR YYSSSPFAYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPASIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVE WESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTQKSLSLSPG* (SEQ ID NO:23).

[0213] The heavy chain CDRs of APOE mAh VH-1 Heavy Chain (IgGl constant domain, G1FES) are bolded and are as follows:

[0214] CDR1 GYIFTDY SEQ ID NO: 48

[0215] CDR2 FPGSGVSY SEQ ID NO: 49

[0216] CDR3 YYSSSPFAY SEQ ID NO: 50

[0217] The human IgGl (FES) region is double underlined and has at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G* (SEQ ID NO: 39). dd. APOE mAb VH-1 Heavy Chain (IgG4 constant domain, G4P)

[0218] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYFCAR YYSSSPFAY WGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCL VKD YFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIA VEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLG* (SEQ ID NO:24).

[0219] The heavy chain CDRs of APOE mAb VH-1 Heavy Chain (IgG4 constant domain, G4P) are bolded and are as follows:

[0220] CDR1 GYIFTDY SEQ ID NO: 48

[0221] CDR2 FPGSGVSY SEQ ID NO: 49

[0222] CDR3 YYSSSPFAY SEQ ID NO: 50

[0223] The human IgG4 (G4P) region is italicized and bolded and has at least 60, 65,70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ S SGL YSLS S VVT VP S S SLGTKT YTCNVDHKP SNTKVDKRVESK YGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG* (SEQ ID NO:40). ee. APOE mAb VH-2

[0224] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFPGSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYYCARYYSSSPFAY WGQGTLVTVSS* (SEQ ID NO:25).

[0225] The heavy chain CDRs of APOE mAh VH-2 are bolded and are as follows:

[0226] CDR1 GYIFTDY SEQ ID NO: 48

[0227] CDR2 FPGSGVSY SEQ ID NO: 49

[0228] CDR3 YYSSSPFAY SEQ ID NO: 50 ff. APOE mAb VH-2 Heavy Chain (IgGl constant domain)

[0229] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFPGSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYYCAR YYSSSPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTOKSLSLSPG* (SEQ ID NO:26).

[0230] The heavy chain CDRs of APOE mAh VH-2 Heavy Chain (IgGl constant domain) are bolded and are as follows:

[0231] CDR1 GYIFTDY SEQ ID NO: 48

[0232] CDR2 FPGSGVSY SEQ ID NO: 49

[0233] CDR3 YYSSSPFAY SEQ ID NO: 50 gg. APOE mAb VH-2 Heavy Chain (IgGl constant domain, G1FES)

[0234] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYYCAR YYSSSPFAY WGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VS NKALPASIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVE WESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTOKSLSLSPG* (SEQ ID NO:27).

[0235] The human IgGl (FES) region is double underlined, and the heavy chain CDRs of APOE mAb VH-2 Heavy Chain (IgGl constant domain, G1FES) are bolded and are as follows:

[0236] CDR1 GYIFTDY SEQ ID NO: 48

[0237] CDR2 FPGSGVSY SEQ ID NO: 49

[0238] CDR3 YYSSSPFAY SEQ ID NO: 50hh. APOE mAb VH-2 Heavy Chain (IgG4 constant domain, G4P)

[0239] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAEKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYYCAR YYSSSPFAY WGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCL VKD YFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIA VEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLG* (SEQ ID NO:28).

[0240] The human IgG4 (G4P) region is italicized and bolded and the heavy chain CDRs of APOE mAb VH-2 Heavy Chain (IgG4 constant domain, G4P) are bolded and are as follows:

[0241] CDR1 GYIFTDY SEQ ID NO: 48

[0242] CDR2 FPGSGVSY SEQ ID NO: 49

[0243] CDR3 YYSSSPFAY SEQ ID NO: 50 ii. APOE mAb VH-3

[0244] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYFCAR YYSSSPFAYWGQGTLVTVSS* (SEQ ID NO:29).

[0245] The heavy chain CDRs of APOE mAb VH-3 are bolded and are as follows:

[0246] CDR1 GYIFTDY SEQ ID NO: 48

[0247] CDR2 FPGSGVSY SEQ ID NO: 49

[0248] CDR3 YYSSSPFAY SEQ ID NO: 50 jj. APOE mAb VH-3 Heavy Chain (IgGl constant domain)

[0249] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYFCAR YYSSSPFAY WGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTQKSLSLSPG* (SEQ ID NO: 30).

[0250] The heavy chain CDRs of APOE mAh VH-3 Heavy Chain (IgGl constant domain) are bolded and are as follows:

[0251] CDR1 GYIFTDY SEQ ID NO: 48

[0252] CDR2 FPGSGVSY SEQ ID NO: 49

[0253] CDR3 YYSSSPFAY SEQ ID NO: 50 kk. APOE mAb VH-3 Heavy Chain (IgGl constant domain, G1FES)

[0254] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFPGSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYFCAR YYSSSPFAYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPASIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVE WESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTOKSLSLSPG* (SEQ ID NO: 31).

[0255] The human IgGl (FES) region is double underlined and the heavy chain CDRs of APOE mAh VH-3 Heavy Chain (IgGl constant domain, G1FES) are bolded and are as follows:

[0256] CDR1 GYIFTDY SEQ ID NO: 48

[0257] CDR2 FPGSGVSY SEQ ID NO: 49

[0258] CDR3 YYSSSPFAY SEQ ID NO: 5011. APOE mAb VH-3 Heavy Chain (IgG4 constant domain, G4P)

[0259] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYFCAR YYSSSPFAY WGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCL VKD YFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIA VEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLG* (SEQ ID NO:32).

[0260] The human IgG4 (G4P) region is italicized and bolded and the heavy chain CDRs of APOE mAb VH-3 Heavy Chain (IgG4 constant domain, G4P) are bolded and are as follows:

[0261] CDR1 GYIFTDY SEQ ID NO: 48

[0262] CDR2 FPGSGVSY SEQ ID NO: 49

[0263] CDR3 YYSSSPFAY SEQ ID NO: 50mm. APOE mAb VH-4

[0264] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYYCAR YYSSSPFAYWGQGTLVTVSS* (SEQ ID NO:33).

[0265] The heavy chain CDRs of APOE mAb VH-4 are bolded and are as follows:

[0266] CDR1 GYIFTDY SEQ ID NO: 48

[0267] CDR2 FPGSGVSY SEQ ID NO: 49

[0268] CDR3 YYSSSPFAY SEQ ID NO: 50 nn. APOE mAb VH-4 Heavy Chain (IgGl constant domain)

[0269] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYYCAR YYSSSPFAY WGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTQKSLSLSPG* (SEQ ID NO:34).

[0270] The heavy chain CDRs of APOE mAb VH-4 Heavy Chain (IgGl constant domain) are bolded and are as follows:

[0271] CDR1 GYIFTDY SEQ ID NO: 48

[0272] CDR2 FPGSGVSY SEQ ID NO: 49

[0273] CDR3 YYSSSPFAY SEQ ID NO: 50oo. APOE mAb VH-4 Heavy Chain (IgGl constant domain, G1FES)

[0274] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFPGSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYYCARYYSSSPFAYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLHODWLNGI<EYI<CI<VSNKALPASIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALH NHYTOKSLSLSPG* (SEQ ID NO:35).

[0275] The human IgGl (FES) region is double underlined and the heavy chain CDRs of APOE mAh VH-4 Heavy Chain (IgGl constant domain, G1FES are bolded and are as follows:

[0276] CDR1 GYIFTDY SEQ ID NO: 48

[0277] CDR2 FPGSGVSY SEQ ID NO: 49

[0278] CDR3 YYSSSPFAY SEQ ID NO: 50 pp. APOE mAb VH-4 Heavy Chain (IgG4 constant domain, G4P)

[0279] One embodiment provides a monoclonal antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYINWVRQAPGQGLEWMGWIFP GSGVSYYAESFKGRVTLTVDKSSSTAYMELSRLRSDDTATYYCAR YYSSSPFAY WGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCL VKD YFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNEFYTQKSLSLSLG* (SEQ ID NO:36).

[0280] The human IgG4 (G4P) region is italicized and bolded and the heavy chain CDRs of APOE mAh VH-4 Heavy Chain (IgG4 constant domain, G4P) are bolded and are as follows:

[0281] CDR1 GYIFTDY SEQ ID NO: 48

[0282] CDR2 FPGSGVSY SEQ ID NO: 49

[0283] CDR3 YYSSSPFAY SEQ ID NO: 50

[0284] In some embodiments, the variable domains are combined according to Table1 showing the APOE mAb variants.

[0285] Tablet : APOE mAb Variants -Combination of Variable Domains.b. Human and Variant Antibodies

[0286] In some embodiments, the antibodies are variant antibodies. Many nonhuman antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized variant antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

[0287] Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production (Jakobovits A, et al., Proc Natl Acad Sci U S A., 15;90(6):2551-5 (1993)) Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.

[0288] Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the variant antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The variant antibody optimally also will contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0289] Methods for creating variant non-human antibodies are well known in the art. Generally, a variant antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody variant creation techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Creating the variant antibodies can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a variant form of a nonhuman antibody (or a fragment thereof) is a chimeric antibody or fragment, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, variant antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies (Almagro, et al. Front. Biosci. 13(5): 1619-33 (2008)).

[0290] The choice of human variable domains, both light and heavy, to be used in making the variant antibodies is very important in order to reduce antigenicity. According to the “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the variant antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different variant antibodies.

[0291] It is further important that antibodies be variant with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, variant antibodies can be prepared by a process of analysis of the parental sequences and various conceptual variant products using three dimensional models of the parental and variant sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, z.e., theanalysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0292] The antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the present compositions include fluorescent, enzymatic and radioactive markers. c. Single-Chain Antibodies

[0293] In some embodiments, the antibodies are single-chain antibodies. Methods for the production of single-chain antibodies are well known to those of skill in the art (Bird RE, et al., Science. 242(4877):423-6 (1988). Erratum in: Science 1989 Apr 28;244(4903):409; Huston, J S et al., PNAS 85,16: 5879-83 (1988)) . A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. d. Monovalent Antibodies

[0294] In some embodiments, the antibodies are monovalent antibodies. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen.

[0295] The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab’fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. e. Hybrid Antibodies

[0296] In some embodiments, the antibodies are hybrid antibodies. In hybrid antibodies, one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multifunctional valency, z.e., ability to bind at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques. Such hybrids may, of course, also be formed using chimeric chains. f. Conjugates or Fusions of Antibody Fragments

[0297] In some embodiments, the antibodies are conjugates or fusions of antibody fragments. The targeting function of the antibody can be used therapeutically by coupling the antibody or a fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent.

[0298] Such coupling of the antibody or fragment with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, or by linking the antibody or fragment to a nucleic acid such as an siRNA, comprising the antibody or antibody fragment and the therapeutic agent.

[0299] In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the site of treatment for longer periods of time. For example, it may be desirable to maintain titers of the antibody in the circulation or in the location to be treated for extended periods of time. Antibodies can be engineered with Fc variants thatextend half-life, e.g., using Xtend™ antibody half-life prolongation technology (Xencor, Monrovia, CA). In other embodiments, the half-life of the anti-DNA antibody is decreased to reduce potential side effects. The conjugates disclosed can be used for modifying a given biological response. The drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.

[0300] In other embodiments, the APOE antibodies comprise and additional motif to enhance penetration through the blood-brain barrier. Such motifs are selected from the group consisting of transferrin receptor, CD98hc, basigin , Glutl extracellular domain (ECD) 1, ECD2, ECD3, ECD4, ECD5 and ECD6, IGF-1R, INSR ECD1, and INSRECD2.

[0301] Protein- and antibody-based therapeutics delivery into the brain offer huge potential in treating a broad spectrum of central nervous system (CNS) diseases, including cancer (e.g., glioblastoma (GBM) and glioma), neurodegenerative diseases (e.g., Alzheimer’s disease (AD), cerebral amyloid angiopathy (CAA), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD)), autoimmune diseases (e.g., multiple sclerosis), nervous system diseases (e.g., amyotrophic lateral sclerosis), and genetic disorders (e.g., lysosomal storage diseases (LSDs)). However, a significant hurdle for protein and antibody therapeutics to enter the brain is the blood-brain barrier (BBB). BBB is formed by a continuous monolayer of brain endothelial cells (BECs), pericytes, and astrocytes. The continuous non-fenestrated capillaries integrity is maintained by tight junctions (such as occludin, claudins, and other junctional adhesion molecules) sealing the BECs together. Key nutrients, such as amino acids, glucose, and iron, are transported across the BBB by specific carrier receptors. The tight regulation of peripheral substances in the CNS maintains the homeostasis of the CNS. Lipophilic molecules <400 Da can diffuse through the BBB, with macromolecules showing severely restricted transportation. Beyond BECs, the integrity of BBB is also maintained by the pericytes and astrocytes surrounding the BECs. Astrocyte end-feet and pericytes cover up to 99% of the basal capillary membrane. Additional contributions from factors, such as angiopoietin-1 and angiotensin II, also promote the integrity of the BBB. The multicellular neurovascular barrier regulates the passage of macromolecules across the BBB, which poses substantial challenges in targeting the CNS with antibodies and proteins. Thebiology of BBB is thoroughly reviewed elsewhere and therefore is not a focus of this review.

[0302] There are two transport routes for proteins and antibodies across the BBB: uncontrolled non-specific protein brain entry and controlled endogenous transport systems on the BBB. The nonspecific nature of the absorptive-mediated transport greatly limits the therapeutic potential of therapeutic proteins and antibodies, as indiscriminate cellular uptake is not only a major disadvantage in terms of off-target effects, but it also may lead to the suboptimal pharmacokinetic properties of the therapies. Exploring the endogenous transport systems on the BBB is more desirable for the CNS delivery of protein and antibody therapies. Receptor-mediated transcytosis (RMT) and carrier-mediated transport (CMT) are the major endogenous transport systems on the BBB. The substrate- selective CMT is responsible for the delivery of small molecule nutrients that include glucose, amino acids, monocarboxylic acids, hormones, ions, and vitamins. RMT delivers larger molecules via the vesicular trafficking of the ligand-receptor complexes, such as transferrin, insulin, leptin, tumor necrosis factor-alpha (TNFa), and EGF. Therefore, RMT is the most studied approach for delivering the protein and antibody therapies to the brain. RMT uses endogenous receptors expressed on the luminal side of the BBB, which transport macromolecule nutrients, including iron-bound transferrin, insulin, and leptin, into the brain side via vesicular trafficking of the ligand-receptor complexes. The RTM transport route requires binding to the extracellular domain of the receptor, subsequent endocytosis, and transcytosis to the luminal (brain) side of the capillary endothelium into the interstitial space.

[0303] Transferrin receptor (TfR) is a type II transmembrane receptor and is a homodimer linked by disulfide bonds at Cys89 and Cys98. The extracellular domain of TfR consists of three domains, apical domain, helical domain, and protease-like domain. TfR binds iron-bound holo-Tf via the helical and protease-like domains. At neutral pH, iron binds tightly to Tf, and the iron / Tf complex is trafficked intracellularly via TfR. In the low- PH endosomes, iron is released from Tf. The apo-Tf which is complex with TfR is trafficked back to the cell surface. At neutral pH, the low affinity of apo-Tf to TfR results in the release of apo-Tf. TfR is ubiquitously expressed, with erythroid cells and proliferating cells expressing high levels due to the metabolic demand for iron. BECs were reported to express TfR as well. Transferrin was shown to be transported through the BBB, indicating the TfR pathway can naturally transport macromolecules across the BBB.

[0304] The representative amino acid sequence for human Transferrin receptor is:

[0305] CKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKL DSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDF EDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFG HAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPS DWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRD AWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGAT EWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTG QFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTY KELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQY RADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRV EYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLA LATWTIQGAANALSGDVWDIDNEF (SEQ ID NO:56).

[0306] CD98hc forms covalent heterodimers with various large amino acid transporters, including LAT1, and is essential for trafficking these transporters to the cell surface. The expression of CD98hc on brain endothelial cells is high, and prior work has validated it in both mice and cynomolgus monkeys.

[0307] The representative amino acid sequence for human CD98hc is:

[0308] RAPRCRELPAQKWWHTGALYRIGDLQAFQGHGAGNLAGLKGRLDY LSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFDSLLQSAKKKSIRVIL DLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDIENLKDASSFL AEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGSTGEHTKS LVTQYLNATGNRWCSWSLSQARLLTSFLPAQLLRLYQLMLFTLPGTPVFSYGDEI GLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRL SDQRSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQA SDLPASASLPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA (SEQ ID NO:57).

[0309] Basigin (also known as CD147, EMMPRIN, or BSG) is a transmembrane glycoprotein with multiple functions. It's a type I integral membrane receptor protein belonging to the immunoglobulin superfamily. Basigin plays roles in various biological processes, including immune responses, cell adhesion, and angiogenesis..

[0310] The representative amino acid sequence for human Basigin receptor is:EPGTVFTTVEDLGSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFK VDSDDQWGEYSCVFLPEPMGTANIQLHGPPRVKAVKSSEHINEGETAMLVCKSE S VPP VTDW AWYKITD SEDKALMNGSESRFF VS S SQGRSELHIENLNMEADPGQ Y RCNGTSSKGSDQAIITLRVRSHLA (SEQ ID NO:58)

[0311] GLUT1 (glucose transporter type 1) is a protein that facilitates the transport of glucose across cell membranes. It's encoded by the SLC2A1 gene. GLUT1 deficiency, a rare genetic disorder, can cause seizures and other neurological problems due to impaired glucose transport into the brain. Glut 1 has 6 extracellular domains. Each individual domain is suitable for use to enhance penetration through the blood-brain barrier.

[0312] The representative amino acid sequence for human Glut 1 ECD1 is:NAPQKVIEEFYNQTWVHRYGESILPTTLTTLWS (SEQ ID NO:59).

[0313] The representative amino acid sequence for human Glut 1 ECD2 is:SKLGKSFE (SEQ ID NO: 60).

[0314] The representative amino acid sequence for human Glut 1 ECD3 is:DSIMGNKDL (SEQ ID NO:61).

[0315] The representative amino acid sequence for human Glut 1 ECD4 is:STSIFEKAGVQQP (SEQ ID NO:62).

[0316] The representative amino acid sequence for human Glut 1 ECD5 is:ALLEQLPWMS (SEQ ID NO:63).

[0317] The representative amino acid sequence for human Glut 1 ECD6 is:QYVEQLC (SEQ ID NO: 64).

[0318] . The Insulin-like Growth Factor 1 Receptor (IGF-1R) is a protein that plays a crucial role in brain development, growth, and function. Dysregulation of IGF-1R signaling has been linked to a variety of neurological disorders, particularly those related to neurodegeneration and cognitive impairment.

[0319] The representative amino acid sequence for human IGF-1R is:DVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPF TLYRIDIHSCNHEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDPVFFYVQAKTGYENFIH (SEQ ID NO:65).

[0320] INSR (insulin receptor) dysfunction, particularly insulin resistance in the brain, is implicated in several neurological disorders. Brain insulin resistance can contribute to symptoms like altered eating behavior, mood instability, cognitive impairment, and impaired thermoregulation. It's also linked to the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. INSR has 2 extracellular domains.

[0321] The representative amino acid sequence for human INSR ECD1 is:HLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMI TDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNIT RGSVRIEKNNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTN CPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPT KCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQ YVUTNNKCIPECPSGYTMNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRG CTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRG ETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEV SGTKGRQERNDIALKTNGDQASCENELLKFSYIRTSFDKILLRWEPYWPPDFRDL LGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLM RGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSS QIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTWSPPFESE DSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRKTSS GTGAEDPRPS (SEQ ID NO:66).

[0322] The representative amino acid sequence for human INSR ECD2 is:SLGDVGNVTVAVPTVAAFPNTSSTSVPTSPEEHRPFEKVVNKESLVISGLRHFTGY RIELQACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQ EPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRA TSLAGNGSWTEPTYFYVTDYLDVPSNIAK (SEQ ID NO: 67).2. Proteins and Polypeptides a. Protein and Polypeptide Compositions

[0323] The immunomodulatory or binding agent can be a APOE protein, polypeptide, or fusion protein. For example, the immunomodulatory agent or binding moiety can be anisolated or recombinant protein or polypeptide, or functional fragment, variant, or fusion protein thereof of APOE.

[0324] The APOE protein or polypeptide, or functional fragment, variant, or fusion protein thereof can be an agonist or an antagonist. For example, in some embodiments an antagonist of APOE is a APOE polypeptide or a fragment or fusion protein thereof that binds to a ligand of APOE. The polypeptide can be a soluble fragment, for example the extracellular domain of APOE, or a functional fragment thereof, or a fusion protein thereof. In some embodiments, a soluble ligand of APOE may serve as an antagonist, decreasing APOE mediated signal transduction.

[0325] The activity of a protein or polypeptide of APOE, or any fragment, variant or fusion protein thereof can be determined using functional assays that are known in the art, and include the assays discussed below. Typically, the assays include determining if the protein, polypeptide or fragment, variant or fusion protein thereof increases (z.e., agonist) or decreases (z.e., antagonist) signaling through the APOE receptor. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (z.e., agonist) or decreases (z.e., antagonist) the immune response associated with APOE. Typically, the assays include determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (z.e., agonist) or decreases (z.e., antagonist) signaling through APOE. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof decreases (z.e., agonist) or increases (z.e., antagonist) an immune response regulated by APOE. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (z.e., antagonist) the apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells resulting in reduced self-renewal capacity of AML and ALL stem cells.

[0326] Nucleic acid and polypeptide sequences for APOE are known in the art and exemplary protein and peptide sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare any protein or polypeptide of APOE, or any fragment, variant, or fusion protein thereof. Generally, the proteins, polypeptides, fragments, variants, and fusions thereof of APOE are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence can be replaced by the signalsequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide APOE proteins with and without a signal sequence are disclosed. It is understood that in some cases, the mature protein as it is known or described in the art, z.e., the protein sequence without the signal sequence, is a putative mature protein. During normal cell expression, a signal sequence can be removed by a cellular peptidase to yield a mature protein. The sequence of the mature protein can be determined or confirmed using methods that are known in the art. i. Fragments

[0327] As used herein, a fragment of APOE refers to any subset of the polypeptide that is at least one amino acid shorter than full length protein. Useful fragments include those that retain the ability to bind to their natural ligand or ligands. A polypeptide that is a fragment of any full-length APOE typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural ligand respectively as compared to the full-length protein.

[0328] Fragments of APOE include cell free fragments. Cell free polypeptides can be fragments of full-length, transmembrane, polypeptides that may be shed, secreted or otherwise extracted from the producing cells. Cell free fragments of polypeptides can include some or all of the extracellular domain of the polypeptide, and lack some or all of the intracellular and / or transmembrane domains of the full-length protein. In one embodiment, polypeptide fragments include the entire extracellular domain of the full- length protein. In other embodiments, the cell free fragments of the polypeptides include fragments of the extracellular domain that retain biological activity of full-length protein. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and / or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both. In some embodiments the extracellular domain is the only functional domain of the fragment (e.g., the ligand binding domain). ii. Variants

[0329] Variants of APOE, and fragments thereof are also provided. In some embodiments, the variant is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to SEQ ID NO:2. Useful variants include those that increase biological activity,as indicated by any of the assays described herein, or that increase half-life or stability of the protein. The protein and polypeptides of APOE, and fragments, variants, and fusion proteins thereof can be engineered to increase biological activity. For example, in some embodiments, a APOE polypeptide, protein, or fragment, variant or fusion thereof has been modified with at least one amino acid substitution, deletion, or insertion that increases a function thereof.

[0330] Finally, variant polypeptides can be engineered to have an increased half-life relative to wildtype. These variants typically are modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. The variants can be modified to adjust for effects of affinity for the receptor on the half-life of proteins, polypeptides, fragments, or fusions thereof at serum and endosomal pH. iii. Fusion Proteins

[0331] Fusion polypeptides have a first fusion partner including all or a part of a human or mouse APOE polypeptide fused to a second polypeptide directly or via a linker peptide sequence that is fused to the second polypeptide. In one embodiment, the ECD of human or mouse APOE or a fragment thereof is fused to a second polypeptide. The fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker domain can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide or second polypeptide) of the fusion protein. Similarly, the domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide, second polypeptide or peptide / polypeptide linker domain) of the fusion protein. In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0332] Fusion proteins disclosed herein are of formula I:N-R1-R2-R3-C wherein “N” represents the N-terminus of the fusion protein, “C” represents the C-terminus of the fusion protein. In some embodiments, “Ri” is a polypeptide or protein of APOE or fragment or variant thereof, “R2” is an optional peptide / polypeptide linker domain, and“R3” is a second polypeptide. Alternatively, R3 may be a polypeptide or protein of APOE, or fragment or variant thereof and Ri may be a second polypeptide. In some embodiments, the APOE polypeptide is the extracellular domain.

[0333] Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric / homomultimeric or heterodimeric / heteromultimeric.

[0334] In some embodiments, the fusion protein includes the extracellular domain of APOE, or a fragment or variant thereof, fused to an Ig Fc region. Recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain or a fragment or variant thereof to the Fc region of human IgGl, IgG2, IgG3 or IgG4 or mouse IgG2a, or other suitable Ig domain, as described previously (Chapoval, et al., Methods Mol. Med., 45:247-255 (2000)). iv. Polypeptide Modifications

[0335] The polypeptides and fusion proteins may be modified by chemical moieties that may be present in polypeptides in a normal cellular environment, for example, phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, sumoylation and ubiquitylation. Fusion proteins may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

[0336] The polypeptides and fusion proteins may also be modified by chemical moieties that are not normally added to polypeptides in a cellular environment. For example, the disclosed fusion proteins may also be modified by covalent attachment of polymer chains, including, but not limited to, polyethylene glycol polymer (PEG) chains (i.e., pegylation). Conjugation of macromolecules to PEG has emerged recently as an effective strategy to alter the pharmacokinetic (PK) profiles of a variety of drugs, and thereby to improve their therapeutic potential. PEG conjugation increases retention of drugs in the circulation by protecting against enzymatic digestion, slowing filtration by the kidneys and reducing the generation of neutralizing antibodies. In addition, PEG conjugates can be used to allow multimerization of the fusion proteins.

[0337] Modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is cyclization of the protein.

[0338] Examples of chemical derivatives of the polypeptides include lysinyl and amino terminal residues derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; (9-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may be selectively modified by reaction with carbodiimides (R — N=C=N— R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia. Fusion proteins may also include one or more D-amino acids that are substituted for one or more L-amino acids. v. Modified Binding Properties

[0339] Binding properties of the proteins, polypeptides, fragments, variants and fusions thereof are relevant to the dose and dose regimen to be administered. In one embodiment the disclosed proteins, polypeptides, fragments, variants and fusions thereof have binding properties to APOE or an APOE ligand that demonstrate a higher term, or higher percentage, of occupancy of a binding site (e.g., on the ligand) relative to other receptor molecules that bind thereto. In other embodiments, the disclosed proteins, polypeptides, fragments, variants and fusions thereof have reduced binding affinity to APOE relative to wildtype protein.

[0340] In some embodiments the proteins, polypeptides, fragments, variants and fusions thereof have a relatively high affinity for APOE and may therefore have a relatively slow off rate. In other embodiments, the proteins polypeptides, fragments, variants and fusions thereof are administered intermittently over a period of days, weeks or months to dampen immune responses which are allowed to recover prior to the next administration, which may serve to alter the immune response without completely turning the immune response on or off and may avoid long term side effects.3. Isolated Nucleic Acid Molecules

[0341] Isolated nucleic acid sequences encoding the APOE proteins, polypeptides, fragments, variants and fusions thereof are disclosed herein. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally- occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.

[0342] An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

[0343] Nucleic acids encoding the proteins, polypeptides, fragments, variants and fusions thereof may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the mammal from which the nucleic acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.

[0344] Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence encoding a polypeptide or protein of APOE. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification canimprove, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5- methyl-2’-deoxycytidine or 5-bromo-2’ -deoxy cytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

[0345] Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N.R., ed., BIOS Scientific Publishers, Oxford, 2008).4. Vectors and Host Cells

[0346] Vectors encoding the proteins, polypeptides, fragments, variants and fusions thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0347] Nucleic acids in vectors can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer can also be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase can transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0348] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0349] An expression vector can include a tag sequence. Tag sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A. In one embodiment, a nucleic acid molecule encoding one of the disclosed polypeptides is present in a vector containing nucleic acids that encode one or more domains of an Ig heavy chain constant region, for example, having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl chain.

[0350] Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells bytechniques including, for example, calcium phosphate co-precipitation, DEAE-dextran- mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell) can be used to, for example, produce the proteins, polypeptides, fragments, variants and fusions thereof described herein.

[0351] The vectors described can be used to express the proteins, polypeptides, fragments, variants and fusions thereof in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one embodiment, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.

[0352] In vivo nucleic acid therapy can be accomplished by direct transfer of a functionally active DNA into mammalian somatic tissue or organ in vivo. For example, nucleic acids encoding polypeptides disclosed herein can be administered directly to lymphoid tissues. Alternatively, lymphoid tissue specific targeting can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs) such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue specific TREs are known in the art.

[0353] Nucleic acids may also be administered in vivo by viral means. Nucleic acid molecules encoding fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art. Other virus vectors may also be used, including recombinant adenoviruses and vacciniavirus, which can be rendered non-replicating. In addition to naked DNA or RNA, or viral vectors, engineered bacteria may be used as vectors.

[0354] Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles and polycations such as asialoglycoprotein / polylysine.

[0355] In addition to virus- and carrier-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA and particle-bombardment mediated gene transfer.5. Small Molecules

[0356] The immunomodulatory agent can be a small molecule. Small-molecule agonists and antagonists APOE are known in the art or can be identified using routine screening methods.

[0357] In some embodiments, screening assays can include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on particular classes of compounds suspected of modulating the level of APOE. Assays can include determinations of APOE-mediated signaling activity. Other assays can include determinations of nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.C. Antibody-Drug Conjugates

[0358] Antibody-drug conjugates of the invention are comprised of an antibody or fragment thereof directed to APOE (described above); a linker that links a payload to the antibody; and may further include an optional moiety that binds to one protein of a group consisting of transferrin receptor, CD98hc, basigin, Glutl extracellular domain (ECD) 1, ECD2, ECD3, ECD4, ECD5 and ECD6, IGF-1R, INSR ECD1, and INSR ECD2.1. Linkers

[0359] An ADCs of the invention comprises an antibody to APOE and at least one drug(s), whereby the antibody and the at least one drug are conjugated by a linker. The term “linker,” as used herein, refers to a chemical moiety that may be bifunctional or multifunctional, and is used to attach an antibody to a drug moiety. A linker may include one conjugating component or may include multiple components.

[0360] For example, the linker may include a spacer, which is a moiety that extends the drug linkage to avoid, for example, shielding the active site of the antibody or improvingthe solubility of the ADC. Other examples of components of linkers include a stretcher unit and an amino acid unit.

[0361] The linker described herein may be cleavable, non-cleavable and hydrophilic or hydrophobic.

[0362] In certain embodiments, the cleavable linker is cleavable under intracellular or extracellular conditions, by which an active agent is released from an antibody constructactive agent conjugate in the intracellular environment.

[0363] The cleavable linker can be cleaved by a cleaving agent present in an intracellular environment (e.g., lysosomes, endosomes, or caveolea). The cleavable linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not being limited to, a lysosomal or endosomal protease. Generally, the peptidyl linker has a length of at least two amino acids or a length of at least three amino acids. Cleaving agents may include MMP9, cathepsin B, cathepsin D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release an active drug in target cells (e.g., see Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67- 123). The most common are peptidyl linkers cleavable by enzymes present in antigenexpressing cells. For example, peptidyl linkers cleavable by thiol -dep endent protease cathepsin-B, which is highly expressed in the antigen presenting cell, may be used (e.g., a Phe-Leu or Gly-Phe-Leu-Gly linker). Other examples of these linkers are described in, for example, U.S. Patent No.6, 214, 345. In addition, the peptidyl linker cleavable by an intracellular protease may be, for example, a Val-Cit linker, a Phe-Lys linker (e.g., see U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker), or a Vai-Ala linker. The Val-Cit linker or the Vai-Ala linker may contain a pentafluorophenyl group and may contain a succinimide group or a maleimide group. Alternatively, the Val-Cit linker or the Vai-Ala linker may contain a pentafluorophenyl group, may contain a 4-aminobenzoic acid (PABA) group and a maleimide group, and may contain a PABA group and a succinimide group. Another linker may be acetyl cholinesterase / butryl cholinesterase (AChE / BChE). A further linker may be an a-secretase cleavable linker.

[0364] In addition, a cleavable linker may be easily hydrolyzed in a pH-sensitive manner, i.e., at certain pH values. Generally, the pH-sensitive linker may be hydrolyzed under acidic conditions. For example, acid-labile linkers that can be hydrolyzed inlysosomes (e.g., hydrazone, semi carb azone, thiosemicarbazone, cis-aconic amides, orthoesters, acetals, and ketals) may be used (e.g., see: U.S. Patent NOs. 5,122,368, 5,824,805, and 5,622,929; and Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67- 123; and Neville et al., 1989, Biol. Chem. 264: 14653-14661). These linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH 5.5, which is the approximate pH of lysosomes, or less than pH 5.0. Examples of hydrolysable linkers include thioether linkers (e.g., thioethers attached to a therapeutic agent via an acylhydrazone bond) (e.g., see U.S. Patent No.5, 622, 929).

[0365] Also, the linker is cleavable under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers including N-succinimidyl-5- acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N- succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), and N-succinimidyl-oxycarbonyl- alpha-methyl-alpha-(2-pyridyl-thio)toluene)- (SMPT), and those that can be formed using SPDB and SMPT (e.g., see: Thorpe et al., 1987, Cancer Res.47: 5924-5931; and U.S. Patent No.4, 880, 935).

[0366] In addition, the linker may be a malonate linker (Johnson et al., 1995, Anticancer Res.15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1299-1304), a 3'-N-amide analogue (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12), a P-glucuronide linker (Jeffery et al., 2006, Bioconjug Chem. 17(3):832- 40), or a P-galactoside linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376- 382).

[0367] The non-cleavable linker may be a maleimidocaproyl linker. The maleimidocaproyl linker may include N-maleimidomethylcyclohexane-1 -carboxylate. The maleimidocaproyl linker may contain a succinimide group. The maleimidocaproyl linker may contain a pentafluorophenyl group. The linker may be a combination of a maleimide group and one or more polyethylene glycol molecules. The linker may be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. The linker may be a maleimide-PEG4 linker. The linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. The linker may be a combination of a pentafluorophenyl group and a maleimidocaproyl linker containing one or more polyethylene glycol molecules. The linker may contain a maleimide linked to a polyethylene glycol molecule, wherein the polyethylene glycol allows for morelinker flexibility or allows longer linkers to be used. The linker may be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker.

[0368] In certain embodiments, the linker may be a cleavable linker.

[0369] In certain embodiments, the linker may be a protease-cleavable linker, an acid- cleavable linker, a disulfide linker, a self-immolative linker or a self-stabilizing linker, a malonate linker, a maleimidobenzoyl linker, a 3'-N-amide analogue, a P-glucuronide linker, or a P-galactoside linker.

[0370] In certain embodiments, the protease cleavable linker may include a thiolreactive spacer or a dipeptide, and more specifically, the protease cleavable linker may include a thiol -reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, or a p- amino-benzyloxycarbonyl spacer.

[0371] In certain embodiments, the acid-cleavable linker may be a hydrazine linker or a quaternary ammonium linker.

[0372] Exemplary antibody drug conjugates are disclosed in US 10,583,197, US 9,993,568, US 9,951,072, US 9,919,057, US 9,669,107, US 11,413,353, US 11,173,214, US 11,167,040, US 10,980,890, US 10,583,197, US 10,383,949, US 10,273,235, US 10,183,997, and US 10,118,965, the contents of each of which is fully incorporated by reference herein.

[0373] Multiple methods are commonly used for conjugating drugs to antibodies: i) alkylation of reduced interchain cysteine disulfides through an enzymatically non-cleavable maleimido or simple and cleavable disulfide linker, ii) acylation of lysines by cleavable linear amino acids and iii) prenylation of a linker to a CAAX sequence, wherein C is a cysteine residue, A is an aliphatic amino acid, and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. An exemplary CAAX sequence is CVIM.

[0374] In one aspect, a linker covalently attaches an antibody to a drug moiety. An ADC is prepared using a linker having reactive functionality for binding to the antibody and the drug. For example, a cysteine thiol, or an amine, e.g., N-terminus or amino acid side chain such as lysine, of the antibody may form a bond with a functional group of the linker.

[0375] In one aspect, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method at page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.

[0376] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0377] Suitable linkers include, for example, cleavable and non-cleavable linkers. A linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), proteasesensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020). A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalaninelysine (phe-lys) linker.

[0378] Linkers are preferably stable extracellularly in a sufficient manner to be therapeutically effective. Before transport or delivery into a cell, the ADC is preferably stable and remains intact, i.e. the antibody remains conjugated to the drug moiety. Linkers that are stable outside the target cell may be cleaved at some efficacious rate once inside the cell. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety; and (iii) maintain the therapeutic effect, e.g., cytotoxic effect, of a drug moiety.

[0379] In one embodiment, the linker is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the drug from the antibody in the intracellular environment to be therapeutically effective. In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0380] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2- pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio)toluene), SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press. 1987. See also U.S. Pat. No. 4,880,935).

[0381] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, which is present in the intracellular environment (e.g., within a lysosome or endosome or caveolca). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see. e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers that are cleavable by enzymes that are present in B7-H4-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes.In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val- Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0382] In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1305-12).

[0383] In certain embodiment herein, the ADC does not internalize into cell, yet linkers described in previous examples can be used to release locally payload to increase synaptic and neural functions or decrease inflammation.

[0384] In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. See U.S. Publication No. 20050238649 incorporated by reference herein in its entirety. An ADC comprising a non-cleavable linker may be designed such that the ADC remains substantially outside the cell and interacts with certain receptors on a target cell surface such that the binding of the ADC initiates (or prevents) a particular cellular signaling pathway.

[0385] In some embodiments, the linker is substantially hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used to reduce the extent to which the drug may be pumped out of resistant cells through MDR (multiple drug resistance) or functionally similar transporters.

[0386] In other embodiments, upon cleavage, the linker functions to directly or indirectly inhibit cell growth and / or cell proliferation. For example, in some embodiments, the linker, upon cleavage, can function as an intercalating agent, thereby inhibiting macromolecular biosynthesis (e.g., DNA replication, RNA transcription, and / or protein synthesis).

[0387] In other embodiments, the linker is designed to facilitate bystander killing (the killing of neighboring cells) through diffusion of the linker-drug and / or the drug alone to neighboring cells. In other embodiments, the linker promotes cellular internalization.

[0388] The presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond, enhancing the potency of the ADC. Thus, in one embodiment,the linker includes a sterically hindered disulfide linkage. A sterically hindered disulfide refers to a disulfide bond present within a particular molecular environment, wherein the environment is characterized by a particular spatial arrangement or orientation of atoms, typically within the same molecule or compound, which prevents or at least partially inhibits the reduction of the disulfide bond. Thus, the presence of bulky (or sterically hindering) chemical moieties and / or bulky amino acid side chains proximal to the disulfide bond prevents or at least partially inhibits the disulfide bond from potential interactions that would result in the reduction of the disulfide bond.

[0389] Notably, the aforementioned linker types are not mutually exclusive. For example, in one embodiment, the linker used in the ADCs described herein is a non- cleavable linker that promotes cellular internalization.

[0390] In some embodiments, the ADC has the following formula (formula I):Ab-(L-D)n(I) or a pharmaceutically acceptable salt or solvate thereof; wherein Ab is the antibody, e.g., anti-APOE and (L-D) is a Linker-Drug moiety. The Linker-Drug moiety is made of L- which is a Linker, and -D, which is a drug moiety having, for example, cytostatic, cytotoxic, or otherwise therapeutic activity against APOE and n is an integer from 1 to 20.

[0391] In some embodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is 1.

[0392] In some embodiments, the -D moieties are the same. In yet another embodiment, the -D moieties are different.

[0393] In some embodiments, a linker component comprises an “amino acid unit.” In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21 :778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N- methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). An aminoacid unit may comprise amino acid residues that occur naturally and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0394] In one embodiment, the amino acid unit is valine-citrulline (vc or val-cit). In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.

[0395] Another approach for the generation of ADCs involves the use of heterobifunctional cross-linkers which link the anti-APOE to the drug moiety. Examples of cross-linkers that may be used include N-succinimidyl 4-(5-nitro-2-pyridyldithio)- pentanoate or the highly water-soluble analog N-sulfosuccinimidyl 4-(5-nitro-2- pyridyldithio)-pentanoate. N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N- succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), and N-sulfosuccinimidyl-4-(5- nitro-2-pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2- pyridyldithio)pentanoate (SMNP), N-succinimidyl -4-(5-N,N-dimethylcarboxamido-2- pyridyldithio) butyrate (SCPB) or N-sulfosuccinimidyl4-(5-N,N-dimethylcarboxamido-2- pyridyldithio) butyrate (SSCPB)). The antibodies may be modified with the cross-linkers N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, N-sulfosuccinimidyl 4-(5-nitro-2- pyridyldithio)-pentanoate, SPDB, SNPB, SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of a particular drug that contains a thiol moiety to give excellent yields of an ADC (see also U.S. Pat. No. 6,913,748, incorporated by reference herein).

[0396] In one embodiment, charged linkers (also referred to as pro-charged linkers) are used to conjugate anti-APOE antibodies to drugs to form ADCs. Charged linkers include linkers that become charged after cell processing. The presence of a charged group(s) in the linker of a particular ADC or on the drug after cellular processing provides several advantages, such as (i) greater water solubility of the ADC, (ii) ability to operate at a higher concentration in aqueous solutions, (iii) ability to link a greater number of drug molecules per antibody, potentially resulting in higher potency, (iv) potential for the charged conjugate species to be retained inside the target cell, resulting in higher potency, and (v) improved sensitivity of multidrug resistant cells, which would be unable to exportthe charged drug species from the cell. Examples of some suitable charged or pro-charged cross-linkers and their synthesis are shown in FIGS. 1 to 10 of U.S. Pat. No. 8,236,319, and are incorporated by reference herein. Preferably, the charged or pro-charged cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary amine substituents that significantly increase the solubility of the ADCs, especially for ADCs with 2 to 20 conjugated drugs. Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell.

[0397] Additional examples of linkers that can be used with the compositions and methods include valine-citrulline; maleimidocaproyl; amino benzoic acids; p- aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers; maleimidocaproyl- polyethylene glycol (MC(PEG)6-OH); N-methyl-valine citrulline; N-succinimidyl 4-(N- mal eimidom ethyl )cy cl ohexane-1 -carboxylate (SMCC); N-Succinimidyl 4-(2- pyridyldithiojbutanoate (SPDB); and N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP) (See also US 2011 / 0076232). Another linker for use includes an avidin-biotin linkage to provide an avidin-biotin-containing ADC (See also U.S. Pat. No. 4,676,980, PCT publication Nos. WO 1992 / 022332 A2, WO 1994 / 016729A1, WO 1995 / 015770A 1, WO 1997 / 031655 A2, WO 1998 / 035704A1, WO 1999 / 019500A1, WO2001 / 09785 A2, WO200 1 / 090198 Al, W02003 / 093793A2, W02004 / 050016A2, W02005 / 081898A2, W02006 / 083562A2, W02006 / 089668A1, W02007 / 150020 Al, WO2008 / 135237A1, WO2010 / 111198A1, WO2011 / 057216A1, WO2011 / 058321 Al, WO2012 / 027494 Al, and EP77671B1), wherein some such linkers are resistant to biotinidase cleavage. Additional linkers that may be used include a cohesin / dockerin pair to provide a cohesion-dockerin- containing ADC (See PCT publication Nos. W02008 / 097866A2, W02008 / 097870A2, W02008 / 103947A2, and W02008 / 103953A2).

[0398] Additional linkers may contain non-peptide polymers (examples include, but are not limited to, polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lactic acid)), PLGA (poly(lactic acid-glycolic acid)), and combinations thereof, wherein a preferred polymer is polyethylene glycol) (See also PCT publication No. WO2011 / 000370). Additional linkers are also described in WO 2004-010957, U.S. Publication No. 20060074008, U.S. Publication No. 20050238649, and U.S. Publication No. 20060024317, each of which is incorporated by reference herein in its entirety).

[0399] The conjugation of the drug to the antibody via a linker can be accomplished by any technique known in the art. A number of different reactions are available for covalent attachment of drugs and linkers to antibodies. This may be accomplished by reaction of the amino acid residues of the antibody, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody. Also available for attachment of drugs to antibodies is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the antibody. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to antibodies. Other techniques are known to the skilled artisan and within the scope of the disclosure.

[0400] In certain embodiments, an intermediate, which is the precursor of the linker, is reacted with the drug under appropriate conditions. In certain embodiments, reactive groups are used on the drug or the intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with the anti- APOE antibodies under appropriate conditions. The synthesis and structure of exemplary linkers, stretcher units, amino acid units, self-immolative spacer units are described in U.S. Patent Application Publication Nos. 20030083263, 20050238649 and 20050009751, each if which is incorporated herein by reference.

[0401] Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation / analysis technique LC / MS.

[0402] In one aspect, the antibody drug conjugate has a structure represented by Formula la or pharmaceutically acceptable salt thereof:Formula la whereinAb is an anti-APOE antibody; each instance of W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'-, - PO2NR’-, or -NR’C(O)-; each instance of R1and R" is independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, heteroaryl or aryl; each instance of Z is independently C1-8 alkyl, halogen, cyano, or nitro; each instance of nl and n2 is independently an integer from 1 to 10, preferably 1 to 4; each instance of n3 is an integer from 0 to 3, preferably 0; each instance of B is independently a therapeutically active substance (e.g., a drug or diagnostic agent), preferably a drug; andY is an alkylene or heteroalkylene, preferably a C1-50 alkylene or C1-50 heteroalkylene, comprising one or a combination thereof selected from the following:(i) comprises one or more unsaturated bonds;(ii) comprises a heteroarylene;(iii) is substituted with at least one C1-20 alkyl; or(iv) comprises at least one isoprenyl group having a structure represented by Formula II:Formula II wherein n4 is an integer from 1 to 20, preferably 1 to 4.

[0403] In certain embodiments, Ab is an anti-APOE antibody comprising a light chain having an amino acid sequence of SEQ ID NO: 15, and a heavy chain having an amino acid sequence of SEQ ID NO:33.

[0404] In certain embodiments, W is -C(O)NR'-, further wherein the C is directly bonded to the phenyl ring of Formula la, and NR' is bonded to Y.

[0405] In certain embodiments, Y comprises a peptide and the peptide comprises at least one hydrophilic amino acid, preferably an amino acid having a side chain having a moiety that bears a charge at neutral pH in aqueous solution (e.g., an amine, guanidine, or carboxyl moiety), most preferably each amino acid of the peptide is independently selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[0406] In certain embodiments, Y is covalently bonded to the antibody by a thioether bond, and the thioether bond comprises a sulfur atom of a cysteine of the antibody.

[0407] In certain embodiments, Y comprises an oxime and: the oxygen atom of the oxime is on the side of Y that is linked to W and the carbon atom of the oxime is on the side of Y that is linked to Ab; or the carbon atom of the oxime is on the side of Y that is linked to W and the oxygen atom of the oxime is on the side of Y that is linked to Ab.

[0408] In certain embodiments, Y comprises a connection unit represented by Formula III or Formula IV:-(CH2)r(V(CH2)P)q-Formula III,-(CH2CH2X)W-Formula IV;V is a single bond, -O-, -S-, -NR1-, -C(O)NR2-, -NR3C(O)-, -NR4SO2-, or -SO2NR5-, preferably -O-;X is -O-, Ci-8 alkylene, or -NR1-, preferably -O-;R1to R5are each independently hydrogen, Ci-6 alkyl, Ci-6 alkyl C6-20 aryl, or Ci-6 alkyl C3- 20 heteroaryl; r is an integer from 1 to 10, preferably 2; p is an integer from 0 to 12, preferably 2; q is an integer from 1 to 20, preferably 2, 5, or 11; and w is an integer from 1 to 20, preferably 6 to 20.

[0409] In certain embodiments, Y comprises -(CH2CH2O)w-, -O(CH2CH2O)W-, or - (CH2CH2O)WCH2-, wherein w is an integer from 1 to 20, preferably 2 to 10.

[0410] In certain embodiments, Y comprises a moiety represented by Formula V, VI, VII, VIII or IX:Formula V,Formula VI,Formula VII,Formula VIIIFormula IXLi is a single bond or C1-30 alkylene; andR11is hydrogen or C1-10 alkyl.

[0411] In certain embodiments, Y is linear. As used herein, the term “linear”, when used in the context of variable Y, refers to a unit that couples the antibody to a single therapeutically active agent (e.g., a drug or diagnostic agent) via an unbranched covalent moiety (e.g., a via C1-50 alkylene). The term “linear”, when used in the context of variable Y, does not preclude substitution (e.g., alkyl, aryl, or heteroaryls) on Y, provided that said substituents are not therapeutically active agents or coupled to further active agents.

[0412] In other embodiments, Y is branched. As used herein, the term “branched”, when used in the context of variable Y, refers to a unit that couples the antibody to multiple therapeutically active agents (e.g., drugs and / or diagnostic agents) via a covalent moiety. For example, Y may comprise an alkylene that splits at a branching point into multiple alkylene chains, each of which covalently links the antibody to one or more therapeutically active agents (e.g., drugs).

[0413] In certain embodiments, Y comprises: i) a branching unit covalently coupled to Ab by a primary linker; ii) a first branch, which couples a first therapeutically active substance, via a first cleavage group, to the branching unit; and iiia) a second branch, which couples a second therapeutically active substance, via a second cleavage group, to the branching unit; or iiib) a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.

[0414] In certain embodiments, at least one branching unit has a structure representedwhereinG1, G2, G3is each independently a bond,R30is hydrogen or C1-30 alkyl;R40is hydrogen or L5-COOR50;R50is hydrogen or C1-30 alkyl; andL2, L3, L4, and L5are each independently a bond or alkylene.

[0415] In certain embodiments, the branching unit is a nitrogen atom. In other embodiments, the branching unit is an amide and the primary linker comprises the carbonyl of the amide. In yet other embodiments, the branching unit is an amide and the secondary linker comprises the carbonyl of the amide. In certain preferred embodiments, the branching unit is lysine.

[0416] In certain embodiments wherein Y is covalently bonded to the antibody by a thioether bond, the antibody comprises an amino acid motif recognizable by an isoprenoid transferase at the C-terminus of the antibody, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

[0417] In certain embodiments, the isoprenoid transferase is famesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

[0418] In certain embodiments, the amino acid motif has a CY1Y1X sequence, further wherein:C is cysteine; each Yi independently is an aliphatic amino acid;X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine; and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

[0419] In certain embodiments, each Yi is independently selected from alanine, isoleucine, leucine, methionine, and valine.

[0420] In certain embodiments, the conjugate comprises at least one of 1 to 20 amino acids between the antibody and the amino acid motif, and at least one of the amino acids is glycine.

[0421] In one embodiment, glucuronide-based linker connects drug units to an antibody in which its glucuronide unit comprises a glycosidase recognition site that is cleavable by an enzyme having P-glucuronidase activity thereby releasing free drug. Glucuronide-based linkers improve the solubility of ADCs and exhibit sufficient serum stability to provide targeted delivery of a conjugated drug to a targeted cell.2. Payload

[0422] Anti-APOE antibodies may be conjugated to at least one payload. The payloads of the invention are selected from the group consisting of an activator of pro- synaptic growth, a neuronal stimulator and an anti-inflammatory agent.

[0423] Activators of pro-synaptic growth are well known in the art and include EphB2 small molecule agonists, ephrin-B, IGF1, 5-HT or 5-HT2A agonists, Ngrl inhibitors, rapamycin, TrkB agonists, and other activators of pro-synaptic growth pathways.

[0424] Neuronal stimulators are well known in the art and include sodium or calcium channel agonists, memantine and donazepil.

[0425] Anti-inflammatory agents are well known in the art and include IL- lb inhibitors, IL-6 inhibitors, TNFalpha inhibitors, type 1 IFN inhibitors and corticosteroids.

[0426] Other examples of payloads include cholinesterase and acetylcholinesterase inhibitors, protein degraders, glycopeptides and oligonucleotides. Cholinesterase and acetylcholinesterase inhibitors include, for example, donepezil, rivastigmine, galantamine, tacrine and NMDA antagonist. Protein degraders include, for example, phagocytic inducers such as Gas6 or Hexokinase-2 inhibitors. Glycopeptides include, for example, mannose-6-phosphate glycopeptides for routing to lysosomes. Oligonucleotides include, for example, double stranded siRNA for targeting RNA encoding APOE.3. Optional Moiety Enhances Crossing of the Blood-Brain Barrier

[0427] The antibody-drug conjugates of the invention also optionally comprise a moiety to enhance penetration through the blood-brain barrier selected from the group consisting of transferrin receptor, CD98hc, basigin, Glutl extracellular domain (ECD) 1, ECD2, ECD3, ECD4, ECD5 and ECD6, IGF-1R, INSR ECD1, and INSR ECD2, and are well known in the art. Such moieties are described above.D. Pharmaceutical Compositions

[0428] Pharmaceutical compositions including the disclosed immunomodulatory agents are provided. Pharmaceutical compositions containing the immunomodulatoryagent can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0429] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected. For the disclosed immunomodulatory agents, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. For the disclosed immunomodulatory agents, generally dosage levels of 0.001 to 20 mg / kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.

[0430] In certain embodiments, the immunomodulatory agent is administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the immunomodulatory agent composition which is greater than that which can be achieved by systemic administration. The immunomodulatory agent compositions can be combined with a matrix as described above to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.1. Formulations for Parenteral Administration

[0431] In some embodiments, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16 Ed ISBN: 0- 912734-04-3, latest edition, incorporated herein by reference in its entirety, provides acompendium of formulation techniques as are generally known to practitioners. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.2. Formulations for Oral Administration

[0432] In some embodiments the compositions are formulated for oral delivery. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are incorporated herein by reference. The compositions may be prepared in liquid form, or may be in dried powder (e.g, lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the compositions. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g, U.S. Patent No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or chemically modified forms thereof) and inert ingredients which protect peptide in the stomach environment, and release of the biologically active material in the intestine.

[0433] The agents can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety permits uptake into the blood stream from the stomach or intestine, or uptake directly into the intestinal mucosa. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical usage. Other moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1, 3-dioxolane and poly-1, 3,6- tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al. (1982) J. AppL Biochem. 4: 185-189],

[0434] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

[0435] Controlled release oral formulations may be desirable. The agent can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release is based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.

[0436] For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. In some embodiments, the release will avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate(CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.3. Formulations for Topical Administration

[0437] The disclosed immunomodulatory agents can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.

[0438] Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

[0439] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

[0440] Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.

[0441] Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations may require the inclusion of penetration enhancers.4. Controlled Delivery Polymeric Matrices

[0442] The immunomodulatory agents disclosed herein can also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and thepeptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

[0443] Either non-biodegradable or biodegradable matrices can be used for delivery of fusion polypeptides or nucleic acids encoding the fusion polypeptides, although in some embodiments biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.

[0444] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13- 22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Se , 35:755-774 (1988).

[0445] The devices can be formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.III. Methods of ManufactureA. Methods of Making Antibodies

[0446] The disclosed antibodies can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, an antibody is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment,the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL and one VH region in the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, most recent edition).

[0447] APOE deficient (“knockout) mice or wild type mice can be utilized for the generation of high affinity mAbs against APOE using proprietary immunization techniques.

[0448] The disclosed antibodies can be modified by recombinant means to increase greater efficacy of the antibody in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). The modification in amino acids includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesired activities, e.g., complement-dependent cytotoxicity. Frequently, the antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, or fusion proteins of APOE. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0449] For example, suitable antibodies with the desired biologic activities can be identified using in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as the inhibition of tumor growth. The antibodies provided herein can also be useful in diagnostic applications. As capture or nonneutralizing antibodies, they can be screened for the ability to bind to the specific antigenwithout inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.

[0450] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (z.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0451] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0452] Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic peptide. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0453] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0454] A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, z.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0455] Monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0456] Antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

[0457] Methods of making antibodies using protein chemistry are also known in the art. One method of producing proteins comprising the antibodies is to link two or morepeptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or antigen binding fragment thereof via similar peptide condensation reactions.

[0458] For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.B. Methods for Producing Proteins

[0459] The disclosed proteins, polypeptides, fragments, variants and fusions thereof can be manufactured using conventional techniques that are known in the art. Isolated fusion proteins can be obtained by, for example, chemical synthesis or by recombinant production in a host cell. To recombinantly produce a protein, polypeptide, fragment, variant or fusion thereof, a nucleic acid containing a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, ormammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked.

[0460] Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

[0461] In eukaryotic host cells, a number of viral-based expression systems can be utilized to express fusion proteins. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.

[0462] Mammalian cell lines that stably express proteins, polypeptides, fragments, variants or fusions thereof, can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of proteins, polypeptides, fragments, variants or fusions thereof, in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Additional suitable expression systems include the GS Gene Expression System™ available through Lonza Group Ltd.

[0463] Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by metabolic selection, or antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells. Alternatively, a protein, polypeptide, fragment, variant or fusion thereof, can be produced by (a) ligating amplified sequences into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.

[0464] Proteins, polypeptides, fragments, variants or fusions thereof, can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, proteins, polypeptides, fragments, variants or fusions thereof can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, an Fc-fusion polypeptide in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify polypeptides. Fusion proteins can additionally be engineered to contain a secretory signal (if there is not a secretory signal already present) that causes the Proteins, polypeptides, fragments, variants or fusions thereof to be secreted by the cells in which it is produced. The secreted Proteins, polypeptides, fragments, variants or fusions thereof can then conveniently be isolated from the cell media.C. Methods for Producing Isolated Nucleic Acid Molecules

[0465] Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacementamplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878; and Weiss (1991) Science 254: 1292-1293.

[0466] Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3’ to 5’ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, doublestranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also obtained by mutagenesis. Protein-encoding nucleic acids can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992.IV. Assays and Antibody Screening

[0467] One embodiment provides assays for antibody screening. Assays for antibody screening include:

[0468] 1. Analysis of binding affinity of APOE -Fc to ligands in comparison to APOE.

[0469] 2 Functional assays to confirm APOE -Fc prevents signaling by APOE expressing cells. Reporter cells may be utilized for these assays, or primary APOE + cells are another option.A. Phase screening

[0470] 1. Phase I screening: screen for mAb binding to cell lines transfected to express cell surface APOE. Additionally, mAbs should have the capacity to bind endogenously expressed APOE on the surface of primary human cell subsets, or endogenously expressed. These mAbs should be highly specific for APOE.

[0471] 2. Phase II screening: APOE specific mAbs should block the binding ofAPOE to its ligands.

[0472] 3. Phase III screening: Functional assays to confirm that APOE mAbs or combination of mAbs modulate APOE mediated signaling. These assays will utilize cell lines that express endogenous APOE, or primary cells such as human monocytes, macrophages and dendritic cell subsets or any other leukocyte populations that express APOE to assess function in the presence of APOE mAbs. Additionally, reporter cells lines may be used to determine if signaling pathways such as NF-kB (NF-kB reporter) or NF AT (NF AT reporter) are altered following culture with APOE mAbs.

[0473] 4. Phase IV screening: Functional assays to determine if APOE mAbs are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of APOE expressing cell lines. In particular, APOE mAbs will be tested for the ability to deplete through one of these methods leukemia cell lines, known to express APOE on the cell surface. APOE mAbs may also be engineered to deplete APOE expressing cells and tested as described later in this document through known methods.

[0474] 5. Phase V screening: Functional assays to determine if APOE mAbs are capable of delivering or inducing a positive or negative signal (agonist) via APOE into APOE-expressing cells to stimulate or inhibit cellular function, respectively. Cell lines that endogenously express APOE, or transfectants of cell lines, will be assessed for activation or inhibition following culture with APOE. In other assays, reporter cell lines will be used to determine whether APOE mAbs enhance or dampen positive signaling pathways such as NF-kB (NF-kB reporter) or other known cell signaling reporters. Induction of apoptosis in cell lines will also be evaluated

[0475] Phase II and III assays can be used to predict the concentrations of APOE mAb(s) required to block physiological levels of ligands in vivo.V. Method of Use

[0476] The antibodies and antibody-drug conjugates of the invention are used to treat neurodegenerative or neuroinflammatory diseases. The APOE antibodies of the invention remove plaque by targeting it. The antibody-drug conjugates of the invention comprise antibodies directed to APOE and can be linked to payloads that increase synaptic formation to regenerate cognitive function. That is, the anti-APOE removes plaque by targeting it and also deliver a pro-synaptic drug to the area surrounding the plaque to increase synaptic healing and regeneration of cognitive function.

[0477] Alternatively, the antibody-drug conjugates directed to APOE can comprise a neuron stimulating agent to regenerate cognitive function.

[0478] Another alternative for the antibody-drug conjugates directed to APOE can comprise an anti-inflammatory agent to reduce the inflammatory nature of neurodegenerative diseases.

[0479] Exemplary methods are discussed in more detail below.

[0480] Treatment of Neurodegenerative or Neuroinflammatory Diseases

[0481] The disclosed compositions and methods can be used to treat neurodegenerative or neuroinflammatory diseases and conditions. Generally, the agents are used to treat neurodegeneration or neuroinflammation in the subject by administering to the subject an amount of the antibody-drug conjugates that inhibits, reduces or blocks further neurodegeneration or neuroinflammation. The method can reduce or more symptoms of the neurodegeneration or neuroinflammation.

[0482] Antagonists or agonists of APOE-mediated signaling can be used to modulate immune responses in subjects in need of such treatment.

[0483] In one embodiment, the APOE binding moieties induce, promote, or enhance ligand binding to APOE and induce, promote, or enhance proliferation or activation of APOE+ immunosuppressive cells or cause depletion of these cells.

[0484] In one embodiment, the APOE binding moieties inhibit, reduce or block ligand binding to APOE and inhibit, reduce, or block APOE+ immunosuppressive cells or cause depletion of these cells.

[0485] Exemplary methods are discussed in more detail below.A. Immune Response Stimulation1. Therapeutic Strategies

[0486] Methods of inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering a subject an effective amount of a APOE immunomodulatory agent or binding moiety, or cells primed ex vivo with the APOE immunomodulatory agent or binding moiety. The immune response can be, for example, a significant reduction in amyloid plaque burden in in AD and improve cognitive function or inhibiting suppression of the immune response in the tumor microenvironment, resulting in a therapeutic anti-tumor immune response.

[0487] Alternatively, the immunomodulatory agent can stimulate signal transduction through APOE and promote or enhance an immune response.2. Subjects to be Treated a. Treatment of Neurodegenerative or Neuroinflammatory Diseases

[0488] The disclosed compositions and methods can be used to treat neurodegenerative or neuroinflammatory diseases and conditions. Generally, the agents are used to stimulate or enhance an immune response to neurodegeneration or neuroinflammation in the subject by administering to the subject an amount of an immunomodulatory agent, for example an immunomodulatory agent that inhibits, reduces, or blocks APOE expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. The immunomodulatory agent can bind APOE and promote or enhance an immune response by reducing signal transduction through APOE. The method can reduce or more symptoms of the neurodegeneration or neuroinflammation.B. Immune Response Inhibiting1. Therapeutic Strategies

[0489] Methods of reducing or inhibiting an immune response in a subject are provided. Typically the methods include administering a subject an effective amount of a APOE immunomodulatory agent or combinations thereof, or cells primed ex vivo with these immunomodulatory agents. The immune response can be, for example, promoting or enhancing a suppressive immune response. In one embodiment, the disclosed compositions promote, enhance or activate Tregs, increase the production of cytokines such as IL- 10 from Tregs, increase the differentiation of Tregs, increase the number of Tregs, increase the ratio of Tregs within an immune cell population, or increase the survival of Tregs. to provide an immune suppressive response.

[0490] In another embodiment, the immunomodulatory agent promotes a suppressive immune response by inducing, promoting, or enhancing signal transduction through APOE.

[0491] The methods can be used in vivo or ex vivo as immune response-inhibiting therapeutic applications. Thus in some embodiments, the agent, or nucleic acid encoding the agent, is administered directly to the subject. In some embodiments, the agent or nucleic acid encoding the agent, is contacted with cells (e.g., immune cells) ex vivo, and the treat cells are administered to the subject (e.g. adoptive transfer). In general, the disclosedimmunomodulatory agents can be used for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an overactive or inappropriate immune response. The agents can enable a less robust immune response to be possible. The disclosed compositions are useful to reduce or inhibit immune responses involving T cells.

[0492] The immunomodulatory agents utilized for reducing an immune response are typically those that increase APOE expression, ligand binding, crosslinking, APOE mediated signaling, or a combination thereof. For example, the agent can be an agonist of APOE, such as an agonist (stimulating) anti- APOE antibody or antigen binding fragment thereof.C. Anti-APOE4 immunotherapies

[0493] Disclosed herein are anti-APOE immunotherapies wherein treatment with an ApoE antibody enhances the clearance of Ap & ApoE plaques, reduction of inflammation and neuron protection.

[0494] Recent studies have shown that APOE, especially APOE4, binds to Ap, and plays a key role in Ap deposition and clearance. Several studies have shown that simply reducing APOE4 levels lowers brain Ap levels in APP transgenic mice (Bien-Ly N, et al., J Neurosci., 32(14):4803-l 1 (2012); Kim J, et al., J Neurosci. 31(49): 18007-12 (2011)). Studies have also shown that other approaches such as blocking AP-APOE4 interaction can also lead to beneficial effects, prompting the development of strategies to either reduce the availability of APOE4 or prevent its toxic interactions.

[0495] The idea behind anti-APOE4 antibodies follows that of anti-Ap antibodybased therapies. These antibodies are able to cross the blood brain barrier (BBB) and neutralize the negative effects of APOE4, even if only a small amount of antibodies can effectively enter the brain (Kim J, etal., J Exp Med. 209(12):2149-56 (2012)). APOE4 has already been implicated in Ap deposition, and along with other amyloid-associated proteins it is found in Ap deposits. Figure 1 illustrates the idea is that if isoform-specific antibodies can sequester pathogenic forms of APOE, it can prevent Ap build-up in the brain. Studies in mouse models have shown that anti-APOE antibodies can efficiently inhibit the formation of Ap deposits when introduced before the onset of pathology (Kim J, et al., 2023 supra). Kim et al showed that these antibodies were able to attenuate plaque burden when introduced in mice with pre-existing Ap deposits, suggesting that this antibody couldwork as a therapeutic agent. Other studies also show that topical application of anti-APOE antibodies directly onto the brain prevented deposition of new Ap plaques as well as cleared pre-existing plaques. The fact that these anti-APOE antibodies can disrupt the direct binding of apoE to Ap deposits is very promising, as this might work synergistically with anti Ap immunotherapy in AP0E4 patients to achieve a higher degree of Ap reduction.

[0496] In a 2018 paper, Liao et al. reported that the antibody ‘HAE-4’ that preferentially recognizes the nonlipidated forms of APOE4 / APOE3 over the lipidated versions and is highly effective in preventing Ap deposition by a FcyR-dependent mechanism in an APPIAPOFA mouse model (Liao F, et al., J Clin Invest. 128(5):2144-55 (2018)). This and other studies demonstrated that non-lipidated forms of APOE4 may be preferentially pathogenic and that since the non-lipidated form of APOE4 is a small fraction of the total CNS APOE burden, this antibody would not be titered out by total APOE and could be efficacious at a lower or less frequent dose (Borchelt DR., J Clin Invest. 128(5): 1734-6 (2018)).

[0497] Provided herein are APOE immunomodulatory agents that modulate APOE expression, ligand binding, crosslinking, APOE-mediated signaling, or a combination thereof. In one embodiment, the disclosed APOE immunomodulatory agents include anti- APOE antibodies and antigen-binding fragments thereof having a variable light chain domain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; and a variable heavy chain domain having at least 99% sequence identity to selected from the group consisting of SEQ ID NOs: 21, 25, 29 and 33. In another embodiment, the disclosed APOE immunomodulatory agents include APOE fusion proteins and binding fragments thereof having a light chain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and a heavy chain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID Nos: 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, and 36. In yet another embodiment, the APOE immunomodulatory agent is a APOE antibody-drug conjugate comprising an APOE antibody or fragment disclosed herein, a linker, a payload, and an optional moiety, wherein the antibody binds to APOE, and wherein the optional moiety is selected from the group consisting of transferrin receptor, CD98hc, basigin, Glutl extracellular domain (ECD) 1, ECD2, ECD3, ECD4, ECD5 and ECD6, IGF-1R, INSR ECD1, and INSRECD2.

[0498] Also disclosed in a pharmaceutical composition comprising an APOE immunomodulatory agent disclosed herein, wherein the APOE immunomodulatory agent binds to amyloid-associated APOE aggregates contained within amyloid plaques in a subject’s brain parenchyma and brain vasculature. Parenchymal and vascular fibrillar amyloid deposits are a key characteristic of conditions like Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). These deposits, composed of amyloid beta (AP) protein, can form senile plaques in brain tissue (parenchymal) and accumulate on the walls of blood vessels (vascular). This accumulation can lead to various neurological and vascular complications. Particularly, the distribution and amount of amyloid deposits correlate with the severity of AD and other related disorders.

[0499] Studies have shown that vascular and parenchymal amyloid deposits are not entirely identical. Specifically, antibodies may exhibit different affinities for the epitopes of these deposits, indicating that they may be structurally or compositionally distinct. The specificity of antibody binding can have implications for the efficacy and safety of antiamyloid therapies. The immunomodulatory agents provided herein target both the vascular and parenchymal amyloid deposits, providing an immunotherapeutic approach that can effectively reduce amyloid burden in a subject.

[0500] VI. Combination Therapies

[0501] The disclosed immunomodulatory agents can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the immunomodulatory agent and the additional therapeutic agent are administered separately, but simultaneously. The immunomodulatory agent and the additional therapeutic agent can also be administered as part of the same composition. In other embodiments, the immunomodulatory agent and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regime.

[0502] The subject can be administered a first therapeutic agent 1, 2, 3, 4, 5, 6, or more hours, or 1, 2, 3, 4, 5, 6, 7, or more days before administration of a second therapeutic agent. In some embodiments, the subject can be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days prior to a first administration of second agent. The immunomodulatory agent can be the first or the second therapeutic agent.

[0503] The immunomodulatory agent and the additional therapeutic agent can be administered as part of a therapeutic regimen. For example, if a first therapeutic agent can be administered to a subject every fourth day, the second therapeutic agent can be administered on the first, second, third, or fourth day, or combinations thereof. The first therapeutic agent or second therapeutic agent may be repeatedly administered throughout the entire treatment regimen.

[0504] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for treatment of HIV or Hepatitis B or C), anti-parasites (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof (including variant, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatories, ligands that bind to Toll-Like Receptors (including but not limited to CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, other molecules that deactivate or down-regulate suppressor or regulatory T-cells, and the like.

[0505] The additional therapeutic agents are selected based on the condition, disorder or disease to be treated. For example, the immunomodulatory agent can be co-administered with one or more additional agents that function to enhance or promote an immune response or reduce or inhibit an immune response.VII. Kits

[0506] The disclosed APOE immunomodulatory agents can be packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity. The agent can be supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. For example, the agent can be supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, or at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent can be stored at between 2 and 8°C in their original container and are typically administered within 12 hours, orwithin 6 hours, or within 5 hours, or within 3 hours, or within 1 hour after being reconstituted.

[0507] In an alternative embodiment, agent can be supplied in liquid form in a hermetically sealed container indicating the quantity and concentration. In some embodiments, the liquid form of the agent supplied in a hermetically sealed container including at least 1 mg / ml, or at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml of the agent.

[0508] Pharmaceutical packs and kits including one or more containers filled with agent are also provided. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The pharmaceutical pack or kit can also include one or more containers filled with one or more of the ingredients of the disclosed pharmaceutical compositions. Optionally associated with such contained s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0509] Kits designed for the above-described methods are also provided. Embodiments typically include one or more APOE immunomodulatory agents. In particular embodiments, a kit also includes one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers. In other embodiments, a kit also includes one or more anti-inflammatory agents useful for the treatment inflammatory and autoimmune diseases, in one or more containers.

[0510] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0511] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.EXAMPLESExample 1: APOE Antibodies and Heavy and Light Chains Sequences Thereof

[0512] Materials and Methods

[0513] Mouse anti-human APOE monoclonal antibodies: APOE injected recombinant APOE4 with complete Freund’s adjuvant into mice. For an initial screening of antibodies, supernatants from hybridoma cells were added to 96-well plates coated with recombinant APOE4, and the APOE mAb that bound to APOE4 was detected using antimouse IgG HRP. The antibodies that performed well in the initial screening were further characterized and selected for in vivo studies. For the in vivo study, antibodies were generated from cultured hybridoma cells and purified on a protein G column. All APOE mAb utilized in vivo contained similar amounts of endotoxin as compared with control antibodies.

[0514] RACE: RACE (Rapid Amplification of cDNA Ends) identification of the heavy and light chains was performed according to the following protocol: (1) mRNA denaturing, (2) cDNA synthesis, (3) 5 ’RACE Reaction, (4) analyzed PCR results (on an agarose gel to visualize the amplified DNA fragment - the correct antibody variable region DNA fragments should have a size between 500-700 base pairs, (5) TOPO cloned PCR positive bands; (6) PCR-amplified TOPO clones, followed by gel electrophoresis and recovery from agarose gel, sequencing of clones, and CDR analysis using sequencing data (CDR regions were defined using VBASE2).

[0515] Results

[0516] Antibodies were cloned using RACE methods. After sequencing cloned DNA fragments, antibody sequence analysis identified one heavy chain and one light chain for antibody samples. The antibody nucleotide sequences, heavy and light chain sequences and CDRs are provided as SEQ ID NOs: 41-50 above. APOE mAb was humanized and the variant sequences are provided as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 29 and 33. Fusion proteins are provided as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22- 24, 26-28, 30-32, and 34-36.Example 2: Binding of APOE mAb to Native & Heat-induced APOE4 Aggregates

[0517] Materials and Methods

[0518] APOE binding ELISAs. Recombinant APOE was coated in PBS overnight. After washes with PBS, the wells were blocked with BSA-PBS, washed once with PBSand subsequently loaded with APOE mAbs at serial concentrations. Bound APOE mAb were detected with HRP-labeled goat anti-mouse IgG and visualized. For experiments where APOE aggregation was first induced by heating, recombinant APOE4 was first heat treated overnight at 40C as first described in Liao, et al. JCI (2018). To normalize the amount of APOE coated to the ELISA plates from the supernatant and pellet fractions after heating and ultracentrifugation, the relative amounts of APOE were first assessed by SDS- PAGE followed by a Coomassie blue stain. Heat-induced APOE4 aggregates were denatured and then diluted for coating onto ELISA plates alongside freshly-thawed protein. After overnight incubation, the wells were washed with PBS. ELISA experiments were performed with native APOE and APOE aggregates using APOE mAb.

[0519] Results

[0520] APOE mAb binding to APOE4 was enhanced following APOE4 heat treatment. Figure 2 shows the comparison of binding of APOE mAb to native and heat- induced APOE4 aggregates. The APOE mAb bound to both forms of APOE; however the binding of APOE mAB to heat-induced APOE4 aggregates was dramatically enhanced (FIG. 2A). Binding of APOE mAb to plate-bound APOE4 was dose-responsive and enhanced following APOE4 heat-denaturation. APOE mAb binds neither native nor heat- treated forms of APOE2 (FIG. 2B).

[0521] Because APOE mAb binding for APOE4 is enhanced following heat treatment, APOE mAb likely binds preferentially to aggregated protein as is generated by this protocol. The reproducibility of this function and its possible relevance to the therapeutic MOA of APOE mAb in binding amyloid-associated APOE aggregates led to the use of this assay as a platform for screening humanized APOE mAb variants in subsequent experiments.Example 3: APOE mAb Appears to Bind ApoE Intermediate Increased at Higher Temperatures

[0522] Materials and Methods

[0523] Samples of recombinant human APOE4 protein were heat-treated overnight at indicated temperatures. The following day, heat-treated APOE4 protein was coated alongside freshly-thawed, ‘native’ APOE4 on the wells of ELISA plates. Coated wells were treated with a serial dilution of APOE mAb antibody, whose dose-dependent binding was then measured using ELISA.

[0524] Results

[0525] APOE mAb binding to APOE4 was enhanced by heat-treatment, to a point. APOE4 heat treatment at temperatures of 55C or higher led to a loss of APOE mAb binding (FIGs. 3A-3B). APOE mAb binds APOE4 with higher affinities as APOE is pre-treated at progressively higher temperatures (FIG. 3 A). Beyond 55 degrees of heat treatment, APOE mAb binding to heat-treated APOE4 is lost. This trend is demonstrated in FIG. 3B for a single concentration of APOE mAb, where labels demonstrate hypothesized structural transitions of APOE4 during heat treatment at various temperatures.

[0526] APOE4 may bind preferentially to a three-dimensional APOE4 epitope that is exposed by heat treatment around physiological temperatures of humans prior to complete denaturation. This may reflect the tendency of APOE mAb to bind preferentially to APOE aggregates contained within amyloid plaques in patient brain samples.Example 4: APOE mAb Humanization

[0527] Twenty humanized variants transiently expressed and preliminary expression was evaluated by SDS PAGE (FIGs. 4B and 4C) and Octet (FIG. 4A).Example 5: Humanized Variants and Parent Molecule Bind Comparably to ApoE4

[0528] Materials and Methods

[0529] Humanized APOE mAb variants 2, 6, 12, and 16 were expressed from stable CHO cultures and purified using Protein G. Recombinant human APOE4 was heat treated overnight at 40C as first described in Liao, et al. JCI (2018). Treated protein was coated alongside freshly-thawed, native protein on assay plates which were then treated serial dilutions of purified antibodies. Antibody binding to plate-coated ligand was measured by ELISA.

[0530] Results

[0531] Purified, humanized APOE mAb variants exhibited a selectivity in binding to heat-treated APOE4 that was functionally comparable to the parent antibody. FIGs. 5A-5E shows binding curves generated by treated heat-treated or native APOE4 with serial dilutions of humanized APOE mAb variant while FIG. 5F shows modeled EC50s for binding to native or heat-treated APOE4 were calculated for each antibody and plotted alongside values for the parent antibody. EC50s for binding to native and heat-denatured APOE4 were comparable to the parent antibody for all humanized APOE mAb variants.Thus, all variants were considered functionally comparable with regard to target binding in vitro.Example 6: Comparable Staining of Plaque- Associated ApoE

[0532] Materials and Methods

[0533] Humanized APOE mAb variants 2 and 16 were used to stain brain tissue from amyloidogenic 5XFAD; hAPOE4+ / + mice (He, Z., et al., Nat Med A, 29-38 (2018)). Tissues were co-stained for parenchymal and vascular fibrillar amyloid deposits using X- 34. Antibody binding to amyloid-associated APOE was detected using an anti-human secondary antibody, and the resulting signal localization was compared with X-34 to assess the ability of NCI 81 variants to bind amyloid-associated APOE.

[0534] Results

[0535] As observed for the parent molecule, Variants 2 and 16 were observed to selectively bind X-34-associated APOE4 deposits in both brain parenchyma and brain vasculature. APOE mAb parent binds to APOE4 colocalized with both parenchymal and vascular amyloid (CAA) (FIGs. 6A-6I). APOE mAb variant 16 (see Table 1 (SEQ ID NO: 15 and 33) binds similarly to APOE4 co-deposited with parenchymal and vascular amyloid. APOE- / - tissues were included as native controls. Based on regional selectivity and specific colocalization patterns, humanized APOE mAb Variant 16 shows comparable function to the parent antibody in selectively binding APOE4 co-deposited with amyloid in tissue from an Alzheimer’s disease model.Example 7: Cross-reactivity to Cynomolgus ApoE

[0536] Materials and Methods

[0537] Human APOE4 or cynomolgus macaque APOE-His fusion proteins were coated on assay plates, which were then treated with purified, titrated humanized APOE mAb Variant 16. Antibody binding to plate-coated ligand was measured by ELISA.

[0538] Results

[0539] Antibody binding data to either human or cyno APOE was used to generate binding curves. Plotted antibody binding curves were overlapping for both ligands tested. FIG. 7 shows the antibody binding signal for APOE mAb Variant 16 binding to 6xHis- fusions of native human APOE4 (black) or cyno APOE (red). Binding curve overlap demonstrates that APOE mAb Variant 16 binds to cyno APOE-His with comparableaffinity to human AP0E4-His. This suggests that cynomolgus macaque is a suitable model organism for further testing APOE mAh Variant 16 pharmacokinetics and toxicology in IND-enabling studies.Example 8: ApoE mAb does not Bind to Plasma ApoE

[0540] Materials and Methods

[0541] Plasma was collected from mice expressing human APOE isoforms APOE3 and APOE4. Plasma samples were coated in serial dilutions on ELISA plates alongside titrated recombinant APOE3 and APOE4. An antibody for total APOE (‘Abeam’) was used to establish equivalent concentrations between plasma and recombinant APOE, which were then used as reference concentrations to interrogate APOE selectivity of APOE mAb.

[0542] Results

[0543] Pan-APOE antibody binding to coated samples showed titratable increases in immobilized APOE ligand. APOE titration curves for plasma and recombinant proteins were used to select comparable APOE3 concentrations for comparison of APOE mAb variant 16 antibody binding. At equivalent concentrations of plasma-derived and recombinant APOE3, APOE mAb variant 16 binding was highly enriched for recombinant protein. FIGs. 8A-8B show ELISA signals from a pan-APOE antibody was used to establish equivalent coating concentrations between plasma APOE3 (FIG. 8A) and recombinant APOE3 (FIG. 8B). FIG. 8C shows APOE mAb variant 16 binding data compared at the plasma and recombinant protein dilutions selected in top panel. APOE mAb variant 16 does not bind plasma APOE, suggesting that APOE lipidation may be a determining factor in APOE mAb binding. While explaining in part the favorable pharmacokinetics of APOE mAb when compared to other antibodies that bind plasma APOE (see Liao, et al., JCI 2018), these data suggest that lack of binding to plasma APOE may also aid bioavailability to CNS tissues.Example 9: Identification of Potential Antibody Residues Involved in Enhanced Binding to Folding Intermediate

[0544] Material and Methods

[0545] APOE mAb variants were expressed from stable CHO cultures. Recombinant human APOE4 was heat treated overnight at 40°C. Heat-treated protein was coated alongside freshly-thawed, native protein on assay plates which were then treated serialdilutions of purified antibodies. Antibody binding to plate-coated ligand was measured by ELISA.

[0546] Results

[0547] A single point mutation in the APOE mAb Variant 16 variable region abrogates binding selectivity for treated APOE4 while preserving the interaction with native protein. Modeled dose-response curves for binding to native or heat-denatured APOE4 were calculated for APOE mAb parent (FIG. 9A) and a point mutant (FIG. 9B). Point mutation of APOE mAb Variant 16 led to overlapping binding curves for native and heat-treated ligands compared to the parent molecule. A critical tyrosine residue in a CDR of APOE mAb mediates binding selectivity for the heat-treated protein. This same residue may underlie the interaction with the pathological, aggregated form of APOE targeted by APOE mAb in disease models.Example 10: APOE4 antibodies bind to APOE4 aggregates induced by overnight heat-treatment at 40 C

[0548] APOE4 antibodies of the invention bind to APOE4 aggregates induced by overnight heat-treatment at 40 C. To define the epitope region of antibody binding, several APOE4 fragments were synthesized, to include a first peptide fragment of amino acids 150- 160 and a second peptide fragment of amino acids 140-160. The full-length APOE4 protein, the first peptide fragment and the second peptide fragment were heat-treated at 40 C overnight. The heat-treated samples were coated on ELISA plates and then probed with anti-APOE4 mAb variant 16 to compare binding to the 3 different samples. As shown in Figure 10 A, the antibody bound to heat-treated full length APOE4 tighter than untreated full-length APOE4. Figure 10B shows that the antibody bound the first peptide fragment (APOE4 amino acids 150-160) with about the same affinity. Figure 10C shows that the antibody binds to the second peptide (APOE4 amino acids 140-160) with much higher affinity that either the full-length protein or the first peptide fragment. This suggests that a portion of the antibody binding epitope contained in amino acids 150-160 is both necessary and sufficient for antibody binding. However, the addition of amino acids 140-150 of peptide 2 (amino acids 140-160), shows that those amino acids are necessary to maximize binding affinity of the antibodies.

[0549] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequencesubmissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

What is claimed is:

1. An apolipoprotein E (APOE) immunomodulatory agent that modulates APOE expression, ligand binding, crosslinking, APOE mediated signaling, or a combination thereof, comprising: a. an anti-APOE antibody and antigen-binding fragments thereof; b. an APOE fusion protein and binding fragments thereof; or c. an APOE antibody-drug conjugate; wherein the immunomodulatory agent immunospecifically binds to APOE having at least 99% sequence identity to SEQ ID NO: 51, 52, 53, 54, or 55.

2. The APOE immunomodulatory agent of claim 1, wherein the anti-APOE antibody and antigen-binding fragments thereof comprise: a. a variable light chain domain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; and b. a variable heavy chain domain having at least 99% sequence identity to selected from the group consisting of SEQ ID NOs: 21, 25, 29 and 33.

3. The APOE immunomodulatory agent of claim 2, wherein a. the variable light chain domain of the anti-APOE antibody and antigenbinding fragments thereof comprise complementarity-determining regions (CDRs) selected from CDRs consisting of at least 99% sequence identity to SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, or combinations thereof, and b. the variable heavy chain domain of the anti-APOE antibody and antigenbinding fragments thereof comprise complementarity-determining regions (CDRs) selected from CDRs consisting of at least 99% sequence identity to SEQ ID NO: 48, 49, 50, or combinations thereof.

4. The APOE immunomodulatory agent of claim 1, wherein the APOE fusion protein and binding fragments thereof comprise: a. a light chain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; andb. a heavy chain having at least 99% sequence identity to sequences selected from the group consisting of SEQ ID Nos: 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, and 36.

5. The APOE immunomodulatory agent of claim 4, wherein a. the light chain domain of the APOE fusion protein and binding fragments thereof comprise complementarity-determining regions (CDRs) selected from CDRs consisting of at least 99% sequence identity to SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, or combinations thereof b. the heavy chain domain of the APOE fusion protein and binding fragments thereof comprise complementarity-determining regions (CDRs) selected from CDRs consisting of at least 99% sequence identity to SEQ ID NO: 48, 49, 50, or combinations thereof.

6. The APOE immunomodulatory agent of claim 2, wherein the APOE antibody and fragments thereof s variable light chain domain and variable heavy chain domain are selected from a group of sequences having: a. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; b. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; c. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; d. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; e. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33;f. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; g. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; h. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; i. the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, 25, 29, or 33; or j . the variable light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19 and variable heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: SEQ ID NO:21, 25, 29, or 33.

7. The APOE immunomodulatory agent of claim 4, wherein the fusion protein and binding fragments thereof have the light chain and heavy chain selected from a group of sequences having: a. the light chain with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; b. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:4 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; c. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:6 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36;- I l l -d. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:8 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; e. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 10 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; f. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 12 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; g. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 14 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; h. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 16 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; i. the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO: 18 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36; or j . the light chain with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:20 and heavy chain domains with at least 95%, 96%, 97%, 98%, 99% and 100% sequence identity to SEQ ID NO:22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, or 36.

8. The APOE immunomodulatory agent of claim 2 wherein a light chain with at least 99% sequence identity to SEQ ID NO: 15 and heavy chain domains with at least 99% sequence identity to SEQ ID NO: 33.

9. An APOE antibody-drug conjugate comprising a. an APOE antibody or fragment thereof of claim 2; b. a linker; c. a payload; and d. an optional moiety, wherein the antibody binds to APOE, and wherein the optional moiety is selected from the group consisting of transferrin receptor, CD98hc, basigin , Glutl extracellular domain (ECD) 1, ECD2, ECD3, ECD4, ECD5 and ECD6, IGF-1R, INSR ECD1, and INSR ECD2.

10. The APOE antibody-drug conjugate of claim 9, wherein the linker is selected from an extracellular protease cleavable linker, a cathepsin cleavable linker, or a non-cleavable linker.

11. The APOE antibody-drug conjugate of claim 9, wherein the payload is selected from a pro-synaptic agent, a neurostimulatory agent, or a pro-inflammatory agent.

12. The APOE antibody-drug conjugate of claim 11, wherein the pro-synaptic agent is selected from EphB2 small molecule agonists, ephrin-B, IGF1, 5-HT or 5-HT2A agonists, Ngrl inhibitors, rapamycin, or TrkB agonists.

13. The APOE antibody-drug conjugate of claim 11, wherein the neurostimulatory agent is selected from a sodium channel agonist, a calcium channel agonist, memantine, or donazepil.

14. The APOE antibody-drug conjugate of claim 9, wherein the payload is selected from an anti-inflammatory agent selected from an IL-lb inhibitor, an IL-6 inhibitor, a TNF alpha inhibitor, a type 1 IFN inhibitor, or a corticosteroid.

15. The APOE antibody-drug conjugate of claim 9, wherein the optional moiety is selected from a transferrin receptor, CD98hc, basigin, or Glutl.

16. A pharmaceutical composition for treating a neurodegenerative disease in a subject in need thereof, comprising the APOE immunomodulatory agents of claims 1-15.

17. The pharmaceutical composition of claim 16, wherein the APOE immunomodulatory agent is selected from a. a therapeutically effective amount of the APOE antibody or antigen binding fragments thereof; b. a therapeutically effective amount of the APOE fusion protein and binding fragments thereof; or c. a therapeutically effective amount of the antibody-drug conjugate of claim 5; wherein the pharmaceutical composition is administered to the subject to treat a neurodegenerative disease.

18. The pharmaceutical composition of claim 16, wherein the neurodegenerative disease is selected from Alzheimer’s disease, cerebral amyloid angiopathy, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington’s disease.

19. The pharmaceutical composition of claim 16, wherein the APOE immunomodulatory agent binds to amyloid-associated APOE aggregates contained within amyloid plaques in a subject’s brain parenchyma and brain vasculature to reduce APOE4 levels and lower the brain amyloid protein levels in the subject’s brain.

20. A method of treating a neurodegenerative disease comprising administering to a subject in need thereof the pharmaceutical composition of claim 16, wherein the pharmaceutical composition binds to amyloid-associated APOE aggregates contained within amyloid plaques in the subject’s brain parenchyma and brain vasculature to reduce APOE4 levels and lower the brain amyloid protein levels.

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