Anti-apoe antibodies

Abstract

To provide compositions and methods for effectively treating at least one symptom or sign of A plaque or cerebral amyloid angiopathy (CAA) associated symptoms, or for decreasing amyloid plaque load or CAA load.SOLUTION: An isolated anti-ApoE antibody comprises (a) a light chain variable region comprising a specific amino acid sequence, and / or (b) a heavy chain variable region comprising a consensus sequence of another specific amino acid sequence. The antibody may be a monoclonal antibody or antibody fragment.SELECTED DRAWING: None

Description

[Technical field]

[0001] Government Rights This invention was made with Government support under award R01AG047644 given by the National Institutes of Health. The Government has certain rights in the invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 414,413, filed October 28, 2016, and U.S. Provisional Patent Application No. 62 / 533,336, filed July 17, 2017, each of which is incorporated by reference in its entirety.

[0003] FIELD OF THE PRESENT APPLICATION The present invention relates to anti-ApoE antibodies and compositions thereof. The present invention also relates to compositions and methods for delaying and / or preventing Aβ amyloidosis. The present invention also relates to compositions and methods for delaying and / or preventing Aβ plaque-related symptoms and / or cerebral amyloid angiopathy (CAA)-related symptoms, such as Alzheimer's disease (AD) or CAA-related symptoms in a subject. In particular, the present invention relates to regulating the concentration of amyloid beta (Aβ) in the brain of a subject.

[0004] Sequence Listing Reference A paper copy of the sequence listing and a computer readable form of the same are attached below and are incorporated herein by reference. The information recorded in computer readable form is identical to the sequence listing as set forth in accordance with 37 CFR § 1.821(f). [Background technology]

[0005] Alzheimer's disease (AD) is the most common cause of dementia and a growing public health problem. It is currently estimated to affect over 5 million people in the United States, and is expected to increase to 13 million by 2050. Alzheimer's disease leads to memory loss, loss of cognitive function, and ultimately loss of independence. This inflicts significant personal and financial damage on subjects and families. Due to the severity of the disease and its increasing prevalence in the population, there is an urgent need to develop better treatments.

[0006] Cerebral amyloid angiopathy (CAA) occurs in approximately 90% of individuals who develop AD, and occurs independently of AD in some individuals. CAA can lead to ischemic and hemorrhagic strokes, which can cause severe disability or death. Currently, there is no treatment for CAA.

[0007] Biochemical, genetic and animal model evidence implicates amyloid beta (Aβ) as the pathogenic peptide in AD and most cases of CAA. Neuropathological and neurochemical features of AD include synaptic loss and selective neuronal cell death, reduction of specific neurotransmitters, and the presence of abnormal proteinaceous deposits within neurons (neurofibrillary tangles) and in the extracellular space (cerebrovascular, diffuse and senile plaques). Features of CAA include the accumulation of fibrillar forms of Aβ in perforating and pial arterioles on the surface of the cerebral cortex. CAA can lead to ischemic or hemorrhagic stroke. The main component of the plaques found in AD and CAA is Aβ, a peptide of 38-43 amino acid sequence cleaved from the amyloid precursor protein (APP).

[0008] Throughout life, soluble Aβ is secreted primarily by neurons, but also by other cell types. Excessive Aβ deposition may result from increased Aβ synthesis (e.g., as occurs in familial early-onset AD and some familial early-onset CAA), decreased Aβ clearance in the brain, or increased Aβ fibril formation. The lack of convincing evidence that Aβ overproduction occurs in the more common late-onset forms of AD suggests that insufficient Aβ clearance may drive Aβ deposition and amyloid plaque formation as well as CAA.

[0009] The apolipoprotein E gene (ApoE) remains the most widely replicated genetic risk factor for late-onset AD and CAA, with carriers of the ε4 allele having a 3-15-fold higher risk of disease onset and earlier age of disease onset. ApoE4 carriers represent over 60% of the population with AD, while the ε2 allele is the least represented in AD and may be protective in some populations. Human ApoE isoforms differ at amino acid positions 112 or 158 (ε2 has Cys112, Cys158; ε3 has Cys112, Arg158; ε4 has Arg112, Arg158). Mouse ApoE and human ApoE share approximately 70% amino acid homology. Amino acid differences in human ApoE isoforms affect protein structure and stability. For example, ApoE4 has a greater tendency toward an unstructured molten globule state compared to ApoE2 and ApoE3, making it more likely to form aggregates.

[0010] In the brain, ApoE is primarily produced by glia and functions primarily to distribute cholesterol and lipids to neurons. The majority of ApoE in the CNS is found on HDL-like lipoprotein particles, and ApoE lipidation is regulated by the ABCA1 and ABCG1 transporters. ABCA1 knockout mice crossed with transgenic AD mice produce poorly lipidated ApoE, and these mice have increased Aβ plaques. Conversely, the brains of AD transgenic mice overexpressing ABCA1 contain well lipidated apoE lipoprotein particles and have reduced Aβ plaque deposition. Binding of ApoE to LDLR, LRP1, apoER2, and VLDLR, which are expressed on various neuronal cell types, allows uptake of lipoprotein particles via receptor-dependent endocytosis, processing of lipid cargo, and recycling of unbound receptors. An amino acid difference at position 158 of ApoE2 results in reduced receptor binding to the LDLR, and individuals with the ε2 / ε2 genotype are at increased risk of type III hyperlipoproteinemia.

[0011] The deposition of Aβ plaques in AD patients is ApoE isoform dependent (E4>E3>E2), and the extent of the interaction between ApoE and Aβ and the function of their interaction remain controversial. Compelling evidence suggests that the differences in Aβ deposition are secondary to differences in apoE isoform-dependent Aβ clearance and aggregation. Clearance of Aβ monomers from the brains of AD transgenic mice is also ApoE isoform dependent (E2>E3>E4), and recent studies suggest that ApoE4 may potentially impede clearance by competing for binding to receptors that function to remove Aβ. Also, the extent of ApoE lipidation may affect the formation of Aβ oligomers, with higher levels in ε4 / ε4 AD patients than ε3 / ε3. The present disclosure describes the discovery that anti-ApoE antibodies with certain characteristics provide a treatment for subjects with Aβ amyloidosis. Summary of the Invention

[0012] Certain aspects of the invention include an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 86, L2 of SEQ ID NO: 30, L3 comprising SEQ ID NO: 88, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 90, H2 comprising SEQ ID NO: 92, H3 comprising SEQ ID NO: 94, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In certain embodiments, the antibody is a humanized antibody.

[0013] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 87, L2 of SEQ ID NO: 30, L3 comprising SEQ ID NO: 89, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 91, H2 comprising SEQ ID NO: 93, H3 comprising SEQ ID NO: 95, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibodies are contemplated herein. In some embodiments, the antibodies are humanized antibodies.

[0014] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 78, L2 of SEQ ID NO: 24, L3 comprising SEQ ID NO: 25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 80, H2 comprising SEQ ID NO: 82, H3 comprising SEQ ID NO: 84, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0015] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 79, L2 of SEQ ID NO: 24, L3 comprising SEQ ID NO: 25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 81, H2 comprising SEQ ID NO: 83, H3 comprising SEQ ID NO: 86, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibodies are contemplated herein. In some embodiments, the antibodies are humanized antibodies.

[0016] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, L3 comprising SEQ ID NO: 107, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 109, H2 comprising SEQ ID NO: 111, H3 comprising SEQ ID NO: 113, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0017] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, L3 comprising SEQ ID NO: 108, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 110, H2 comprising SEQ ID NO: 112, H3 comprising SEQ ID NO: 114, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0018] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO:23, L2 of SEQ ID NO:24, L3 comprising SEQ ID NO:25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO:26, H2 comprising SEQ ID NO:27, H3 comprising SEQ ID NO:28, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0019] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO:29, L2 of SEQ ID NO:30, L3 comprising SEQ ID NO:31, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO:32, H2 comprising SEQ ID NO:33, H3 comprising SEQ ID NO:34, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0020] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 47, L2 of SEQ ID NO: 24, L3 comprising SEQ ID NO: 25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 48, H2 comprising SEQ ID NO: 49, H3 comprising SEQ ID NO: 50, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0021] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO:51, L2 of SEQ ID NO:24, L3 comprising SEQ ID NO:25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO:52, H2 comprising SEQ ID NO:53, H3 comprising SEQ ID NO:54, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0022] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO:55, L2 of SEQ ID NO:24, L3 comprising SEQ ID NO:25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO:56, H2 comprising SEQ ID NO:57, H3 comprising SEQ ID NO:58, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0023] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO:59, L2 of SEQ ID NO:24, L3 comprising SEQ ID NO:25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO:60, H2 comprising SEQ ID NO:61, H3 comprising SEQ ID NO:62, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0024] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 63, L2 of SEQ ID NO: 30, L3 comprising SEQ ID NO: 64, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 65, H2 comprising SEQ ID NO: 66, H3 comprising SEQ ID NO: 67, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0025] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO:68, L2 of SEQ ID NO:24, L3 comprising SEQ ID NO:25, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO:69, H2 comprising SEQ ID NO:70, H3 comprising SEQ ID NO:71, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0026] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 72, L2 of SEQ ID NO: 73, L3 comprising SEQ ID NO: 74, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 75, H2 comprising SEQ ID NO: 76, H3 comprising SEQ ID NO: 77, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0027] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, L3 comprising SEQ ID NO: 123, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 124, H2 comprising SEQ ID NO: 125, H3 comprising SEQ ID NO: 126, or any combination thereof. Compositions (including but not limited to pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0028] Another aspect of the invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 99, L2 of SEQ ID NO: 100, L3 comprising SEQ ID NO: 101, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 102, H2 comprising SEQ ID NO: 103, H3 comprising SEQ ID NO: 104, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0029] Another aspect of the present invention includes an isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, L3 comprising SEQ ID NO: 117, or any combination thereof; and / or (b) a heavy chain variable region comprising H1 comprising SEQ ID NO: 118, H2 comprising SEQ ID NO: 119, H3 comprising SEQ ID NO: 120, or any combination thereof. Compositions (including, but not limited to, pharmaceutical compositions) comprising the antibody are contemplated herein. In some embodiments, the antibody is a humanized antibody.

[0030] Another embodiment of the present invention includes a method for ameliorating clinical signs of Aβ amyloidosis, comprising administering an effective amount of an anti-ApoE antibody to a living human subject. In another embodiment, the present invention includes a method for effectively treating at least one clinically detectable Aβ plaque-related symptom and / or CAA-related symptom, comprising administering an effective amount of an anti-ApoE antibody to a living human subject. In another embodiment, the present invention includes a method for treating Alzheimer's disease, comprising administering an effective amount of an anti-ApoE antibody to a living human subject. In another embodiment, the present invention includes a method for treating CAA, comprising administering an effective amount of an anti-ApoE antibody to a living human subject. In each of the above embodiments, the anti-ApoE antibody can be an antibody as described above.

[0031] Yet another aspect of the present invention includes a pharmaceutical composition useful for treating at least one clinically detectable Aβ plaque-related condition. The composition comprises a pharmacologic effective amount of an anti-ApoE antibody suitable for administration to a living human subject. The anti-ApoE antibody can be an antibody described above. In some embodiments, the pharmaceutical composition is effectively administered systemically to a living subject.

[0032] Yet another embodiment of the present invention includes a pharmaceutical composition useful for treating at least one clinically detectable symptom associated with CAA. The composition comprises a pharmacologic effective amount of an anti-ApoE antibody suitable for administration to a living human subject. The anti-ApoE antibody can be an antibody described above. In some embodiments, the pharmaceutical composition is effectively administered systemically to a living subject.

[0033] Yet another aspect of the present invention includes a pharmaceutical composition useful for treating Alzheimer's disease. The composition comprises a pharma- ceutical effective amount of an anti-ApoE antibody suitable for administration to a living human subject. The anti-ApoE antibody can be an antibody described above. In some embodiments, the pharmaceutical composition is effectively administered systemically to a living subject.

[0034] Yet another embodiment of the present invention includes a pharmaceutical composition useful for treating CAA. The composition comprises a pharmacologic effective amount of an anti-ApoE antibody suitable for administration to a living human subject. The anti-ApoE antibody can be an antibody described above. In some embodiments, the pharmaceutical composition is effectively administered systemically to a living subject.

[0035] Yet another aspect of the invention includes a kit comprising a container containing a pharmaceutical composition comprising a pharma- ceutical effective amount of an anti-ApoE antibody suitable for administration to a living human subject, and any medical device used for said administration. The anti-ApoE antibody may be an antibody as described above.

[0036] Other aspects and iterations of the invention are detailed below. [Brief description of the drawings]

[0037] This application contains at least one photograph executed in color. Copies of this patent application publication containing the color photograph(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] [Figure 1] Figure 1 shows images of Western blots. Brain lysates from ApoE KO mice or mice expressing ApoE3, ApoE4 or mouse ApoE were immunoblotted with GA-50, HJ151 or HJ152. Western blots show that HJ151 is ApoE4 specific, and HJ152 recognizes both ApoE3 and ApoE4. GA-50 is a positive control.

[0039] [Diagram 2] Figure 2 shows an image of a Western blot. Brain lysates from ApoE KO mice or mice expressing ApoE3, ApoE4 or mouse ApoE were immunoblotted with HJ153 or HJ154. Western blots show that HJ153 and HJ154 recognize both ApoE3 and ApoE4.

[0040] [Diagram 3] Figure 3 shows images of brain tissue from 5XFAD APP transgenic mice expressing various human ApoE isoforms stained with biotinylated HJ153 or HJ154 antibodies. The figure shows that these two antibodies stain ApoE in neuropil, astrocytes, and amyloid plaques when present. Brain tissue from ApoE KO mice was used as a negative control.

[0041] [Figure 4]Figure 4 shows some pictures of immunoblots. Samples containing ApoE2, ApoE3 or ApoE4 were immunoprecipitated with HJ152, HJ153 or HJ154. The material immunoprecipitated with anti-ApoE antibodies (indicated as IP) and the solution after immunoprecipitation (indicated as post-IP) were run on SDS-PAGE gel, transferred to nitrocellulose membrane, and then immunoblotted with GA-50 (a commercial anti-ApoE antibody). WUE4, a monoclonal antibody against human ApoE, was used as a positive control. The results showed that HJ152 could immunoprecipitate some ApoE from the samples, but some ApoE still remained in the products after IP. HJ153 and HJ154 could immunoprecipitate all ApoE from the samples.

[0042] [Diagram 5] Figure 5A-H are graphs showing the characteristics of various antibodies. Specifically, Figure 5A-D are graphs showing the results of ELISA using various antibodies. ELISA plates were coated with 0.5 μg / ml of recombinant apoE2 (squares), apoE3 (open circles) or apoE4 (closed circles). Various concentrations of (A) HJ151, (B) HJ153, (C) HJ154 or (D) HJ156 were then loaded onto the plate. Binding was detected using a horseradish peroxidase-labeled goat anti-mouse secondary antibody. The results show that HJ153 and HJ154 bind to ApoE2, ApoE3 and ApoE4 as detected in the ELISA. HJ151 is specific for ApoE4, and HJ156 binds only to ApoE3 and ApoE4. Figure 5E-H are graphs showing the surface plasmon resonance profiles for various antibodies. To detect binding to biotinylated recombinant apoE4 captured on a streptavidin chip, anti-ApoE antibodies were serially diluted 3-fold (starting at 100 nM for HJ153 (E), 1000 nM for HJ151 (F) and HJ1156 (G)). Samples were injected at a flow rate of 30 μl / min. (H) The apparent KD values ​​of HJ151, HJ153 and HJ156 were calculated based on the SPR experiments.

[0043] [Figure 6] Figure 6 is a graph showing that several anti-ApoE antibodies reduce Aβ plaques in APP / PS1-21 E4 / E4 mice after ICV infusion. APP / PS1-21 E4 / E4 mice received intracerebroventricular (ICV) continuous infusion of PBS (negative control), mouse IgG2ab (negative control), HJ5.1 (anti-Aβ antibody, positive control) or anti-apoE antibodies (HJ151, HJ154 and HJ156) starting at 2 months of age. Anti-ApoE or control antibodies (2 mg / ml) were loaded into subcutaneous osmotic minipumps (Alzet, model 2006) and infused through a surgically implanted catheter into the left lateral ventricle (bregma -0.4 mm, 1.0 mm lateral to the midline, 2.5 mm below the skull) at a rate of 1.2 μl / min for 6 weeks. At 3.5 months of age, mice were perfused and sections were stained for Aβ plaques using anti-Aβ antibody HJ3.4B. The percentage of area covered by plaques in the cerebral cortex dorsal to the hippocampus was quantified. Data were analyzed using one-way ANOVA followed by Tukey's post-hoc test (*, p<0.05; **, p<0.01; ***, p<0.001; n=10–12 / group).

[0044] [Figure 7]FIG. 7 is a graph showing that several anti-ApoE antibodies reduce the amount of fibrous plaques after ICV infusion in APP / PS1-21 E4 / E4 mice. APP / PS1-21 E4 / E4 mice received continuous intracerebroventricular (ICV) infusion of PBS (negative control), mouse IgG2ab (negative control), HJ5.1 (anti-Aβ antibody, positive control) or anti-apoE antibodies (HJ151, HJ154 and HJ156) from 2 months of age. Anti-ApoE or control antibodies (2 mg / ml) were loaded into a subcutaneous osmotic minipump (Alzet, model 2006) and infused through a surgically implanted catheter into the left lateral ventricle (bregma-0.4 mm, 1.0 mm lateral to the midline, 2.5 mm below the skull) at a rate of 1.2 μl / min for 6 weeks. At 3.5 months of age, mice were perfused and brain sections were stained for fibrous plaques with thioflavin S. The area covered by plaques in the cerebral cortex dorsal to the hippocampus was quantified. Data were analyzed using one-way ANOVA followed by Tukey's post-hoc test (*, p<0.05; n=10-12 / group).

[0045] [Figure 8]Figure 8A-F are graphs showing that anti-ApoE antibodies reduce insoluble Aβ42 in APP / PS1-21 E4 / E4 mice after ICV infusion. APP / PS1-21 E4 / E4 mice received intracerebroventricular (ICV) continuous infusion of PBS (negative control), mouse IgG2ab (negative control), HJ5.1 (anti-Aβ antibody, positive control) or anti-apoE antibodies (HJ151, HJ154 and HJ156) from 2 months of age. Anti-ApoE antibodies or control antibodies (2 mg / ml) were loaded into subcutaneous osmotic minipumps (Alzet, model 2006) and infused through a surgically implanted catheter into the left lateral ventricle (bregma -0.4 mm, 1.0 mm lateral to the midline, 2.5 mm below the skull) at a rate of 1.2 μl / min for 6 weeks (n=10-12 / group). At 3.5 months of age, mice were perfused and cortical tissues were homogenized sequentially in PBS, Triton, and 5 M guanidine. Aβ levels were measured in the soluble (A, D) and insoluble (B, C, E, F) fractions (Aβ40 data are shown in A–C, and Aβ42 data are shown in D–F). Student's t-test was used to analyze the data. Anti-apoE antibodies reduced Aβ42 levels in the various fractions (*, p<0.05; **, p<0.01).

[0046] [Figure 9] FIG. 9 is a graph showing the body weight of APP / PS1-21 E4 / E4 mice that were intraperitoneally injected with anti-ApoE antibody (injections once a week for 7 weeks).

[0047] [Figure 10] FIG. 10 is a graph showing that HJ151, HJ155 and HJ156 do not significantly reduce soluble Aβ40 in the PBS fraction of brain tissue homogenates from APP / PS1-21 E4 / E4 mice intraperitoneally injected with anti-ApoE antibody (injections once a week for 7 weeks).

[0048] [Figure 11]FIG. 11 is a graph showing that HJ151, HJ155 and HJ156 do not significantly reduce soluble Aβ42 in the PBS fraction of brain tissue homogenates from APP / PS1-21 E4 / E4 mice intraperitoneally injected with anti-ApoE antibody (injections once a week for 7 weeks).

[0049] [Figure 12] FIG. 12 is a graph showing that HJ156 significantly reduced insoluble Aβ40 in the 5 M guanidine fraction of brain tissue homogenates from APP / PS1-21 E4 / E4 mice that were intraperitoneally injected with anti-ApoE antibody (injections once a week for 7 weeks) (*p<0.05).

[0050] [Figure 13] FIG. 13 is a graph showing that HJ156 significantly reduced insoluble Aβ42 in the 5 M guanidine fraction of brain tissue homogenates from APP / PS1-21 E4 / E4 mice that were intraperitoneally injected with anti-ApoE antibody (injections once a week for 7 weeks) (*p<0.05, **p<0.01).

[0051] [Figure 14] Figure 14 is a graph showing the relative antibody concentration in the cortex of APPPS1-21 / APOE4 mice expressing recombinant (r)HJ151, rHJ151 with D265A mutation (Δ), rHJ156 and rHJ156Δ. APPPS1-21 / APOE4 mice were injected with AAV2 / 8 expressing full-length rHJ151 and rHJ156 with or without D265A mutation at P0. At 3.5 months of age, antibody concentrations in the PBS-soluble fraction of the cortex were measured by ELISA. The relative levels of each antibody were calculated by using purified antibodies from its hybridoma as a standard.

[0052] [Figure 15]Figure 15 is a graph showing that HJ151 and HJ156 significantly reduce insoluble Aβ42 in the guanidine fraction of brain tissue homogenates of APP / PS1-21 E4 / E4 mice injected with anti-ApoE antibody expressed in the brain using an adeno-associated virus (AAV) 2 / 8 vector. HJ151 and HJ156 with the D265A mutation (HJ151Δ and HJ156Δ) have no effect.

[0053] [Figure 16] 16 is a graph showing that HJ151 and HJ156 reduce insoluble Aβ40 in the guanidine fraction of brain tissue homogenates of APP / PS1-21 E4 / E4 mice injected with anti-ApoE antibody expressed in the brain using an adeno-associated virus (AAV) 2 / 8 vector. HJ151 and HJ156 with the D265A mutation (HJ151Δ and HJ156Δ) have no effect.

[0054] [Figure 17] 17A-B are graphs showing that HJ151 and HJ156 significantly reduce amyloid-β plaque area (A) and fibrous plaque area (B) in brain sections from APP / PS1-21 E4 / E4 mice injected with anti-ApoE antibodies expressed in the brain using an adeno-associated virus (AAV) 2 / 8 vector. HJ151 and HJ156 with the D265A mutation (HJ151Δ and HJ156Δ) have no effect.

[0055] [Figure 18] Figures 18A-G show unfixed tissue sections (20 μm thick) from APPPS1-21 / apoE4 / 4 (APPPS1-21 / E4), 5XFAD / apoE knockout (5XFAD / EKO) mice and human apoE4 / 4 (human E4) brains stained with biotinylated ("B") antibodies HJ3.4B (A), HJ151B (B), HJ152B (C), HJ153B (D), HJ154B (E), HJ155B (F) and HJ156B (G).

[0056] [Figure 19] Figure 19A-D shows antibody detection of ApoE in human plasma lipoprotein particles. (A) shows a schematic of the plasma binding assay used to demonstrate detection of ApoE in human plasma-derived lipoprotein particles coated on an ELISA plate. (B-D) show graphs of results from the binding assay for various antibodies, respectively. Detection of ApoE is observed with HJ153, HJ154, HJ1510, HJ1511, HJ1517, HJ1530 and HJ1534.

[0057] [Figure 20] Figures 20A-C show antibody detection of ApoE in a competitive ELISA. (A) shows a schematic of the plasma competition assay used to evaluate antibody binding preferences to coated recombinant non-lipidated ApoE4 (recApoE; 0.5 μg / mL) after preincubation in various dilutions of human plasma. (B-C) show graphs of results from the competitive ELISA for various antibodies, respectively. "x ApoE" is an abbreviation for "anti-ApoE antibody."

[0058] [Figure 21] Figures 21A-D show images at two different magnifications of unfixed frozen brain sections from APP / PS1-21 E2 / E2 mice immunostained for ApoE with anti-ApoE antibody HJ156B at 20 μg / ml (A, C) or 50 μg / ml (B, D). Scale bar is 400 μm.

[0059] [Figure 22] Figures 22A-D show images at two different magnifications of unfixed frozen brain sections from APP / PS1-21 E3 / E3 mice immunostained for ApoE with anti-ApoE antibody HJ156B at 20 μg / ml (A, C) or 50 μg / ml (B, D). Scale bar is 400 μm.

[0060] [Diagram 23]Figures 23A-D show images at two different magnifications of unfixed frozen brain sections from APP / PS1-21 E4 / E4 mice immunostained for ApoE with anti-ApoE antibody HJ156B at 20 μg / ml (A, C) or 50 μg / ml (B, D). Scale bar is 400 μm.

[0061] [Figure 24] Figures 24A-D show images of unfixed frozen brain sections from APP / PS1-21 E4 / E4 mice immunostained for ApoE using anti-ApoE msIgG / huFc chimeric antibody HJ153 and biotinylated rabbit anti-human IgG secondary antibody. Primary antibody dilutions of (A) 0.4 μg / mL, (B) 2 μg / mL, (C) 20 μg / mL, and (D) 50 μg / mL were used. Scale bars are 400 μm.

[0062] [Diagram 25] Figures 25A-D show images of unfixed frozen brain sections from APP / PS1-21 E4 / E4 mice immunostained for ApoE using anti-ApoE msIgG / huFc chimeric antibody HJ156 and a biotinylated rabbit anti-human IgG secondary antibody. Primary antibody dilutions of (A) 0.4 μg / mL, (B) 2 μg / mL, (C) 20 μg / mL, and (D) 50 μg / mL were used. Scale bars are 400 μm.

[0063] [Figure 26] Figures 26A-D show images of unfixed frozen brain sections from APP / PS1-21 E4 / E4 mice immunostained for ApoE using anti-ApoE msIgG / huFc chimeric antibody HJ1514 and biotinylated rabbit anti-human IgG secondary antibody. Primary antibody dilutions of (A) 0.4 μg / mL, (B) 2 μg / mL, (C) 20 μg / mL, and (D) 50 μg / mL were used. Scale bars are 400 μm.

[0064] [Figure 27]Figures 27A-D show images of unfixed frozen brain sections from APP / PS1-21 E4 / E4 mice immunostained for ApoE using anti-ApoE msIgG / huFc chimeric antibody HJ1518 and biotinylated rabbit anti-human IgG secondary antibody. Primary antibody dilutions of (A) 0.4 μg / mL, (B) 2 μg / mL, (C) 20 μg / mL, and (D) 50 μg / mL were used. Scale bars are 400 μm.

[0065] [Figure 28] Figures 28A-D show images of unfixed frozen brain sections from APP / PS1-21 E4 / E4 mice immunostained for ApoE with 2 μg / mL of anti-ApoE msIgG / huFc chimeric antibody ((A) HJ153 chimera, (B) HJ156 chimera, (C) HJ1514 chimera, (D) HJ1518 chimera). Scale bar is 400 μm.

[0066] [Figure 29] Figure 29 is a graph showing Aβ deposition (measured as % area of ​​Aβ staining) in APP / PS1-21 E4 / E4 mice treated with weekly intraperitoneal (IP) injections of PBS, HJ16.6 (IgG negative control), and anti-ApoE antibodies HJ151, HJ155, and HJ156 from 2 to 3.5 months of age. All antibodies were administered at 50 mg / kg. Brain sections from 2-month-old untreated mice and 3.5-month-old treated mice were stained with anti-Aβ antibody HJ3.4-biotin. Aβ staining was quantified from the cortex. n=10 per group. *p<0.05 compared to PBS and HJ16.6 groups.

[0067] [Diagram 30]FIG. 30 is a graph showing Aβ plaque counts in APP / PS1-21 E4 / E4 mice treated with weekly intraperitoneal (IP) injections of PBS, HJ16.6 (IgG negative control), and anti-ApoE antibodies HJ151, HJ155, and HJ156 from 2 to 3.5 months of age. All antibodies were administered at 50 mg / kg. Brain sections from 2-month-old untreated mice and 3.5-month-old treated mice were stained with anti-Aβ antibody HJ3.4-biotin. Aβ plaque counts detected by Aβ plaque staining were quantified from the cortex. n=10 per group. *p<0.05 compared to PBS and HJ16.6 groups.

[0068] [Diagram 31] Figure 31 is a graph showing fibrillar Aβ plaque deposition (measured as % area of ​​X-34 staining) in APP / PS1-21 E4 / E4 mice treated with weekly intraperitoneal (IP) injections of PBS, HJ16.6 (IgG negative control), and anti-ApoE antibodies HJ151, HJ155, and HJ156 from 2 to 3.5 months of age. All antibodies were administered at 50 mg / kg. Brain sections from 2-month-old untreated mice and 3.5-month-old treated mice were stained with X-34, a dye that stains fibrous plaques. X-34 was quantified from the cortex. n=10 per group. *p<0.05 compared to PBS and HJ16.6 groups.

[0069] [Diagram 32]FIG. 32 is a graph showing fibrillar Aβ plaque counts in APP / PS1-21 E4 / E4 mice treated with weekly intraperitoneal (IP) injections of PBS, HJ16.6 (IgG negative control), and anti-ApoE antibodies HJ151, HJ155, and HJ156 from 2 to 3.5 months of age. All antibodies were administered at 50 mg / kg. Brain sections from 2-month-old untreated mice and 3.5-month-old treated mice were stained with X-34, a dye that stains fibrous plaques. Fibrillar Aβ plaque counts detected by X-34 staining were quantified from the cortex. n=10 per group. *p<0.05 compared to PBS and HJ16.6 groups.

[0070] [Diagram 33] Figure 33A-F show images of brain sections immunostained for Aβ using an anti-Aβ antibody (HJ3.4-biotin). (A,D) Brain sections from untreated 2-month-old mice before Aβ plaque development. (B,E) Brain sections from 3.5-month-old mice treated with PBS. (C,F) Brain sections from 3.5-month-old mice treated with 50 mg / kg negative control IgG (HJ16.6). Scale bar is 300 μm, n=10 per group.

[0071] [Diagram 34] Figure 34A-F show images of brain sections immunostained for Aβ using an anti-Aβ antibody (HJ3.4-biotin). (A,D) Brain sections from 3.5-month-old mice treated with 50 mg / kg HJ151. (B,E) Brain sections from 3.5-month-old mice treated with 50 mg / kg HJ155. (C,F) Brain sections from 3.5-month-old mice treated with 50 mg / kg HJ156. Scale bar is 300 μm, n=10 per group.

[0072] [Diagram 35]Figure 35A-F show images of brain sections stained with X-34 dye, which recognizes fibrotic plaques. (A,D) Brain sections from untreated 2-month-old mice before Aβ plaque development. (B,E) Brain sections from 3.5-month-old mice treated with PBS. (C,F) Brain sections from 3.5-month-old mice treated with 50 mg / kg negative control IgG (HJ16.6). Scale bar is 300 μm, n=10 per group.

[0073] [Diagram 36] Figure 36A-F show images of brain sections stained with X-34 dye, which recognizes fibrotic plaques. (A,D) Brain sections from 3.5-month-old mice treated with 50 mg / kg HJ151. (B,E) Brain sections from 3.5-month-old mice treated with 50 mg / kg HJ155. (C,F) Brain sections from 3.5-month-old mice treated with 50 mg / kg HJ156. Scale bars are 300 μm, n=10 per group.

[0074] [Figure 37]Figure 37A-F are graphs showing the binding profiles of HJ151, HJ153 and HJ156 with lipidated apoE. (A, B) ELISA after coating of plasma onto plates. Plasma from apoE KO, APOE2, APOE3 and APOE4 mice were coated onto plates. Various concentrations of chi153 and chi156 were loaded. Captured antibodies were detected with HRP goat anti-human IgG antibody. (C, D) ELISA after coating of antibodies onto plates. Plates were coated with HJ153 and HJ156. Plasma from mice with various genotypes was loaded. Captured ApoE was detected with HRP goat polyclonal anti-ApoE. (E) Plasma competition experiments were performed by ELISA format. Recombinant ApoE4 was coated onto plates. Plates were loaded with HJ151 (50 nM), HJ153 (4 nM), and HJ156 (50 nM) preincubated with serially diluted plasma from APOE4 KI mice. Plate-bound HJ15 antibodies were detected with HRP-goat anti-mouse IgG antibody. (F) Plasma antibody concentrations of HJ153, HJ156, or control IgG after IP injection into APOE4 KI or EKO mice. HJ156 was administered at 2, 10, and 50 mg / kg, and plasma samples were collected by submandibular puncture. HJ153 was administered at 10 mg / kg. Control mouse IgG2a (msIgG2a) is anti-Her2 and was administered at 10 mg / kg. Quantification of administered antibodies in plasma was performed by antigen capture ELISA using coated recombinant apoE4 to detect HJ153 or HJ156 and recombinant Her2 to detect control IgG. Peripheral clearance of HJ156 was similar to control IgG with target-mediated clearance observed by day 14. HJ153 showed high clearance, reaching a nadir by 48 hours.

[0075] [Figure 38]Figure 38A-E shows the binding of HJ151 and HJ156 to ApoE4 in amyloid plaques in unfixed mouse brain sections and the specificity of ApoE4 to heat-induced aggregates. (A) Unfixed frozen brain sections from APPPS1 / APOE4 or APPPS1 / APOE KO mice were stained with anti-Aβ antibody HJ3.4, anti-ApoE antibodies HJ151 and HJ156 (scale bar = 400 μm). (B) Binding of HJ151, HJ153 and HJ156 to untreated recombinant ApoE4 (untreated) and ApoE4 incubated at 40°C for 24 h (40C). (C) Incubation of ApoE4 at 40°C for 24 h leads to the formation of aggregates that were recovered in the pellet fraction after ultracentrifugation at 186,000 g for 1 h. 1: untreated ApoE4. 2: ApoE4 incubated at 40°C for 24 h. Supernatants (S) and pellets (P) from ultracentrifugation were separated by SDS-PAGE and stained with Coomassie blue. (D) Binding of HJ156 to different preparations of ApoE4 loaded at the same concentration on an ELISA plate. Untreated Supernatant: Supernatant fraction from ultracentrifugation of untreated ApoE4. 24h 40C Supernatant / Pellet: Supernatant / Pellet fraction of ApoE4 incubated at 40°C for 24 h. (E) Binding of HJ156 to untreated ApoE4 and ApoE4 incubated at 40°C, denatured with 1% SDS or 4 M guanidine hydrochloride, and not denatured.

[0076] [Figure 39]Figure 39A-D are images of HJ151, HJ156 and control antibodies binding to human ApoE4 in live mouse brains. (A) Negative control IgG2ab conjugated with Alexa594, (B) HJ151 and (C) HJ156 were directly applied to the brain surface of live APPPS1-21 / APOE4 mice that were 6 months old, and antibody binding was observed under two-photon microscopy. Amyloid was labeled with methoxy-X04. The middle panel is a high-magnification image of the area in the white box in the top panel. The arrow indicates the plaque. The bottom panel is a high-magnification image of the area in the yellow box in the top panel. The arrow indicates CAA. (D) APPPS1-21 / APOE4 mice were injected once (0 h) or twice (0 and 48 h) with 50 mg / kg body weight (ip) of control huIgG or chi156 and sacrificed 48 h after the last injection. Antibodies in the brain were detected by biotinylated rabbit anti-human IgG followed by DAB (left panel: bar=1 mm, right panel: high magnification image of the indicated area in the left panel. Bar=300 μm).

[0077] [Diagram 40] Figure 40 is a graph showing that HJ156 activates microglia and promotes plaque clearance. Four-month-old APPPS1-21 / APOE4 mice that already had pre-existing plaques were administered short-term treatments of HJ151 and HJ156 antibodies (four doses by IP injection every three days). Stained sections were analyzed for the area of ​​CD45+ microglia relative to the amount of fibrous plaques. HJ151 did not affect the amount of activated microglia. *p<0.05.

[0078] [Diagram 41] Figure 41 shows microglia and plaque staining after acute immunization with HJ156. At 4 months of age, APPPS1 / APOE4 mice were IP-injected with 50 mg / kg body weight of antibody every 3 days 4 times. Mice were sacrificed 24 hours after the last injection, and plaques were stained with X34 and activated microglia were stained with CD45 (bar=1 mm).

[0079] [Diagram 42] Figure 42A-B graphically illustrates the results of a long-term dose-ranging efficacy study. Two-month-old APPPS1-21 / APOE4 mice were injected intraperitoneally 12 times with weekly doses of IgG2ab (50 mg / kg), anti-Aβ HJ3.4 (50 mg / kg) or HJ156 (2, 10 and 50 mg / kg) (n=17-18 / group, mixed sex). Groups of 2-month-old animals (n=12) were harvested and baseline levels of Aβ were determined. At 4.5 months of age (3 days after the last dose), mice were perfused with ice-cold PBS containing 0.3% heparin. Cerebral cortices were homogenized sequentially with cold PBS and 5M guanidine buffer in the presence of 1x protease inhibitor mix. (A) Aβ40 and (B) Aβ42 in the guanidine fraction were determined by ELISA. One-way ANOVA followed by Tucky's post-hoc test was performed to compare different groups. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

[0080] [Diagram 43]Figure 43A-C graphically shows the binding of (A) HJ151, (B) HJ154 and (C) HJ156 antibodies to untreated recombinant and heat-induced aggregates of ApoE2, ApoE3 and ApoE4. Aggregated ApoE was induced by incubating 1 mg / ml concentration of ApoE at 40°C for 24 hours. The aggregates were then recovered in the pellet fraction after ultracentrifugation at 186,000g for 1 hour. For ELISA, untreated ApoE or heat-induced aggregates of ApoE were directly coated onto ELISA plates at 0.5 μg / ml overnight at 4°C. After washing three times with PBS, the wells were blocked with 1% BSA-PBS for 1 hour at room temperature with shaking at 500 rpm. The blocked wells were washed once with PBS and then loaded with antibodies at graded concentrations (starting at 100 nM followed by 5-fold dilutions). Bound antibodies were detected with HRP-conjugated goat anti-mouse IgG (Jackson Immunoresearch) and visualized at OD650 with TMB substrate (reactions were stopped with BioFX stop solution). Results show that HJ151 detected only ApoE4 with some preference for heat-treated ApoE4, HJ154 detected all three isoforms of ApoE regardless of conformation, and HJ156 detected both ApoE3 and ApoE4 with preference for heat-treated ApoE. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Detailed Description Applicant has discovered anti-ApoE antibodies and methods of using the same for treating Aβ amyloidosis, comprising administering to a living subject a therapeutically effective amount of an anti-ApoE antibody that (a) binds to human ApoE4 with a KD of about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, (b) preferentially binds to recombinant alipidated human ApoE4 compared to human plasma-derived ApoE4 or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4, and (c) binds to human ApoE in amyloid plaques in unfixed brain tissue. The present invention includes the discovery that anti-ApoE antibodies having these characteristics provide treatment for subjects with Aβ amyloidosis (collectively referred to herein as "subjects in need of treatment"), including, but not limited to, subjects diagnosed with a disease characterized by brain Aβ plaques, subjects diagnosed with a disease characterized by vascular Aβ plaques in the brain, subjects diagnosed with symptoms associated with Aβ plaques, subjects diagnosed with symptoms associated with CAA, subjects diagnosed with clinical signs of Aβ amyloidosis with or without symptoms associated with Aβ plaques and / or symptoms associated with CAA, subjects diagnosed with Alzheimer's disease, and subjects diagnosed with CAA. Methods for determining clinical signs of Aβ amyloidosis in asymptomatic patients are known in the art and are described below.

[0082] I. Definition The term "subject" refers to a human or a non-human animal that expresses human ApoE.

[0083] As used herein, the terms "treat", "treating" and "treatment" refer to both therapeutic treatment and prophylactic measures, where the objective is to prevent or slow (alleviate) the progression of undesirable physiological changes or disease / disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or complete), whether detectable or not. "Treatment" can also mean prolonging survival compared to that expected assuming no treatment. Those in need of treatment include those already with the disease, condition or disorder, as well as those prone to having the disease, condition or disorder, or those in whom the disease, condition or disorder is to be prevented.

[0084] The term "Aβ" refers to a peptide derived from the carboxy-terminal region of a larger protein called amyloid precursor protein (APP). The gene encoding APP is present on chromosome 21. Aβ exists in many forms, some of which have toxic effects: Aβ peptides typically have a sequence length of 37-43 amino acids, but may have truncations and modifications that change their overall size. They can be found intracellularly or extracellularly in monomeric, oligomeric and aggregated forms, in soluble and insoluble compartments, and may also form complexes with other proteins or molecules. The harmful or toxic effects of Aβ may be due to some or all of the above forms, as well as other forms not specifically mentioned. For example, two such Aβ isoforms are Aβ40 and Aβ42, with the Aβ42 isoform being particularly fibrillogenic or insoluble and associated with disease states.

[0085] "Aβ amyloidosis" is clinically defined as evidence of Aβ deposition in the brain or cerebrovascular, typically in the form of amyloid plaques or CAA. Diseases associated with Aβ amyloidosis include, but are not limited to, pre-symptomatic stages of Alzheimer's disease, Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia, and inclusion body myositis. "At high risk of developing Aβ amyloidosis-related disease" refers to a higher risk than that predicted by the subject's age, family history, genetic status, and other known risk factors.

[0086] "Clinical signs of Aβ amyloidosis" refers to the determination of Aβ deposits known in the art. Clinical signs of Aβ amyloidosis include, but are not limited to, Aβ deposits determined by amyloid imaging (e.g. PiB PET, florbetapir, or other imaging methods known in the art) or by reduction in cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio.See, for example, Klunk WE et al. Ann Neurol 55(3) 2004 and Fagan AM et al. Ann Neurol 59(3) 2006 (which are incorporated herein by reference in their entirety). Clinical indicia of Aβ amyloidosis may also include measurements of Aβ metabolism, particularly Aβ42 metabolism, alone or in comparison with measurements of metabolism of other Aβ variants (e.g., Aβ37, Aβ38, Aβ39, Aβ40, and / or total Aβ), see U.S. Patent Application Nos. 14 / 366831, 14 / 523148, and 14 / 747453, which are incorporated herein by reference in their entireties. Further 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 (all of which are incorporated herein by reference in their entirety).Importantly, subjects with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition.However, subjects with clinical signs of Aβ amyloidosis are at high risk of developing diseases associated with Aβ amyloidosis.

[0087] "Aβ plaque-associated symptoms" or "CAA-associated symptoms" refer to any symptoms caused by or associated with the formation of amyloid plaques or CAA (composed of regularly arranged fibrous aggregates called amyloid fibrils), respectively. Examples of Aβ plaque-associated symptoms include, but are not limited to, neurodegeneration, cognitive impairment, memory impairment, behavioral changes, impaired emotional control, seizures, impaired nervous system structure or function, and increased risk of developing or worsening Alzheimer's disease or CAA. Neurodegeneration can include changes in neuronal structure (including molecular changes, such as intracellular accumulation of toxic proteins, protein aggregates, and macro-level changes, such as changes in axon or dendrite shape or length, changes in myelin sheath composition, loss of myelin sheath), changes in neuronal function, decreased neuronal function, death of neurons, or any combination thereof. Cognitive impairment can include, but is not limited to, impairments in memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and systematic thinking. Behavioral changes may include, but are not limited to, aggressive behavior or language, impulsivity, reduced inhibitions, apathy, reduced motivation, personality changes, alcohol, tobacco or drug abuse, and other addiction-related behaviors. Disorders of emotional regulation may include, but are 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. Disorders of nervous system structure or function may include, but are not limited to, hydrocephalus, Parkinson's disease, sleep disorders, psychosis, and disorders of balance and coordination. This may include movement disorders such as monoparesis, hemiparesis, quadriplegia, ataxia, ballismus, and tremors. This may also include loss or dysfunction of the senses, including smell, touch, taste, sight, and hearing. This may further include disorders of the autonomic nervous system such as bowel and bladder dysfunction, sexual dysfunction, and disorders of blood pressure and temperature regulation.This can also include hormonal disorders resulting from hypothalamic and pituitary dysfunction, such as deficiencies and dysregulation of growth hormone, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, gonadotropin releasing hormone, prolactin and many other hormones and modulators.

[0088] "ApoE" (NP_000032.1, UniProtKB Identifier P02649) is an apolipoprotein expressed from the APOE gene (e.g., the nucleotide sequence identified as GenBank Accession Number NM_000041 or NCBI Reference Sequence: NC_000019.10) located on chromosome 19, and has three major polymorphic forms: ApoE2 (Cys112, Cys158), ApoE3 (Cys112, Arg158), and ApoE4 (Arg112, Arg158). Unless otherwise specified, "ApoE" refers to "human ApoE" and includes functional fragments. "Recombinant ApoE" refers to ApoE encoded by a nucleic acid introduced into a system (e.g., a prokaryotic cell, a eukaryotic cell, or a cell-free expression system) that supports expression of the nucleic acid and its translation into a protein. Methods for producing recombinant proteins are well known in the art, and the production of recombinant ApoE disclosed herein is not limited to a particular system. However, one skilled in the art will recognize that the choice of system can affect how "lipid-free" ApoE is produced when recombinantly produced. As used herein, the term "alipidated ApoE" refers to ApoE that is recombinantly produced in prokaryotic cells.

[0089] As used herein, the term "antibody" is used in the broadest sense to encompass a variety of antibodies and antibody-like structures, including, but not limited to, full-length monoclonal, polyclonal, and multispecific (bispecific, trispecific, etc.) antibodies, as well as heavy chain antibodies and antibody fragments, provided that they exhibit the desired antigen-binding activity. The antibody domains involved in antigen binding are referred to as "variable regions" or "variable domains" and are described in detail later in this document. A single variable domain may be sufficient to confer antigen-binding specificity. Preferably, but not necessarily, antibodies useful in the present invention are recombinantly produced. The antibodies may be glycosylated or non-glycosylated, although glycosylated antibodies may be preferred. An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity as measured by methods known in the art.

[0090] In addition to the antibodies described herein, it may be possible to design antibody mimetics or aptamers that function substantially similarly to the antibodies of the invention using methods known in the art. An "antibody mimetic" refers to a polypeptide or protein that can specifically bind to an antigen but is not structurally related to an antibody. An antibody mimetic has a molecular weight of about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. Aptamers are a class of small nucleic acid ligands consisting of RNA or single-stranded DNA oligonucleotides that have high specificity and affinity for their targets. Aptamers interact with and bind to their targets through structural recognition, a process similar to the antigen-antibody reaction process. Aptamers typically have a molecular weight smaller than antibodies, about 8 to 25 kDa.

[0091] The terms "full-length antibody" and "intact antibody" may be used interchangeably and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain including an Fc region as defined herein. The basic structural unit of a native antibody comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). Light chains are classified as gamma, mu, alpha, and lambda. Heavy chains are classified as gamma, mu, alpha, delta, and epsilon, which determine the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. The amino-terminal portion of each light and heavy chain contains a variable region (VL and VH, respectively) of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids in sequence, and in the case of the heavy chain also includes a "D" region of about 10 amino acids in sequence. Intact antibodies are properly cross-linked by disulfide bonds, as is known in the art.

[0092] The variable domains of the heavy and light chains of an antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated using a VH or VL domain from an antibody that binds to the antigen, and a library of complementary VL or VH domains, respectively, may be screened (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0093] "Framework region" or "FR" refers to the portion of a variable domain other than the hypervariable region (HVR) portion. The FR of a variable domain generally consists of four FR domains FR1, FR2, FR3 and FR4. Thus, the sequence of the HVRs and FRs generally appears in the following sequence: FR1-HVR1-FR2-HVR2-FR3-HVR3-FR4. As is known in the art, the FR domains of the heavy and light chains may differ.

[0094] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of the variable domain that differ significantly in sequence (also commonly referred to as "complementarity determining regions" or "CDRs") and / or that form structurally defining loops ("hypervariable loops"). Generally, antibodies have six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). As used herein, "HVRs derived from a variable region" refer to HVRs that have no more than two amino acid substitutions compared to the corresponding HVRs in the original variable region. Examples of HVRs include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b) and / or (c), as defined herein below for the various antibodies of the disclosure. In this specification, unless otherwise specified, the numbering of the HVR portions and other portions (e.g., FR portions) in the variable domains is according to Kabat et al., supra.

[0095] The term "Fc region" is used herein to define an immunoglobulin heavy chain C-terminal region that includes at least a portion of the constant region. This term encompasses native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region is from Cys226 or Pro230 to the heavy chain carboxyl terminus. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In this document, unless otherwise specified, the numbering of the amino acid sequence of the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0096] A "variant Fc region" comprises an amino acid sequence that may differ from that of a native Fc region by one or more amino acid substitutions and / or by altered glycosylation patterns compared to a native Fc region or the Fc region of the original polypeptide. In one example, a variant Fc region may have from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in a native sequence Fc region or the Fc region of the original polypeptide. A variant Fc region may have at least about 80% identity, at least about 90% identity, or at least about 95% identity to a native sequence Fc region or the Fc region of the original polypeptide.

[0097] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single chain forms of antibodies and higher order variants thereof; single domain antibodies; and multispecific antibodies formed from antibody fragments.

[0098] Single chain forms of antibodies and higher forms thereof include, but are not limited to, single domain antibodies, single chain variant fragments (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), diabodies, and triabodies and tetrabodies. ScFvs include heavy and light chain variable regions linked by a linker. In many, but not all, instances, the linker can be a peptide. The linker peptide is preferably about 5-30 amino acids long, or about 10-25 amino acids long. Typically, the linker allows for stabilization of the variable regions without interfering with proper folding and formation of the active binding site. In preferred embodiments, the linker peptide is rich in glycine and serine or threonine. ScFvs can be used to facilitate phage display, or can be used for flow cytometry, immunohistochemistry, or as targeting domains. Methods for preparing and using ScFv are known in the art. ScFv can also be conjugated to human constant regions (e.g., heavy chain constant domains derived from IgG domains such as IgG1, IgG2, IgG3 or IgG4, or heavy chain constant domains derived from IgA, IgM or IgE). Diabodies, triabodies and tetrabodies and higher variants are typically created by varying the length of the linker peptide from zero to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be created using self-assembly units linked to variable domains.

[0099] "Single domain antibody" refers to an antibody fragment consisting of a single variable antibody domain monomer.

[0100] Multispecific antibodies include bispecific antibodies, trispecific antibodies, or antibodies with quadruple or greater specificity. Multispecific antibodies may be made by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. The chains may be linked by covalent bonds.

[0101] A "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. A "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology well known in the art, or can be produced by recombinant technology, phage display technology, synthetic technology, or a combination of such technologies, and other techniques readily known in the art. Furthermore, monoclonal antibodies can be labeled with a detectable label, immobilized on a solid phase, and / or conjugated to a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[0102] "Heavy chain antibody" refers to an antibody consisting of two heavy chains. The heavy chain antibody may be an IgG-like antibody from camel, llama, alpaca, shark, etc., or an IgNAR from cartilaginous fish.

[0103] "Humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response after administration in humans, but maintains the same binding specificity and affinity as the original. A humanized antibody binds to the same or similar epitope as its non-human antibody. The term "humanized antibody" encompasses antibodies that are partially or entirely composed of amino acid sequences derived from the human antibody series, due to alterations in the sequence of an antibody with a non-human hypervariable region ("HVR"). The simplest of such alterations may consist of simply replacing the mouse constant regions with the constant regions of a human antibody, resulting in a human / mouse chimera that may have sufficiently low immunogenicity to be acceptable for pharmaceutical use. Preferably, the variable regions of the antibody are also humanized, by techniques that are by now well known in the art. For example, one, several or all six non-human HVRs may be maintained and the framework regions of the variable regions replaced with the corresponding human framework regions. For example, to restore or improve the specificity or affinity of a humanized antibody, some framework portions may be replaced with the corresponding portions derived from a non-human VL or VH domain (e.g., the non-human antibody from which the HVR portions are derived). A substantially human framework region has at least about 75% identity (i.e., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% sequence identity) with a known human framework sequence. HVRs may also be randomly mutated to maintain or improve antigen binding activity and affinity when the framework regions are fully human or substantially human. As mentioned above, it is sufficient to use antibody fragments for use in the methods of the present invention. Furthermore, as used herein, the term "humanized antibody" refers to an antibody that comprises substantially human framework regions, at least one HVR derived from a non-human antibody, and any constant regions present therein are substantially human. A substantially human constant region has at least about 90% identity (i.e., about 90%, about 95% or about 99% sequence identity) with a known human constant sequence.Hence, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding sequences of one or more series of human immunoglobulins.

[0104] If desired, the design of humanized immunoglobulins can be performed as described below or using similar methods known to those skilled in the art (see, for example, Almagro, et al. Front. Biosci. 2008, 13(5):1619-33). The mouse antibody variable regions are aligned against the sequences of the most similar human series (e.g., using BLAST or a similar algorithm). The CDR portions from the mouse antibody sequence are grafted into the similar human "acceptor" series. One or more positions close to the CDR or in the framework (e.g., Vernier positions) can then be reverted to the original mouse amino acid to give the humanized antibody a similar binding affinity to the original mouse antibody. Typically, several versions of the humanized antibody with different reversion mutations are generated and tested experimentally for activity. The humanized antibody variant with the closest properties to the original mouse antibody and with the fewest mouse framework reversions is selected as the final humanized antibody candidate.

[0105] II. Anti-ApoE antibody The anti-ApoE antibodies disclosed herein can be described or specified with respect to the epitope they recognize or bind. The portion of the target polypeptide that specifically interacts with the antigen-binding domain of the antibody is the "epitope". ApoE can have any number of epitopes depending on the origin of the protein (e.g., mouse, rat, cynomolgus monkey, human, etc.), the isoform (e.g., ApoE2, ApoE3, ApoE4), the conformational state of the isoform (e.g., fibrillar, aggregated, insoluble, soluble, monomeric, oligomeric, oxidized, post-translationally modified, etc.), and the location of the isoform (e.g., intracellular, extracellular, complexed with other proteins or molecules in particles, in amyloid plaques, etc.). It should further be noted that the "epitope" on ApoE can be a linear epitope or a conformational epitope, and in either case can include non-polypeptide elements, for example, the epitope can include carbohydrate or lipid side chains. The term "affinity" refers to a measure of the strength of binding of a particular epitope with the antigen-binding site of an antibody.

[0106] As used herein, "anti-ApoE antibody" refers to an isolated antibody that binds to recombinant ApoE4 or ApoE4 isolated from human brain with an affinity constant or affinity of interaction (KD) of about 0.1 pM to about 10 μM, preferably about 0.1 pM to about 1 μM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of an antibody for an antigen are known in the art and are further described in the Examples. Anti-ApoE antibodies useful in the present invention include those suitable for administration to a subject in a therapeutically effective amount.

[0107] The anti-ApoE antibodies disclosed herein can also be described or specified in terms of cross-reactivity. The term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen; a measure of relatedness between two different antigenic substances. That is, an antibody is cross-reactive if it binds to an epitope other than the one that led to its generation. A cross-reactive epitope usually contains many of the same complementary structural features as the epitope that led to the generation of the antibody, and in some cases may actually fit better than the original epitope. For example, an antibody has some degree of cross-reactivity when it binds a related but non-identical epitope (e.g., an epitope that is at least about 85%, at least about 90%, or at least about 95% identical (calculated by methods known in the art) to the reference epitope). An antibody can be said to have little or no cross-reactivity if it does not bind an epitope that is less than about 95%, less than about 90%, or less than about 85% identical to the reference epitope. An antibody can be considered "highly specific" for an epitope if it does not bind any other analogs, orthologs, or homologs of that epitope.

[0108] The epitope that the anti-ApoE antibody of the present disclosure binds to may be unique to ApoE4, may be common to ApoE4 and other ApoE isoforms (e.g., ApoE2 and / or ApoE3), or may be an ApoE4 epitope that is related but not identical to the epitope of another isoform. In some embodiments, the anti-ApoE antibody does not preferentially bind to ApoE2, ApoE3, or ApoE4. In some other embodiments, the anti-ApoE antibody preferentially binds to ApoE4 or preferentially binds to ApoE3 and ApoE4. An antibody that preferentially binds to an ApoE isoform binds to that isoform more readily than a different ApoE isoform. "Preferentially binds" means that the antibody specifically binds to an epitope of a first antigen more readily than to another epitope of the first antigen or another epitope of a second antigen. In one example, an antibody can be considered to preferentially bind a first epitope if it binds to a first epitope with a smaller off rate (k(off)) than to a second epitope. In another example, an antibody can be considered to preferentially bind a first epitope if it binds to a first antibody with a smaller dissociation constant (KD) than to a second epitope. In yet another example, an antibody binds to an ApoE isoform with at least about 10-, 50-, or 100-fold lower effective concentration (EC) for one ApoE isoform compared to other isoforms, as measured by ELISA or similar assay. 50 ), the antibody can be considered to preferentially bind to that ApoE isoform. Alternatively, the EC 50 If the difference is less than 10-fold, it can be considered that the antibody does not preferentially bind to those isoforms.

[0109] Another aspect of the isolated anti-ApoE antibody of the present disclosure is that it binds to human ApoE in amyloid plaques in unfixed brain tissue. "Unfixed brain tissue" refers to brain tissue that is not fixed with paraformaldehyde or other fixatives. The anti-ApoE antibody can be considered to "bind to human ApoE in amyloid plaques in unfixed brain tissue" when its staining pattern is consistent with binding to Aβ in brain parenchymal amyloid plaques or Aβ in brain perivascular deposits in the form of CAA.

[0110] A further aspect of the isolated anti-ApoE antibody of the present disclosure is that it preferentially binds to recombinant lipid-free human ApoE4 compared to human plasma-derived ApoE4 or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4. Briefly, this can be measured by (a) coating recombinant lipid-free human ApoE4 onto an ELISA plate, (b) incubating the anti-ApoE antibody in the presence of various dilutions of human plasma or plasma from a transgenic mouse expressing human ApoE4 (e.g., 20-fold to 100-fold dilutions of plasma containing about 1-5 μM ApoE) to prepare pre-incubated antibody-plasma mixtures for each dilution, (c) adding the pre-incubated antibody-plasma mixture to the ELISA plate of (a) and allowing it to equilibrate with the recombinant lipid-free ApoE4 coated on the plate, and (d) measuring the binding of the anti-ApoE antibody in the mixture to the recombinant lipid-free ApoE4 coated on the plate. Antibodies that preferentially bind to recombinant non-lipidated ApoE4 may show similar binding to the plate at plasma dilutions between about 1000-fold and about 20-fold (i.e., the binding signal does not decrease by more than 20%, preferably not more than 10%). A more detailed description is given in Example 13. In some embodiments, the isolated anti-ApoE antibody does not specifically bind to plasma-derived ApoE. Clearance (CL) is a pharmacokinetic parameter that indicates the efficiency of irreversible elimination of a drug from the systemic circulation, and is expressed as the volume of blood / plasma / serum that is cleared of the drug per unit time. An anti-ApoE antibody does not specifically bind to plasma-derived ApoE, for example, if it is administered to a human ApoE-expressing animal and its clearance value is less than 25 times that of an isotype control antibody (the dose of both antibodies administered to the animal is similar). See, for example, Figure 37F and Example 19.

[0111] A further aspect of the isolated anti-ApoE antibody of the present disclosure is that it may or may not have a variant Fc region. For example, the Fc region may be modified to increase or decrease its affinity for the Fc receptor of microglial cells and / or to have an altered glycosylation pattern.

[0112] Further aspects of the anti-ApoE antibodies of the disclosure are described in more detail below.

[0113] (a) Anti-ApoE antibodies that preferentially bind to ApoE3 and ApoE4: Group I In another aspect, the anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 94. In some embodiments, the heavy chain variable region further comprises SEQ ID NO: 90 and / or SEQ ID NO: 92. In some other embodiments, the heavy chain variable region further comprises SEQ ID NO: 91 and / or SEQ ID NO: 93. In some of the above embodiments, the antibody has a light chain variable region comprising SEQ ID NO: 88 or SEQ ID NO: 89. The light chain variable region may further comprise (a) SEQ ID NO: 86 or SEQ ID NO: 87; and / or (b) SEQ ID NO: 30.

[0114] In another aspect, the anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 95. In some embodiments, the heavy chain variable region further comprises SEQ ID NO: 90 and / or SEQ ID NO: 92. In some other embodiments, the heavy chain variable region further comprises SEQ ID NO: 91 and / or SEQ ID NO: 93. In some of the above embodiments, the antibody has a light chain variable region comprising SEQ ID NO: 88 or SEQ ID NO: 89. The light chain variable region may further comprise (a) SEQ ID NO: 86 or SEQ ID NO: 87; and / or (b) SEQ ID NO: 30.

[0115] In another aspect, the anti-ApoE antibody is selected from Table A. TIFF2025023998000001.tif37162 TIFF2025023998000002.tif219170 TIFF2025023998000003.tif219170 TIFF2025023998000004.tif219170 TIFF2025023998000005.tif23170

[0116] In certain exemplary embodiments, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO:3, or a VH having one or more HVRs from SEQ ID NO:4. The HVRs from SEQ ID NO:3 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO:29, L2 of SEQ ID NO:30, L3 of SEQ ID NO:31, or any combination thereof (e.g., antibodies 1-7 of Table B). The HVRs from SEQ ID NO:4 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO:32, H2 of SEQ ID NO:33, H3 of SEQ ID NO:34, or any combination thereof (e.g., antibodies 8-14 of Table B). The antibodies comprising one or more HVRs from SEQ ID NO:4 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO:3. The HVRs can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:29, L2 of SEQ ID NO:30, L3 of SEQ ID NO:31, or any combination thereof (e.g., antibodies 15-63 of Table B). In various embodiments above, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:3, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:4. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 3, 4, 29, 30, 31, 32, 33 and 34, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0117] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 17, or a VH having one or more HVRs from SEQ ID NO: 18. The HVRs from SEQ ID NO: 17 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 63, L2 of SEQ ID NO: 30, L3 of SEQ ID NO: 64, or any combination thereof (e.g., antibodies 64-70 of Table B). The HVRs from SEQ ID NO: 18 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 65, H2 of SEQ ID NO: 66, H3 of SEQ ID NO: 67, or any combination thereof (e.g., antibodies 71-77 of Table B). The antibodies comprising one or more HVRs from SEQ ID NO: 18 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 17. The HVR can be L1, L2, L3, or any combination thereof. In preferred embodiments, the VL can comprise L1 of SEQ ID NO:63, L2 of SEQ ID NO:30, L3 of SEQ ID NO:64, or any combination thereof (e.g., antibodies 78-126 of Table B). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:17, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:18. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 17, 18, 63, 30, 64, 65, 66 and 67, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0118] In some embodiments, each of the above exemplary antibodies may also have a variant Fc region, including but not limited to, a variant Fc region that has been modified to alter its native interaction with microglial FcR.

[0119] In some other embodiments, the isolated antibodies of Group I recognize an epitope listed in Tables 4-7, for example, as described for exemplary antibodies HJ152 or HJ1514.

[0120] TIFF2025023998000006.tif213163 TIFF2025023998000007.tif219170 TIFF2025023998000008.tif219170 TIFF2025023998000009.tif54170

[0121] (b) Group II: Anti-ApoE antibodies that preferentially bind to ApoE3 and ApoE4 In another aspect, the anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 84. In some embodiments, the heavy chain variable region further comprises SEQ ID NO: 80 and / or SEQ ID NO: 82. In some other embodiments, the heavy chain variable region further comprises SEQ ID NO: 81 and / or SEQ ID NO: 83. In some of the above embodiments, the antibody has a light chain variable region comprising SEQ ID NO: 25. The light chain variable region may further comprise (a) SEQ ID NO: 78 or SEQ ID NO: 79, and / or (b) SEQ ID NO: 24.

[0122] In another aspect, the anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 85. In some embodiments, the heavy chain variable region further comprises SEQ ID NO: 80 and / or SEQ ID NO: 82. In some other embodiments, the heavy chain variable region further comprises SEQ ID NO: 81 and / or SEQ ID NO: 83. In some of the above embodiments, the antibody has a light chain variable region comprising SEQ ID NO: 25. The light chain variable region may further comprise (a) SEQ ID NO: 78 or SEQ ID NO: 79, and / or (b) SEQ ID NO: 24.

[0123] In another aspect, the anti-ApoE antibody is selected from Table C. TIFF2025023998000010.tif41162 TIFF2025023998000011.tif219170 TIFF2025023998000012.tif219170 TIFF2025023998000013.tif219170 TIFF2025023998000014.tif17170

[0124] In certain exemplary embodiments, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO:9, or a VH having one or more HVRs from SEQ ID NO:10. The HVRs from SEQ ID NO:9 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO:47, L2 of SEQ ID NO:24, L3 of SEQ ID NO:25, or any combination thereof (e.g., antibodies 1-7 in Table D). The HVRs from SEQ ID NO:10 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO:48, H2 of SEQ ID NO:49, H3 of SEQ ID NO:50, or any combination thereof (e.g., antibodies 8-14 in Table D). The antibodies comprising one or more HVRs from SEQ ID NO:10 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO:9. The HVRs can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:47, L2 of SEQ ID NO:24, L3 of SEQ ID NO:25, or any combination thereof (e.g., antibodies 15-63 of Table D). In various embodiments above, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:9, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:10. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 85%, 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 9, 10, 47, 24, 25, 48, 49 and 50, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0125] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 11, or a VH having one or more HVRs from SEQ ID NO: 12. The HVRs from SEQ ID NO: 11 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 51, L2 of SEQ ID NO: 24, L3 of SEQ ID NO: 25, or any combination thereof (e.g., antibodies 64-70 in Table D). The HVRs from SEQ ID NO: 12 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 52, H2 of SEQ ID NO: 53, H3 of SEQ ID NO: 54, or any combination thereof (e.g., antibodies 71-77 in Table D). The antibodies comprising one or more HVRs from SEQ ID NO: 12 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 11. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:51, L2 of SEQ ID NO:24, L3 of SEQ ID NO:25, or any combination thereof (e.g., antibodies 78-126 of Table D). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:11, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:12. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 85%, 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 11, 12, 51, 24, 25, 52, 53 and 54, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0126] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 13, or a VH having one or more HVRs from SEQ ID NO: 14. The HVRs from SEQ ID NO: 13 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 55, L2 of SEQ ID NO: 24, L3 of SEQ ID NO: 25, or any combination thereof (e.g., antibodies 127-133 of Table D). The HVRs from SEQ ID NO: 14 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 56, H2 of SEQ ID NO: 57, H3 of SEQ ID NO: 58, or any combination thereof (e.g., antibodies 134-140 of Table D). The antibodies comprising one or more HVRs from SEQ ID NO: 14 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 13. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:55, L2 of SEQ ID NO:24, L3 of SEQ ID NO:25, or any combination thereof (e.g., antibodies 141-189 of Table D). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:13, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:14. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 13, 14, 55, 24, 25, 56, 57 and 58, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0127] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 15, or a VH having one or more HVRs from SEQ ID NO: 16. The HVRs from SEQ ID NO: 15 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 59, L2 of SEQ ID NO: 24, L3 of SEQ ID NO: 25, or any combination thereof (e.g., antibodies 190-196 in Table D). The HVRs from SEQ ID NO: 16 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 60, H2 of SEQ ID NO: 61, H3 of SEQ ID NO: 62, or any combination thereof (e.g., antibodies 197-203 in Table D). The antibodies comprising one or more HVRs from SEQ ID NO: 16 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 15. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:59, L2 of SEQ ID NO:24, L3 of SEQ ID NO:25, or any combination thereof (e.g., antibodies 204-252 of Table D). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:15, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:16. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 15, 16, 59, 24, 25, 60, 61 and 62, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0128] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 19, or a VH having one or more HVRs from SEQ ID NO: 20. The HVRs from SEQ ID NO: 19 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 68, L2 of SEQ ID NO: 24, L3 of SEQ ID NO: 25, or any combination thereof (e.g., antibodies 253-259 in Table D). The HVRs from SEQ ID NO: 20 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 69, H2 of SEQ ID NO: 70, H3 of SEQ ID NO: 71, or any combination thereof (e.g., antibodies 206-266 in Table D). The antibodies comprising one or more HVRs from SEQ ID NO: 20 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 19. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:68, L2 of SEQ ID NO:24, L3 of SEQ ID NO:25, or any combination thereof (e.g., antibodies 267-315 of Table D). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:19, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:20. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 19, 20, 68, 24, 25, 69, 70 and 71, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0129] In some embodiments, each of the above exemplary antibodies may also have a variant Fc region, including but not limited to, a variant Fc region that has been modified to alter its native interaction with microglial FcR.

[0130] In some other embodiments, the isolated antibodies of Group II recognize an epitope listed in Tables 4-7, for example, as described for exemplary antibodies HJ155, HJ156, HJ159, HJ1513, or HJ1518.

[0131] TIFF2025023998000015.tif134162 TIFF2025023998000016.tif219170 TIFF2025023998000017.tif219170 TIFF2025023998000018.tif219170 TIFF2025023998000019.tif219170 TIFF2025023998000020.tif219170 TIFF2025023998000021.tif219170 TIFF2025023998000022.tif219170 TIFF2025023998000023.tif77170

[0132] (c) Group III: Anti-ApoE antibodies that do not preferentially bind to ApoE4, ApoE3, or ApoE2 In another aspect, the anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 113. In some embodiments, the heavy chain variable region further comprises SEQ ID NO: 109 and / or SEQ ID NO: 111. In some other embodiments, the heavy chain variable region further comprises SEQ ID NO: 110 and / or SEQ ID NO: 112. In some of the above embodiments, the antibody has a light chain variable region comprising SEQ ID NO: 107. The light chain variable region may further comprise (a) SEQ ID NO: 105, and / or (b) SEQ ID NO: 106.

[0133] In another aspect, the anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 114. In some embodiments, the heavy chain variable region further comprises SEQ ID NO: 109 and / or SEQ ID NO: 111. In some other embodiments, the heavy chain variable region further comprises SEQ ID NO: 110 and / or SEQ ID NO: 112. In some of the above embodiments, the antibody has a light chain variable region comprising SEQ ID NO: 108. The light chain variable region may further comprise (a) SEQ ID NO: 105, and / or (b) SEQ ID NO: 106.

[0134] In another aspect, the anti-ApoE antibody is selected from Table E. TIFF2025023998000024.tif199166 TIFF2025023998000025.tif219170 TIFF2025023998000026.tif219170 TIFF2025023998000027.tif71170

[0135] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 115, or a VH having one or more HVRs from SEQ ID NO: 116. The HVRs from SEQ ID NO: 115 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, L3 of SEQ ID NO: 117, or any combination thereof (e.g., antibodies 1-7 of Table F). The HVRs from SEQ ID NO: 116 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 118, H2 of SEQ ID NO: 119, H3 of SEQ ID NO: 120, or any combination thereof (e.g., antibodies 8-14 of Table F). The antibodies comprising one or more HVRs from SEQ ID NO: 116 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 115. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 of SEQ ID NO: 117, or any combination thereof (e.g., antibodies 15-63 of Table F). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 115, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 116. In each of the above embodiments, the anti-ApoE antibodies can optionally include one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 85%, 90%, 95% or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 115, 116, 105, 106, 117, 118, 119 and 120, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0136] In another exemplary embodiment, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 121, or a VH having one or more HVRs from SEQ ID NO: 122. The HVRs from SEQ ID NO: 121 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, L3 of SEQ ID NO: 123, or any combination thereof (e.g., antibodies 64-70 of Table F). The HVRs from SEQ ID NO: 122 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 124, H2 of SEQ ID NO: 125, H3 of SEQ ID NO: 126, or any combination thereof (e.g., antibodies 71-77 of Table F). The antibodies comprising one or more HVRs from SEQ ID NO: 122 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 121. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 of SEQ ID NO: 123, or any combination thereof (e.g., antibodies 78-126 of Table F). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 121, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 122. In each of the above embodiments, the anti-ApoE antibodies can optionally include one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 85%, 90%, 95% or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 121, 122, 105, 106, 123, 124, 125 and 126, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0137] In some embodiments, each of the above exemplary antibodies may also have a variant Fc region, including but not limited to, a variant Fc region that has been modified to alter its native interaction with microglial FcR.

[0138] In some other embodiments, the isolated antibodies of Group III recognize an epitope listed in Tables 4-7.

[0139] TIFF2025023998000028.tif171161 TIFF2025023998000029.tif219170 TIFF2025023998000030.tif219170 TIFF2025023998000031.tif98170

[0140] (d) an anti-ApoE antibody that preferentially binds to ApoE4 over ApoE3 or ApoE2; In exemplary embodiments, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO:1, or a VH having one or more HVRs from SEQ ID NO:2. The HVRs from SEQ ID NO:1 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO:23, L2 of SEQ ID NO:24, L3 of SEQ ID NO:96, or any combination thereof (e.g., antibodies 1-7 of Table G). The HVRs from SEQ ID NO:2 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO:26, H2 of SEQ ID NO:27, H3 of SEQ ID NO:28, or any combination thereof (e.g., antibodies 8-14 of Table G). The antibodies comprising one or more HVRs from SEQ ID NO:2 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO:1. The HVRs can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:23, L2 of SEQ ID NO:24, L3 of SEQ ID NO:96, or any combination thereof (e.g., antibodies 15-63 of Table G). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:1, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:2. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 1, 2, 23, 24, 96, 26, 27 and 28, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0141] In some embodiments, each of the above exemplary antibodies may also have a variant Fc region, including but not limited to, a variant Fc region that has been modified to alter its native interaction with microglial FcR.

[0142] In some other embodiments, the isolated antibodies of Group IV recognize an epitope listed in Tables 4-7, for example, as described for exemplary antibody HJ151.

[0143] TIFF2025023998000032.tif106164 TIFF2025023998000033.tif219170 TIFF2025023998000034.tif44170

[0144] (e) a further anti-ApoE antibody that does not preferentially bind to ApoE2, ApoE3 or ApoE4. In exemplary embodiments, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO:21, or a VH having one or more HVRs from SEQ ID NO:22. The HVRs from SEQ ID NO:21 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO:72, L2 of SEQ ID NO:73, L3 of SEQ ID NO:74, or any combination thereof (e.g., antibodies 1-7 of Table H). The HVRs from SEQ ID NO:22 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO:75, H2 of SEQ ID NO:76, H3 of SEQ ID NO:77, or any combination thereof (e.g., antibodies 8-14 of Table H). The antibodies comprising one or more HVRs from SEQ ID NO:22 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO:21. The HVRs can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO:72, L2 of SEQ ID NO:73, L3 of SEQ ID NO:74, or any combination thereof (e.g., antibodies 15-63 of Table H). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:21, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:22. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 21, 22, 72, 73, 74, 75, 76 and 77, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0145] In exemplary embodiments, the anti-ApoE antibodies of this group comprise a VL having one or more HVRs from SEQ ID NO: 97, or a VH having one or more HVRs from SEQ ID NO: 98. The HVRs from SEQ ID NO: 97 can be L1, L2, L3, or any combination thereof. In some embodiments, the VL can comprise L1 of SEQ ID NO: 99, L2 of SEQ ID NO: 100, L3 of SEQ ID NO: 101, or any combination thereof (e.g., antibodies 64-70 of Table H). The HVRs from SEQ ID NO: 98 can be H1, H2, H3, or any combination thereof. In some embodiments, the VH can comprise H1 of SEQ ID NO: 102, H2 of SEQ ID NO: 103, H3 of SEQ ID NO: 104, or any combination thereof (e.g., antibodies 71-77 of Table H). An antibody comprising one or more HVRs from SEQ ID NO: 98 can further comprise a light chain variable region (VL) comprising one or more HVRs from SEQ ID NO: 97. The HVR can be L1, L2, L3, or any combination thereof. In certain preferred embodiments, the VL can comprise L1 of SEQ ID NO: 99, L2 of SEQ ID NO: 100, L3 of SEQ ID NO: 101, or any combination thereof (e.g., antibodies 78-126 of Table H). In various of the above embodiments, the antibody can be a humanized antibody, or the antibody can have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 97, and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 98. In each of the above embodiments, the anti-ApoE antibody can optionally comprise one or more constant regions, or portions of constant regions, that are substantially human (i.e., have at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also includes the corresponding nucleic acid sequences of SEQ ID NOs: 97, 98, 99, 100, 101, 102, 103 and 104, which can be readily determined by one of skill in the art and which can be incorporated into a vector or other larger DNA molecule, e.g., a chromosome, for expression of the antibodies of the present disclosure.

[0146] In some embodiments, each of the above exemplary antibodies may also have a variant Fc region, including but not limited to, a variant Fc region that has been modified to alter its native interaction with microglial FcR.

[0147] In some other embodiments, the isolated antibodies of Group V recognize an epitope listed in Tables 4-7, for example, as described for exemplary antibody HJ1526.

[0148] TIFF2025023998000035.tif189164 TIFF2025023998000036.tif219170 TIFF2025023998000037.tif219170 TIFF2025023998000038.tif82170

[0149] (f) Group V In another aspect, an anti-ApoE antibody competitively inhibits the binding of a reference antibody to its epitope. An antibody is said to competitively inhibit the binding of a reference antibody to a particular epitope if it preferentially binds to the epitope such that it inhibits the binding of the reference antibody to the epitope by at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. In some embodiments, an anti-ApoE antibody competitively inhibits the binding of HJ152 and / or HJ1514 to human ApoE. In some other embodiments, an anti-ApoE antibody competitively inhibits the binding of HJ155, HJ156, HJ159, HJ1513 and / or HJ1518 to human ApoE. In still other embodiments, an anti-ApoE antibody competitively inhibits the binding of HJ151 to human ApoE. In still other embodiments, the anti-ApoE antibody competitively inhibits the binding of HJ1526 to human ApoE. In another embodiment, the anti-ApoE antibody competitively inhibits the binding of HJ158 to human ApoE. In another embodiment, the anti-ApoE antibody competitively inhibits the binding of HJ1531 and / or HJ1536 to human ApoE.

[0150] III. Treatment Methods The present disclosure provides a method for treating Aβ amyloidosis, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ApoE antibody. The present disclosure also provides a method for treating a subject diagnosed with a disease characterized by Aβ plaques in the brain, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ApoE antibody. The present disclosure also provides a method for treating a subject diagnosed with a disease characterized by vascular Aβ plaques in the brain, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ApoE antibody. The present disclosure also provides a method for inhibiting the progression of a disease characterized by Aβ plaques in the brain, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ApoE antibody. The present disclosure also provides a method for treating a subject diagnosed with Alzheimer's disease, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ApoE antibody. The present disclosure also provides a method for treating a subject diagnosed with CAA, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ApoE antibody. Suitable anti-ApoE antibodies are described in Section II. In embodiments in which the subject is a human, the anti-ApoE antibody is compatible for administration to a living human subject (eg, is humanized).

[0151] In one embodiment, the present disclosure provides a method for preventing or slowing the progression of a disease characterized by Aβ plaques in the brain. The method comprises administering a therapeutically effective amount of an anti-ApoE antibody to a subject in need. Suitable anti-ApoE antibodies include those disclosed herein. The progression of a disease characterized by Aβ plaques in the brain includes the worsening of clinical signs of Aβ amyloidosis, symptoms associated with Aβ plaques, or symptoms associated with CAA, and can be assessed by methods known in the art and described herein. In an exemplary embodiment, the clinical sign is the deposition of amyloid plaques.

[0152] In another aspect, the present disclosure provides a method for improving clinical signs of Aβ amyloidosis. The method comprises administering a therapeutically effective amount of an anti-ApoE antibody to a subject in need. Suitable anti-ApoE antibodies include those disclosed herein. Non-limiting examples of improving clinical signs of Aβ amyloidosis can include a reduction in amyloid plaque deposition, stabilization of amyloid plaque deposition (i.e., no further increase), an increase in CSF Aβ42 concentration, an increase in CSF Aβ42 / Aβ40 ratio, a decrease in Aβ42 / Aβ40 peak time ratio (e.g., approaching 1) measured by stable isotope labeling kinetics, a decrease in Aβ42 / Aβ40FTR ratio (e.g., approaching 1) measured by stable isotope labeling kinetics, and a change in the ratio of relative Aβ42 label to relative Aβ40 label after stable isotope labeling (e.g., approaching 1). In each of the above embodiments, the improvement (i.e., change) of clinical signs is at least statistically significant. In some embodiments, the change can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% compared to untreated subjects or negative control treated subjects. In some embodiments, the change can be at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% compared to untreated subjects or negative control treated subjects. In some other embodiments, the change can be at least 100, 125, 150, 200, 250, 300, 350, 400 or 450% compared to untreated subjects or negative control treated subjects.

[0153] In another aspect, the present disclosure provides a method for reducing amyloid plaque load in the brain of a subject. The method comprises administering a therapeutically effective amount of an anti-ApoE antibody to a subject in need thereof. Suitable anti-ApoE antibodies include those disclosed herein. The method of the present invention can reduce amyloid plaque load in the hippocampus of a subject and / or reduce amyloid plaque load in the cerebral cortex of a subject. In each of the above aspects, amyloid plaque load can be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to untreated subjects or subjects treated with a negative control. In some embodiments, amyloid plaque load may be reduced by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to untreated subjects or subjects treated with a negative control, hi other embodiments, amyloid plaque load may be reduced by at least 100, 125, 150, 200, 250, 300, 350, 400, or 450% compared to untreated subjects or subjects treated with a negative control.

[0154] In another aspect, the present disclosure provides a method for reducing the amount of CAA deposition in the brain of a subject. The method comprises administering a therapeutically effective amount of an anti-ApoE antibody to a subject having Aβ in a fibrillar form in the perforating and / or leptomeningeal arteriole on the cerebral cortex surface. Suitable anti-ApoE antibodies include those disclosed herein. The method of the present invention can reduce the amount of CAA deposition in the perforating and / or leptomeningeal arteriole on the cerebral cortex surface of a subject. In each of the above aspects, the amount of CAA deposition can be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% compared to untreated subjects or negative control treated subjects. In some embodiments, amyloid plaque burden may be reduced by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to untreated subjects or subjects treated with a negative control, hi other embodiments, amyloid plaque burden may be reduced by at least 100, 125, 150, 200, 250, 300, 350, 400, or 450% compared to untreated subjects or subjects treated with a negative control.

[0155] Another aspect is a method for the treatment of insoluble Aβ in the brain of a subject in need thereof. 42 In one embodiment, the method comprises administering to a subject a therapeutically effective amount of an anti-ApoE antibody. ... 40 In another embodiment, the method further comprises selectively reducing insoluble Aβ40 levels, reducing insoluble Aβ42 levels, or a combination thereof, in the brain of the subject relative to soluble Aβ40, Aβ42 levels, or a combination thereof.

[0156] Aβ levels can be assessed by any suitable method known in the art, for example, by analyzing Aβ by one or more methods selected from the following: Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectrometry (MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SEMI-TOE), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS / MS), and laser densitometry. In vivo imaging of Aβ is particularly suitable for evaluating amyloid plaque deposition. Non-limiting examples of in vivo imaging methods include positron emission tomography (PET), single photon emission computed tomography (SPECT), near infrared (NIR) optical imaging, or magnetic resonance imaging (MRI). Suitable contrast agents are also known in the art (e.g., PIB).

[0157] In another aspect, the present disclosure provides a method for improving Aβ plaque-related symptoms and / or CAA-related symptoms in a subject. The method comprises administering to a subject having at least one Aβ plaque-related symptom and / or at least one CAA-related symptom an anti-ApoE antibody that specifically binds to ApoE in a therapeutically effective amount. Suitable anti-ApoE antibodies include those disclosed herein. Non-limiting examples of improvement of Aβ plaque-related symptoms are described above. In some embodiments, improvement of Aβ plaque-related symptoms can include neurodegeneration, cognitive impairment, behavioral changes, impaired emotional control, and / or reduced seizures. In each of the above embodiments, the improvement (i.e., change) of symptoms is at least statistically significant. In some embodiments, the change can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to untreated subjects or negative control-treated subjects. In some embodiments, the change can be at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% compared to untreated subjects or subjects treated with a negative control, while in some other embodiments, the change can be at least 100, 125, 150, 200, 250, 300, 350, 400 or 450% compared to untreated subjects or subjects treated with a negative control.

[0158] In each of the above embodiments, suitable anti-ApoE antibodies are described in Section II. In some embodiments, the anti-ApoE antibody is (a) selected from the antibodies listed in Tables A-H, (b) an anti-ApoE antibody that competitively inhibits binding of HJ152 and / or HJ1514 to human ApoE, or (c) an anti-ApoE antibody that competitively inhibits binding of HJ155, HJ156, HJ159, HJ1513 and / or HJ1518 to human ApoE. In some embodiments, the anti-ApoE antibody is selected from the antibodies listed in Tables A-B, or an anti-ApoE antibody that competitively inhibits binding of HJ152 and / or HJ1514 to human ApoE. In some embodiments, the anti-ApoE antibody is selected from the antibodies listed in Tables C-D, or is an anti-ApoE antibody that competitively inhibits the binding of HJ155, HJ156, HJ159, HJ1513 and / or HJ1518 to human ApoE. In some embodiments, the anti-ApoE antibody is selected from the antibodies listed in Tables E-F, or is an anti-ApoE antibody that competitively inhibits the binding of HJ1531 and / or HJ1536 to human ApoE. In some embodiments, the anti-ApoE antibody is selected from the antibodies listed in Table G, or is an anti-ApoE antibody that competitively inhibits the binding of HJ151 to human ApoE. In some embodiments, the anti-ApoE antibody is selected from the antibodies listed in Table H, or is an anti-ApoE antibody that competitively inhibits the binding of HJ158 or HJ1526 to human ApoE.

[0159] The anti-ApoE antibodies disclosed herein may also be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a biological response modifier, a pharmaceutical, or PEG. In some embodiments, the therapeutic agent may be a drug, a radioisotope, a lectin, or a toxin. Conjugates that are immunotoxins have been widely discussed in the art. Toxins can be coupled to antibodies by conventional coupling techniques, or immunotoxins with protein toxin moieties can be produced as fusion proteins. When anti-ApoE antibodies conjugated with radioisotopes are used in immunotherapy, certain isotopes can be selected depending on factors such as white blood cell distribution and stability and release properties. Depending on the autoimmune response, several emitters can be used. Radioisotopes that emit alpha and beta particles are commonly used in immunotherapy. In some embodiments, short-range, high-energy alpha emitters, e.g. 212 Examples of radioisotopes that can be attached to the anti-ApoE antibodies disclosed herein for treatment purposes include: 123 I, 124 I, 125 I, 131 I, 89 Zr, 90 Y, 67 Cu, 64 Cu, 111 In, 212 Bi, 212 At, 211 Pb, 47 Sc, 109 Pd and 188 Other therapeutic agents that can be coupled to the anti-ApoE antibodies, as well as ex vivo and in vivo treatment protocols, will be known to, or can be readily ascertained by, those of skill in the art.

[0160] The administration of anti-ApoE antibody or a composition comprising anti-ApoE antibody is carried out using standard effective methods, including peripheral (i.e., not by administration to the central nervous system) or local to the central nervous system. Peripheral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, including direct to the central nervous system (CNS), includes, but is not limited to, administration through lumbar, intraventricular, or intraparenchymal catheters, or administration using surgically implanted controlled release formulations.

[0161] Pharmaceutical compositions for effective administration are carefully designed to be compatible with the chosen mode of administration, and pharma- ceutically acceptable excipients, such as suitable dispersants, buffers, surfactants, preservatives, solubilizers, osmotic regulators, stabilizers, etc., are used as necessary. The latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, incorporated herein by reference in its entirety, provides an overview of formulation methods as generally known to practitioners. It may be particularly useful to alter the solubility of the antibodies useful in the present invention by rendering them more lipophilic, for example, by encapsulation in liposomes or blocking polar groups.

[0162] The concentration of the antibody in the formulation to be administered is an effective amount, ranging from a low concentration of about 0.1% by weight to a high concentration of about 15 or about 20% by weight, and may be selected primarily based on fluid volume, viscosity, etc., according to the particular method of administration selected as necessary. A typical composition for injection into a living subject may be prepared to contain 1 ml of sterile buffered water or phosphate buffered saline and about 1-1000 mg of any antibody of the present disclosure or combinations thereof. The formulation may be sterile filtered after preparation or otherwise rendered microbiologically acceptable. A typical composition for intravenous infusion may contain a volume of 1-250 ml of liquid, such as sterile Ringer's solution, and an anti-ApoE antibody at a concentration of 1-100 mg / ml or more. The anti-ApoE antibodies of the present disclosure may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. Lyophilization and reconstitution may result in varying degrees of antibody activity loss (e.g., conventional immunoglobulins tend to have greater activity loss in IgM antibodies than in IgG antibodies). The amount administered is an effective dose and may need to be adjusted to compensate. The pH of the formulation is generally pharmaceutical grade and may be selected to balance the stability (chemical and physical) of the antibody and the comfort of the subject upon administration. Generally, a pH of 4 to 8 is acceptable. The dose may vary from individual to individual based on the size, weight and other physio-biological characteristics of the individual to receive effective administration.

[0163] As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., an anti-ApoE antibody) that provides a measurable beneficial effect (i.e., a significant effect) in the subject to which it is administered. The therapeutically effective amount or dose of a compound administered according to the present invention can be determined using standard clinical techniques and can vary depending on the circumstances of the case, including the antibody administered, the route of administration, the condition being treated, and other considerations. A typical dose can include about 0.01 mg / kg to about 100 mg / kg of an anti-ApoE antibody described herein. Dosages can range from about 0.05 mg / kg to about 50 mg / kg, more preferably from about 0.1 mg / kg to about 25 mg / kg. Dosage frequency can be daily, or once, twice, three times, or more than once a week or month, as needed for effective treatment of the condition.

[0164] The timing of administration of treatment for a disease and the duration of treatment can be determined according to the circumstances of the case. The duration of treatment can range from a single administration on a one-time basis to lifelong treatment.

[0165] The above-mentioned method, with appropriate adjustment, is believed to be the most convenient, most suitable and effective for the administration of a protein such as a humanized antibody, but other effective administration methods, such as intracerebroventricular administration, transdermal administration and oral administration, may be performed in the present invention using a dosage form appropriate therefor. Furthermore, one skilled in the art may use the polynucleotide of the present invention encoding any of the above antibodies instead of using the protein material itself. For example,

[0166] Additionally, it may be advantageous to use controlled release formulations using biodegradable films and matrices, or osmotic mini-pumps, or delivery systems based on dextran beads, alginate or collagen.

[0167] IV. Diagnostic or follow-up methods The present disclosure also provides anti-ApoE antibodies conjugated to a detectable signal (i.e., a measurable substance or a substance that gives off a measurable signal). Non-limiting examples include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals when using positron emission tomography, and non-radioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use in diagnosis according to the present disclosure. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131 I, 111 In or 99 The signals emitted by such substances can be measured, for example, by single photon emission computed tomography (SPECT) or positron emission tomography (PET).

[0168] Anti-ApoE antibodies conjugated to a detectable signal may be used diagnostically, e.g., as part of a clinical testing procedure to monitor the onset or progression of a neurodegenerative disease, e.g., to determine the efficacy of certain therapeutic and / or prophylactic treatment methods. Based on the level of progression of the neurodegenerative disease, the treatment of the patient can be adjusted.

[0169] In some embodiments, a method is provided for assessing disease progression in a subject treated for a neurodegenerative disease characterized by cerebral amyloid plaques or vascular amyloid plaques in the brain, comprising: (a) administering an anti-ApoE antibody disclosed herein labeled with a substance that produces a measurable signal as described herein (i.e., a "labeled anti-ApoE antibody"), wherein the signal is measured in the patient after administration; (b) administering labeled anti-ApoE or an antigen-binding fragment thereof at one or more time intervals after administration of (a), wherein the signal is measured in the patient after administration; (c) assessing disease progression in the patient based on the change in the signal measured in the patient at the one or more time intervals after administration of (a), wherein an increase in the signal indicates the progression of the neurodegenerative disease in the patient. In some embodiments, the subject is treated with an unlabeled form of the same anti-ApoE antibody. In some embodiments, the subject is treated with an anti-ApoE antibody that competitively inhibits the binding of the labeled anti-ApoE antibody to human ApoE. In some embodiments, the subject is treated with an anti-ApoE antibody that does not competitively inhibit the binding of the labeled anti-ApoE antibody to human ApoE. In some embodiments, the subject is treated with other agents known in the art.

[0170] In some other embodiments, a method is provided for assessing disease progression in a subject treated for a neurodegenerative disease characterized by cerebral amyloid plaques or vascular amyloid plaques in the brain, comprising: (a) administering an anti-ApoE antibody disclosed herein labeled with a substance that produces a measurable signal as described herein (i.e., a "labeled anti-ApoE antibody"), where the signal is measured in the patient after administration; (b) assessing the disease state of the subject based on a comparative study of the signal measured in the subject and the signal measured after administration of the labeled antibody or antigen-binding fragment thereof to one or more control subjects, where a higher signal generated in the patient compared to the control subject is associated with an increase in cerebral amyloid plaques; (c) treating the patient with a treatment method appropriate for the patient's disease state. A "control subject" refers to any healthy subject (or subjects), a subject or subjects with a different degree of disease, or the subject itself at a time when the disease state was earlier. In some embodiments, the treatment method is an unlabeled form of the same anti-ApoE antibody. In some embodiments, the treatment is an anti-ApoE antibody that competitively inhibits the binding of the labeled anti-ApoE antibody to human ApoE. In some embodiments, the treatment is an anti-ApoE antibody that does not competitively inhibit the binding of the labeled anti-ApoE antibody to human ApoE. In some embodiments, the treatment is with an anti-Aβ antibody, an anti-tau antibody, a γ-secretase inhibitor, a β-secretase inhibitor, a cholinesterase inhibitor, an NMDA receptor antagonist, or other agents known in the art.

[0171] The present disclosure also provides uses of the anti-ApoE antibodies disclosed herein for measuring the amount of brain amyloid plaques in a subject, for assessing disease progression in a patient being treated for a neurodegenerative disease, or for treating a neurodegenerative disease characterized by brain amyloid plaques in a patient in need of treatment.

[0172] V. Pharmaceutical Compositions The present disclosure encompasses pharmaceutical compositions comprising the anti-ApoE antibodies disclosed in Section II in a manner that facilitates administration and enhances stability. For example, the anti-ApoE antibodies of the present disclosure may be combined with at least one pharma- ceutically acceptable carrier or excipient to form a pharmaceutical composition that is suitably and effectively administered to a living subject, such as a suitable subject (i.e., a "subject in need of treatment" or a "subject in need thereof"). Methods for preparing and administering the anti-ApoE antibodies of the present disclosure to a subject in need thereof are well known or easily determined by those skilled in the art. The route of administration of the anti-ApoE antibodies may be, for example, peripheral, oral, parenteral, inhaled, or topical.

[0173] Pharmaceutical compositions for effective administration will be carefully designed to suit the selected method of administration and will appropriately use pharma- ceutically acceptable excipients, such as compatible carriers, dispersing agents, buffers, surfactants, preservatives, solubilizers, osmotic pressure regulators, stabilizers, and the like.

[0174] Non-limiting examples of pharma- ceutically acceptable carriers include saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, wool fat, or combinations thereof.

[0175] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents can be included in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride.

[0176] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0177] Compositions of the present disclosure can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier at the time of use.

[0178] In some embodiments, anti-ApoE antibodies may be formulated for parenteral administration. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, such as saline and buffered media. Excipients for parenteral administration include sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Excipients for intravenous administration include fluid and nutrient replenishers, electrolyte replenishers (e.g., based on dextrose Ringer's solution), and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be included. Formulations for parenteral administration may be a single bolus dose, infusion, or a loading bolus dose followed by a maintenance dose. Such compositions may be administered at specific fixed or variable intervals, for example, once daily, or on an "as needed" basis.

[0179] Some of the pharmaceutical compositions disclosed herein can be administered orally in acceptable dosage forms, such as capsules, tablets, aqueous suspensions or solutions. Some pharmaceutical compositions can be administered by nasal aerosol or inhalation. Such compositions can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0180] The amount of anti-ApoE antibody combined with the carrier material to produce a single dosage form can vary based on the subject of treatment and the particular method of administration. The composition can be administered as a single dose or multiple doses, or as an infusion over a set period of time. The dosage regimen can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0181] The following examples are provided to illustrate preferred embodiments of the present invention.Those skilled in the art should recognize that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention.However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention and still achieve similar results, and therefore, all matters described or shown in the examples and drawings should be interpreted as illustrative and not limiting.

[0182] Working Example The following examples illustrate various aspects of the present invention. EXAMPLES

[0183] A mouse monoclonal antibody against ApoE was produced and sequenced. To produce the antibody, human recombinant ApoE protein (299 amino acids) was injected intraperitoneally (IP) into wild-type mice on a B6C3 background. 100 μg of antigen (in 200 μl PBS+200 μl complete Freund's adjuvant) was injected on days 0, 14, and 28. A final IP injection of 50 μg of antigen in PBS was given 3 days before fusing mouse splenocytes with myeloma cells. Serum was tested by direct ELISA against ApoE4 on days 21 and 35. When the titer was greater than 1:10000, mouse splenocytes were fused with myeloma cells according to standard protocols, and hybridoma clones were then isolated.

[0184] [Table 1] EXAMPLES

[0185] Brain lysates from apoE KO mice or mice expressing human apoE3, human apoE4 or mouse apoE were immunoblotted with HJ151, HJ152, HJ153 and HJ154. As a positive control, a commercially available apoE antibody, GA-50, was used. Figures 1 and 2 show that in Western blots, HJ152, HJ153 and HJ154 recognize both ApoE3 and ApoE4, while HJ151 is specific for ApoE4. EXAMPLES

[0186] Brain tissue from 5XFAD APP transgenic mice expressing various human ApoE isoforms was stained with biotinylated HJ153 and HJ154. Brain tissue from ApoE KO mice was used as a negative control. As shown in Figure 3, HJ153 and HJ154 stain ApoE in neuropil, astrocytes, and amyloid plaques (when amyloid plaques are present). EXAMPLES

[0187] Phosphate-buffered saline lysates of cerebral cortex tissues containing ApoE2, ApoE3 or ApoE4 were subjected to immunoprecipitation with HJ152, HJ153 and HJ154. The antibody-immunoprecipitated material (denoted as IP) and the post-immunoprecipitated solution were loaded onto SDS-PAGE gels, transferred to nitrocellulose membranes and immunoblotted with the commercial apoE antibody GA-50. WUE4, a monoclonal antibody against human apoE, was used as a positive control. As shown in Figure 4, HJ152 can immunoprecipitate some of the apoE from the samples, but some apoE remains in the post-immunoprecipitated samples (IP vs. post-IP). Figure 4 also shows that HJ153 and HJ154 can immunoprecipitate all of the ApoE from the samples. EXAMPLES

[0188] ELISA plates were coated with 0.5 μg / ml of recombinant apoE2, apoE3 or apoE4. Various concentrations of HJ151 (Figure 5A), HJ153 (Figure 5B), HJ154 (Figure 5C) or HJ156 (Figure 5D) were then introduced to the plates. A goat anti-mouse secondary antibody labeled with horseradish peroxidase was used to detect binding. Figures 5A-D show that HJ153 and HJ154 bind to ApoE2, ApoE3 and ApoE4 as detected by ELISA. HJ151 is specific for ApoE4, and HJ156 binds only to ApoE3 and ApoE4. Surface plasmon resonance profiles were also obtained for the various antibodies. For detection of binding with biotinylated recombinant apoE4 captured on a streptavidin chip, anti-ApoE antibodies were serially diluted 3-fold (starting at 100 nM for HJ153 (Figure 5F), 1000 nM for HJ151 (Figure 5E) and HJ156 (Figure 5G)). Samples were injected at a flow rate of 30 μl / min. Figure 5H shows the apparent KD values ​​of HJ151, HJ153 and HJ156 calculated based on SPR studies. EXAMPLES

[0189] APP / PS1-21 E4 / E4 mice received continuous intracerebroventricular (ICV) infusions of PBS (negative control), mouse IgG2 (negative control), HJ5.1 (anti-Aβ antibody, positive control), or anti-apoE antibodies (HJ151, HJ154, or HJ156) starting at 2 months of age (n=10-12 / group). Anti-ApoE or control antibodies (2mg / ml) were infused into a subcutaneous osmotic minipump (Alzet, model 2006) through a surgically implanted catheter into the left lateral ventricle (bregma -0.4mm, 1.0mm lateral to the midline, 2.5mm below the skull) at a rate of 1.2μl / min for 6 weeks.

[0190] Mice were perfused at 3.5 months of age, and brain sections were stained with anti-Aβ antibody HJ3.4B for Aβ plaques and Thioflavin S for fibrous plaques. The percentage of area covered by plaques in the cerebral cortex dorsal to the hippocampus was quantified. Data were analyzed using one-way ANOVA followed by Turkey's post-hoc test. As shown in Figures 6 and 7, ICV injection of several anti-ApoE antibodies resulted in a statistically significant reduction in the percentage of area covered by Aβ plaques and fibrous plaques. The amount of soluble and insoluble Aβ in the cerebral cortex dorsal to the hippocampus was also quantified by ELISA. Briefly, cortical tissue was homogenized sequentially in PBS, Triton, and 5M guanidine, and Aβ levels were measured in each fraction. A statistically significant reduction in insoluble Aβ42 (Triton and guanidine fractions) by HJ151 and HJ156 was observed (Figure 8). EXAMPLES

[0191] APP / PS1-21 E4 / E4 mice were injected intraperitoneally with PBS (negative control), mouse IgG (negative control), or anti-apoE antibodies (HJ151, HJ154, or HJ156) starting at 2 months (n=10 / group). Antibodies were injected once a week at 50 mg / kg. Each mouse was weighed at the time of injection. As shown in Figure 9, there was no difference in weight between treatment groups. Mice were perfused 3 days after the 7th injection. The left hemisphere of the brain was fixed in 4% PFA at 4°C for 48 hours and then transferred to PBS and stored at 4°C. The right hemisphere of the brain was dissected and frozen for biochemical analysis.

[0192] The amount of soluble and insoluble Aβ in the dorsal hippocampal cerebral cortex was measured by ELISA. That is, the cerebral cortex tissue was successively homogenized in PBS and 5 M guanidine, and the Aβ levels in the soluble (PBS) and insoluble (guanidine) fractions were measured. As shown in Figures 10-13, HJ156 significantly reduced insoluble Aβ40 and Aβ42 (p<0.0001), but had no statistically significant effect on soluble Aβ. The levels of insoluble Aβ40 and 42 were also reduced by HJ155 and HJ151, but the reduction was not significant. EXAMPLES

[0193] APP / PS1-21 E4 / E4 mice were injected with AAV2 / 8 vectors producing anti-apoE antibodies (HJ151, HJ151Δ or HJ156) or controls (HJ16.5 and PBS) (n=24 / group). The AAV serotype was AAV2 / 8 and the promoter was chicken β-actin (CBA) promoter. Injections were performed as bilateral intracerebroventricular injections in P0 mice. The HJ151Δ antibody is an HJ151 antibody with a mutation in the Fc domain that prevents binding to Fcγ receptors. The antibody HJ16.5 binds to the human protein PLD3 and not to mouse PLD3 or other mouse proteins. Mice were perfused at 3.5 months of age. The left hemisphere of the brain was fixed in 4% PFA at 4°C for 48 hours and then transferred to 30% sucrose and stored at 4°C. The right hemisphere of the brain was dissected and frozen for biochemical analysis.

[0194] The amount of soluble and insoluble Aβ in the dorsal hippocampal cerebral cortex was measured by ELISA. Briefly, cerebral cortex tissue was homogenized sequentially in PBS and 5 M guanidine, and Aβ levels in the soluble (PBS) and insoluble (guanidine) fractions were measured. As shown in Figures 15 and 16, antibodies HJ151 and HJ156 significantly reduced insoluble Aβ40 and Aβ42, whereas HJ151Δ and HJ156Δ had no effect. None of the antibodies had a significant effect on soluble Aβ40 or Aβ42 (not shown). As shown in Figure 14, the relative antibody concentrations in the cerebral cortex of APPPS1-21 / APOE4 mice expressing recombinant (r)HJ151, rHJ151 with D265A mutation (Δ), rHJ156, and rHJ156Δ were measured by antigen capture ELISA. The relative levels of each antibody were calculated using purified antibodies from that hybridoma as a standard. Similar levels of expression were found, with no statistical differences between the different antibodies.

[0195] Overall, AAV-mediated expression of HJ151 and HJ156 significantly reduced insoluble Aβ40 and Aβ42 in the APP / PS1-21 E4 / E4 mouse model. Mutation of the Fc effector (D265A) completely abolished the ability of HJ151 to reduce insoluble Aβ. These results indicate that clearance of Aβ aggregates by anti-apoE antibodies may require antibody opsonization and phagocytosis steps accompanied by microglia-mediated Aβ clearance. EXAMPLES

[0196] Human apoE from APP / PS1-21 E4 / E4 (APPPS1-21 / E4), 5XFAD / apoE knockout (5XFAD / EKO) mice, and patients who died of amyloid deposition 4 / 4Unfixed tissue sections (20 μm thick) from (human E4) brain were stained with biotinylated antibodies HJ3.4B (anti-Aβ), HJ151B, HJ152B, HJ153B, HJ154B, HJ155B and HJ156B (anti-apoE antibodies) (where "B" stands for "biotinylated"). All mice used were old enough to have significant amounts of Aβ in the cerebral cortex (APPPS1-21 / apoE). 4 / 4 , >6 months of age; 5XFAD / apoE knockout (5XFAD / EKO), >6 months of age). Images of mouse and human sections are all from the cerebral cortex. Results shown in FIG. 18 demonstrate that HJ3.4B, HJ151B, HJ152B, HJ153B, HJ154B, HJ155B and HJ156B upregulate APP / PS1-21 E4 / E4 and human apoE 4 / 4 Figure 1 shows staining of plaques and cerebral amyloid angiopathy in 5XFAD / EKO brains. No staining with anti-apoE specific antibodies is observed in 5XFAD / EKO brains. EXAMPLES

[0197] The affinity of several anti-ApoE antibodies to ApoE4 was determined by surface plasmon resonance using a BIAcore T200. Anti-biotin monoclonal mouse antibody was immobilized on a Biacore Series S CM5 sensor chip. Biotinylated recombinant non-lipidated ApoE4 was then captured on the chip. Serial dilutions of each antibody were injected at a flow rate of 30 μl / min. Each sample was analyzed, for example, for 3 minutes of binding and 10 minutes of dissociation. After each injection, the chip was regenerated with 3M MgCl2 or other appropriate buffer. Binding responses were corrected by subtracting the response units (RU) from a flow cell capturing irrelevant IgG at a similar density. For kinetic analysis, k on and k off A 1:1 Languir model of simultaneous fitting of the above was used. The data are shown in Table 2. Recombinant non-lipidated apoE4 was expressed in soluble form in E. coli and then purified or purchased from a commercial supplier.

[0198] [Table 2] EXAMPLES

[0199] ELISA dose-response curves were performed for several anti-ApoE4 antibodies: plates were coated overnight with full-length recombinant non-lipidated ApoE2, ApoE3 or ApoE4 (0.5 μg / ml). The next day, plates were washed, blocked and then incubated with anti-ApoE antibodies. For secondary detection, goat anti-mouse IgG-HRP antibody was used. Data are shown in Table 3. Recombinant non-lipidated ApoE was expressed in soluble form in E. coli and then purified or purchased from a supplier.

[0200] [Table 3] EXAMPLES

[0201] Anti-ApoE antibodies were tested for the detection of ApoE from human plasma lipoproteins by coating plasma ELISA (Figure 19A). Human plasma (confirmed to have one or more e4 alleles by Western blot assay with HJ151 and containing 1-5 μM ApoE4) was diluted 100-fold in PBS and coated onto half-well ELISA microtiter plates overnight at 4°C. Plates were washed extensively with PBS and blocked with 5% BSA / PBS for 1 h. Antibodies were added to the wells at starting concentrations of 400-500 nM and serial 5-fold dilutions therefrom for 1 h. Antibody binding was detected with an anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP). The detection substrate was TMB and the stop solution was 4N sulfuric acid.

[0202] Compared with HJ153 and HJ154, HJ156 showed low binding to coated human plasma ApoE (Figure 19B). Binding of control IgG and commercial anti-ApoE antibody WUE4 was very low. These data indicate that HJ153 and HJ154 robustly detect lipidated ApoE contained in human plasma-derived lipoprotein particles. Figures 19C and D show the binding profiles of other antibodies to coated human plasma. HJ159, HJ1514, HJ1518, HJ1526 (Figure 19C), HJ158, HJ1531, and HJ1536 (Figure 19D) did not robustly detect lipidated ApoE in plasma at the highest antibody concentration, and only faint detection was observed at the previous dilution step. EXAMPLES

[0203] Antibodies that did not detect immobilized plasma-derived human ApoE were further tested by competitive ELISA to exclude the possibility that epitopes on coated plasma ApoE were not available for binding due to interactions with the plastic wells (Figure 20A). Recombinant non-lipidated ApoE at 0.5 μg / ml in PBS was coated on half-well ELISA microtiter plates overnight at 4°C. Plates were washed extensively with PBS and blocked with 5% BSA / PBS for 1 h. HJ153 and antibody clones that did not detect coated human plasma ApoE were preincubated with 4 nM or 50 nM antibody in a dilution series (starting with a 5-fold dilution and then 2-fold dilutions) of human plasma (containing approximately 1-5 μM ApoE) / 5% BSA / PBS. After 1 h preincubation, the mixtures were added to the blocked plates and incubated for 1 h. Detection of antibodies available for binding to coated non-lipidated recombinant ApoE4 (not competed by ApoE in diluted plasma) was performed using anti-mouse IgG-HRP and TMB. The reaction was stopped with 4N sulfuric acid.

[0204] This competitive ELISA showed titration of 4 nM HJ153 in binding to coated ApoE with increasing plasma dilution (FIG. 20B). Antibodies that did not bind plasma in the previous assay did not show titration of binding to coated recombinant non-lipidated ApoE at either 4 nM or 50 nM antibody concentrations. These results indicate that antibodies that do not detect lipidated ApoE in plasma may preferentially bind recombinant non-lipidated ApoE. EXAMPLES

[0205] Unfixed tissue sections from APP / PS1-21 E2 / E2, APP / PS1-21 E3 / E3, and APP / PS1-21 E4 / E4 mice were stained for ApoE with biotinylated antibody HJ156 ("HJ156B"). Mice were 9 months old at the time of staining. Sections were 20 microns thick. Prior to brain removal, all mice were perfused transcardially with PBS at 4°C. Results are shown in Figures 21-23. All images are from the cerebral cortex. EXAMPLES

[0206] Unfixed tissue sections from APP / PS1-21 E4 / E4 mice were stained for ApoE with anti-ApoEms IgG / huFc chimeric antibody H153, anti-ApoEms IgG / huFc chimeric antibody H156, anti-ApoEms IgG / huFc chimeric antibody H1514, or anti-ApoEms IgG / huFc chimeric antibody H1518. Biotinylated rabbit anti-human IgG antibody was used as the secondary antibody. Mice were 9 months old at the time of staining. Section thickness was 20 microns. All mice were perfused transcardially with PBS at 4°C prior to brain removal. Results are shown in Figures 24-28. All images are from the cerebral cortex. EXAMPLES

[0207] APP / PS1-21 E4 / E4 mice were treated with intraperitoneal injections of PBS (negative control), HJ6.6 (IgG negative control), or anti-apoE antibodies (HJ151, HJ155, and HJ156) beginning at 2 months of age (n=10 / group). Antibodies were injected at 50 mg / kg once a week until 3.5 months of age. Prior to brain removal, mice were perfused transcardially with PBS at 4°C. Brains were fixed in 4% paraformaldehyde. 50-micron-thick sections were cut and stained with the indicated reagents.

[0208] Brain sections from 2-month-old untreated and 3.5-month-old treated mice were immunostained with anti-Aβ antibody HJ3.4-biotin or stained with X-34 (a dye that stains fibrous plaques) to quantitate staining in the cerebral cortex. Treatment of APP / PS1-21 E4 / E4 mice with HJ156 resulted in a statistically significant reduction in Aβ plaque deposition, measured as % Aβ deposit area (Figure 29) or number of plaques (Figure 30). Intraperitoneal administration of HJ155 also resulted in a statistically significant reduction in Aβ plaque number (Figure 30); a reduction in Aβ deposition (measured as % Aβ stained area) was also seen, but not significantly (Figure 29). A similar reduction in the amount and number of fibrillar Aβ plaques was seen by X-34 staining (Figures 31 and 32). Representative images of brain sections are shown in Figures 33-36. EXAMPLES

[0209] To determine whether the ability to recognize the different conformational forms of ApoE in Aβ plaques would lead to more potent plaque-reducing antibodies, HJ151, HJ153 and HJ156 were further characterized.

[0210] HJ153 or HJ156 was coated onto plates, and plasma from ApoE KO (EKO), ApoE2, ApoE3 and ApoE4 mice was added (Figure 37C and Figure 37D). The results showed that HJ153 bound to lipidated ApoE2, ApoE3 and ApoE4 (Figure 37C), but HJ156 did not bind (Figure 37D). Next, non-lipidated ApoE4 was coated onto plates, and HJ151, HJ153 and HJ156 were pre-incubated with a dilution series of plasma from APOE4 mice. The results showed that lipidated ApoE could compete with non-lipidated ApoE for HJ153, indicating that HJ153 bound to both lipidated and non-lipidated forms of ApoE (Figure 37E). On the other hand, lipidated ApoE was unable to compete for binding between nonlipidated apoE and HJ151 or HJ156, indicating that HJ151 and HJ156 preferentially bind to the nonlipidated form of ApoE (FIG. 37E).

[0211] Chimeric versions of HJ153 and HJ156 were also characterized. Plasma from EKO, APOE2, APOE3 and APOE4 KI mice was coated onto ELISA plates (with plasma lipoproteins present). Chimeric versions of HJ153 (chi153) and HJ156 (chi156) were prepared, containing the binding domain of mouse IgG antibody and a human IgG backbone. Chimeric antibodies were added to the plates at various concentrations. Plate-bound chi153 and chi156 were detected with anti-human IgG (Figure 37A, Figure 37B). The results showed that chi153 binds lipidated ApoE2, ApoE3 and ApoE4 (Figure 37A), but chi156 does not (Figure 37B).

[0212] We also compared the pharmacokinetics of HJ153 and HJ156 IP-injected (10 mg / kg body weight) into APOE4 and APOE KO mice (Figure 37F). At 48 hours after injection, HJ153 was below the detection limit in plasma, while HJ156 was present in plasma 14 days after injection (Figure 37F). Our ELISA data shows that HJ156 preferentially binds to non-lipidated ApoE, while HJ153 binds to both lipidated and non-lipidated ApoE. This is consistent with a possible explanation for the pharmacokinetics. HJ153 binds to lipidated ApoE that is abundant in plasma, and therefore the clearance rate of HJ153 is greater. Meanwhile, the fact that HJ156 only binds to non-lipidated ApoE that is less abundant or absent in plasma leads to a lower in vivo clearance of HJ156.

[0213] To examine whether HJ151 and HJ156 bind to any form of ApoE in the brain, unfixed frozen brain sections from APPPS1 / APOE4 or APPPS1 / EKO mice using HJ151 and HJ156 (Figure 38A). In sections from the same brains, Aβ immunostaining with anti-Aβ antibody HJ3.4 confirmed the presence of Aβ plaques. The results showed that HJ151 and HJ156 could bind to ApoE in plaques (Figure 38A). Next, we examined the ability of HJ156 to bind to aggregated forms of ApoE induced by incubation at 40°C. The binding of untreated ApoE with HJ151, HJ153 or HJ156 was compared to the binding of ApoE incubated at 40°C with HJ151, HJ153 or HJ156. HJ153 bound similarly to untreated ApoE or ApoE incubated at 40°C, whereas HJ151 and HJ156 bound preferentially to ApoE incubated at 40°C (Figure 38B). Furthermore, the formation of aggregates by incubation at 40°C was confirmed in the pellet fraction after ultracentrifugation at 186000×g for 1 hour (Figure 38C). When ApoE from the supernatant or the pellet was coated on an ELISA plate and detected with HJ156, APOE from the supernatant showed low binding, whereas ApoE from the pellet showed high binding (Figure 38D). ApoE from the pellet and total incubated ApoE bound equally to HJ156 (Figure 38D), indicating that the binding of HJ156 to ApoE species after exposure to 40°C treatment is to the ApoE species in the pellet. Solubilization of ApoE aggregates reduced the binding of HJ156. Specifically, exposure of ApoE aggregates to 1% SDS or 4M guanidine reduced the binding of HJ156 to ApoE aggregates induced by incubation at 40°C (Figure 38E). Thus, the data indicated that HJ156 preferentially binds to aggregated forms of ApoE compared to monomeric ApoE. It also bound to ApoE in plaques in APPPS1 mouse brain sections.

[0214] To examine whether HJ151 and HJ156 bind to ApoE in the brains of living mice, we performed experiments using in vivo two-photon microscopy. Control mouse IgG antibodies, HJ151 and HJ156, were conjugated to Alexa594 and applied to the brain surface of 6-month-old APPPS1 / APOE4 mice (40 μl at 1 mg / ml). The binding of the antibodies to ApoE was observed through a cranial window using two-photon microscopy (Figure 39A-C). The results showed that HJ151 and HJ156 bind to ApoE in amyloid plaques and CAA. To examine whether peripherally administered ApoE antibodies can enter the brain and bind to ApoE, chi156 or control human IgG was IP injected into APPPS1-21 / APOE4 mice at one or two doses (50 mg / kg body weight). Two days after the last injection, chi156 was detected bound to plaques in mice that had received one or two antibody injections (Figure 39D). Thus, the data demonstrated that HJ156 can enter the brain and bind to ApoE in Aβ plaques in live animals.

[0215] To examine whether HJ151 and HJ156 antibodies can increase activated microglia, the amount of CD45+ microglia after short-term treatment with HJ151 and HJ156 antibodies (IP injection of 4 doses every 3 days) was measured relative to the amount of fibrous plaques in APPPS1-21 / APOE4 mice that already had plaques at 4 months of age (Figure 40A, Figure 41). After short-term passive immunization, HJ151 had no effect on the amount of activated microglia, whereas HJ156 significantly increased CD45+ microglia compared to the control (Figure 40). Since both antibodies can bind to ApoE in plaques, the microglia activation effect after short-term peripheral injection may be related to the potency and dose of the antibody.

[0216] To determine the dose-range efficacy profile of HJ156, HJ156 was IP injected at one of three dose levels (2, 10 and 50 mg / kg) once a week for 2.5 months into APPPS1-21 / APOE4 mice (including both sexes, n=17-18 / group) starting at 2 months of age. Control groups received IgG2ab (50 mg / kg) or anti-Aβ HJ3.4 (50 mg / kg) once a week. Mice were deeply anesthetized and perfused 3 days after the 12th injection. The right hemisphere of the brain was dissected and the amount of soluble and insoluble Aβ in the cerebral cortex dorsal to the hippocampus was measured by ELISA. Cerebral cortical tissue was homogenized sequentially in PBS and 5 M guanidine and Aβ levels in the soluble (PBS) and insoluble (guanidine) fractions were measured. Insoluble Aβ in the brain 40 and Aβ 42 A significant dose-dependent effect of HJ156 on reducing levels was demonstrated (FIGS. 42A-B).

[0217] The data above collectively indicate that a key feature of the disclosed antibodies for Aβ plaque reduction is their ability to selectively bind to the conformationally altered form of ApoE found in amyloid plaques. Given that non-lipidated ApoE represents only a small proportion of total ApoE in vivo, targeting this pool of ApoE may explain the lack of observed effects on ApoE levels or lipid metabolism. Furthermore, preferential binding of such antibodies to non-lipidated ApoE may contribute to their increased plasma half-life compared to anti-ApoE antibodies that bind lipidated ApoE (Figure 37F). This increased half-life may significantly increase the concentration of antibodies that can enter the brain and bind to their target. EXAMPLES

[0218] The epitope binding of various anti-ApoE antibodies was examined in multiple ways. Various sequences of human ApoE were expressed on the yeast surface by yeast display using different plasmids. Various anti-ApoE antibodies were then examined for binding to various ApoE sequences as determined by immunofluorescence of the yeast cell surface (Table 4). Since none of the anti-ApoE antibodies tested here recognize mouse ApoE, the epitopes were further narrowed down by comparison of mouse and human ApoE sequences, as shown in Tables 4 and 5. The data in Tables 6 and 7 were obtained as follows: Yeast cells were transformed with DNA constructs containing various myc-tagged ApoE4 regions. Yeast cells were incubated with HJ15x to detect binding or with anti-myc antibody to detect expression. HJ153 was found not to bind to human ApoE4 expressed in yeast, so it was not evaluated in this study.

[0219] [Table 4]

[0220] [Table 5]

[0221] [Table 6]

[0222] [Table 7] EXAMPLES

[0223] Clearance (CL) is a pharmacokinetic parameter that indicates the efficiency of irreversible elimination of a drug from the systemic circulation and is expressed as the volume of blood / plasma / serum cleared of drug per unit time. Anti-ApoE antibodies that bind lipidated ApoE in plasma will show high clearance due to the very high concentration of lipidated ApoE present in the circulation. On the other hand, antibodies that specifically bind non-lipidated ApoE are expected to show low clearance and other pharmacokinetic properties similar to those of isotype control antibodies that do not bind the antigen. For such antibodies, clearance will not be dose-dependent.

[0224] Prior to the present disclosure, no antibody was known that has the ability to preferentially bind to non-lipidated ApoE over lipidated ApoE. Surprisingly, the inventors have found an antibody that preferentially binds to non-lipidated ApoE over lipidated ApoE. Furthermore, an antibody has been found to bind with higher affinity to aggregated ApoE than to non-aggregated ApoE. Aggregated ApoE can be prepared by oxidation and / or heat treatment of ApoE (e.g., recombinant ApoE). Aggregated ApoE can be separated from non-aggregated ApoE by ultracentrifugation (at least 186000g).

[0225] Mice were administered the antibody at the indicated doses by intraperitoneal injection, and submandibular blood was collected 30 min, 4 h, 1 day, 2 days, 4 days, and 7 days later. Anti-ApoE drug levels in plasma samples were determined by ELISA assay using recombinant apoE4 for HJ156 and human Her2 protein as capture antigens for the control IgG antibody (anti-Her2). Clearance values ​​were calculated based on the overall antibody pharmacokinetics over the entire duration of the experiment.

[0226] [Table 8]

Claims

1. An isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising SEQ ID NO:25; and / or (b) a heavy chain variable region comprising the consensus sequence of SEQ ID NO:

84.

2. The isolated antibody described in claim 1, wherein the consensus sequence of SEQ ID NO:84 is SEQ ID NO:

85.

3. The isolated antibody of claim 1 or 2, wherein the light chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:78 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:80 or SEQ ID NO:

82.

4. 4. The isolated antibody of claim 3, wherein the light chain variable region comprises SEQ ID NO:78, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:80, SEQ ID NO:82 and SEQ ID NO:

84.

5. The isolated antibody of claim 1 or 2, wherein the light chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:79 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:81 or SEQ ID NO:

83.

6. 6. The isolated antibody of claim 5, wherein the light chain variable region comprises SEQ ID NO:79, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:

85.

7. The isolated antibody according to any one of claims 1 to 6, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:

19.

8. The isolated antibody according to any one of claims 1 to 7, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:

20.

9. 9. The isolated antibody of any one of claims 1 to 8, wherein: (a) the amino acid sequence of the light chain variable region is SEQ ID NO: 9 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 10; (b) the amino acid sequence of the light chain variable region is SEQ ID NO: 11 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 12; (c) the amino acid sequence of the light chain variable region is SEQ ID NO: 13 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 14; (d) the amino acid sequence of the light chain variable region is SEQ ID NO: 15 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 16; or (e) the amino acid sequence of the light chain variable region is SEQ ID NO: 19 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

20.

10. 3. The isolated antibody of claim 1 or 2, wherein the consensus sequence of SEQ ID NO:84 or SEQ ID NO:85 is SEQ ID NO:

50.

11. The isolated antibody of claim 10, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:47 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:48 or SEQ ID NO:

49.

12. 11. The isolated antibody of claim 10, wherein the light chain variable region comprises SEQ ID NO:47, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:

50.

13. The isolated antibody of claim 1 or 2, wherein the consensus sequence of SEQ ID NO:84 is SEQ ID NO:

54.

14. The isolated antibody of claim 13, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:51 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:52 or SEQ ID NO:

53.

15. 14. The isolated antibody of claim 13, wherein the light chain variable region comprises SEQ ID NO:51, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:

54.

16. 3. The isolated antibody of claim 1 or 2, wherein the consensus sequence of SEQ ID NO:84 or SEQ ID NO:85 is SEQ ID NO:

58.

17. The isolated antibody of claim 16, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:55 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:56 or SEQ ID NO:

57.

18. 17. The isolated antibody of claim 16, wherein the light chain variable region comprises SEQ ID NO:55, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:

58.

19. 3. The isolated antibody of claim 1 or 2, wherein the consensus sequence of SEQ ID NO:84 or SEQ ID NO:85 is SEQ ID NO:

62.

20. 20. The isolated antibody of claim 19, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:59 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:60 or SEQ ID NO:

61.

21. 20. The isolated antibody of claim 19, wherein the light chain variable region comprises SEQ ID NO:59, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:

62.

22. 3. The isolated antibody of claim 1 or 2, wherein the consensus sequence of SEQ ID NO:84 or SEQ ID NO:85 is SEQ ID NO:

71.

23. 23. The isolated antibody of claim 22, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:68 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:69 or SEQ ID NO:

70.

24. 23. The isolated antibody of claim 22, wherein the light chain variable region comprises SEQ ID NO:68, SEQ ID NO:24 and SEQ ID NO:25; and / or the heavy chain variable region comprises SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:

71.

25. An isolated anti-ApoE antibody comprising: (a) a light chain variable region comprising SEQ ID NO:88; and / or (b) a heavy chain variable region comprising the consensus sequence of SEQ ID NO:

94.

26. 26. The isolated antibody of claim 25, wherein the consensus sequence of SEQ ID NO:88 is SEQ ID NO:89; and / or the consensus sequence of SEQ ID NO:84 is SEQ ID NO:

95.

27. The isolated antibody of claim 25 or 26, wherein the light chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:86 or SEQ ID NO:30; and / or the heavy chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:90 or SEQ ID NO:

92.

28. 28. The isolated antibody of claim 27, wherein the light chain variable region comprises SEQ ID NO:86, SEQ ID NO:30 and SEQ ID NO:88; and / or the heavy chain variable region comprises SEQ ID NO:90, SEQ ID NO:92 and SEQ ID NO:

94.

29. The isolated antibody of claim 25 or 26, wherein the light chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:87 or SEQ ID NO:30; and / or the heavy chain variable region further comprises one or more consensus sequences selected from SEQ ID NO:91 or SEQ ID NO:

93.

30. 30. The isolated antibody of claim 29, wherein the light chain variable region comprises SEQ ID NO:87, SEQ ID NO:30 and SEQ ID NO:89; and / or the heavy chain variable region comprises SEQ ID NO:91, SEQ ID NO:93 and SEQ ID NO:

95.

31. The isolated antibody of any of claims 25 to 30, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 3 or SEQ ID NO:

17.

32. The isolated antibody according to any one of claims 25 to 31, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4 or SEQ ID NO:

18.

33. 33. The isolated antibody of any of claims 25 to 32, wherein: (a) the amino acid sequence of the light chain variable region is SEQ ID NO: 3 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4; or (b) the amino acid sequence of the light chain variable region is SEQ ID NO: 17 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

18.

34. 27. The isolated antibody of claim 25 or 26, wherein the consensus sequence of SEQ ID NO:88 or SEQ ID NO:89 is SEQ ID NO:31, and / or the consensus sequence of SEQ ID NO:94 or SEQ ID NO:95 is SEQ ID NO:

34.

35. 35. The isolated antibody of claim 34, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:29 or SEQ ID NO:30; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:32 or SEQ ID NO:

33.

36. 35. The isolated antibody of claim 34, wherein the light chain variable region comprises SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31; and / or the heavy chain variable region comprises SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:

34.

37. 27. The isolated antibody of claim 25 or 26, wherein the consensus sequence of SEQ ID NO:88 or SEQ ID NO:89 is SEQ ID NO:64; and / or the consensus sequence of SEQ ID NO:94 or SEQ ID NO:95 is SEQ ID NO:

67.

38. 38. The isolated antibody of claim 37, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:63 or SEQ ID NO:30; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:65 or SEQ ID NO:

66.

39. 38. The isolated antibody of claim 37, wherein the light chain variable region comprises SEQ ID NO:63, SEQ ID NO:30 and SEQ ID NO:64; and / or the heavy chain variable region comprises SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:

67.

40. An isolated anti-ApoE antibody comprising a light chain variable region comprising SEQ ID NO:107; and / or a heavy chain variable region comprising the consensus sequence of SEQ ID NO:

113.

41. 41. The isolated antibody of claim 40, wherein the consensus sequence of SEQ ID NO:107 is SEQ ID NO:108; and / or the consensus sequence of SEQ ID NO:113 is SEQ ID NO:

114.

42. 42. The isolated antibody of claim 40 or 41, wherein the light chain variable region further comprises one or more consensus sequences selected from SEQ ID NO: 105 or SEQ ID NO: 106; and / or the heavy chain variable region further comprises one or more consensus sequences selected from SEQ ID NO: 109 or SEQ ID NO:

111.

43. 43. The isolated antibody of claim 42, wherein the light chain variable region comprises SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107; and / or the heavy chain variable region comprises SEQ ID NO:109, SEQ ID NO:111 and SEQ ID NO:

113.

44. 42. The isolated antibody of claim 40 or 41, wherein the light chain variable region further comprises one or more consensus sequences selected from SEQ ID NO: 105 or SEQ ID NO: 106; and / or the heavy chain variable region further comprises one or more consensus sequences selected from SEQ ID NO: 110 or SEQ ID NO:

112.

45. 45. The isolated antibody of claim 44, wherein the light chain variable region comprises SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108; and / or the heavy chain variable region comprises SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:

114.

46. The isolated antibody of any of claims 40 to 45, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:115 or SEQ ID NO:

121.

47. The isolated antibody of any of claims 40 to 46, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:116 or SEQ ID NO:

122.

48. 47. The isolated antibody of any of claims 40-46, wherein: (a) the amino acid sequence of the light chain variable region is SEQ ID NO:115 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:116; or (b) the amino acid sequence of the light chain variable region is SEQ ID NO:121 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

122.

49. 42. The isolated antibody of claim 40 or 41, wherein the consensus sequence of SEQ ID NO:107 or SEQ ID NO:108 is SEQ ID NO:117; and / or the consensus sequence of SEQ ID NO:113 or SEQ ID NO:114 is SEQ ID NO:

120.

50. 50. The isolated antibody of claim 49, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO: 105 or SEQ ID NO: 106; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO: 118 or SEQ ID NO:

119.

51. 51. The isolated antibody of claim 50, wherein the light chain variable region comprises SEQ ID NO:106, SEQ ID NO:106 and SEQ ID NO:117; and / or the heavy chain variable region comprises SEQ ID NO:118, SEQ ID NO:119 and SEQ ID NO:

120.

52. 42. The isolated antibody of claim 40 or 41, wherein the consensus sequence of SEQ ID NO:107 or SEQ ID NO:108 is SEQ ID NO:123; and / or the consensus sequence of SEQ ID NO:113 or SEQ ID NO:114 is SEQ ID NO:

126.

53. 53. The isolated antibody of claim 52, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO: 105 or SEQ ID NO: 106; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO: 124 or SEQ ID NO:

125.

54. 54. The isolated antibody of claim 53, wherein the light chain variable region comprises SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:123; and / or the heavy chain variable region comprises SEQ ID NO:124, SEQ ID NO:125 and SEQ ID NO:

126.

55. An isolated ApoE antibody, wherein the light chain variable region comprises SEQ ID NO:96; and / or the heavy chain variable region comprises SEQ ID NO:

28.

56. 56. The isolated antibody of claim 55, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:23 or SEQ ID NO:24; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:26 or SEQ ID NO:

27.

57. 56. The isolated antibody of claim 55, wherein the light chain variable region comprises SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:96; and / or the heavy chain variable region comprises SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:

28.

58. 58. The isolated antibody of any of claims 55 to 57, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 1; and / or the amino acid sequence of the heavy chain variable region is SEQ ID NO:

12.

59. 58. The isolated antibody of any of claims 55 to 57, wherein the light chain variable region is SEQ ID NO:1 and the heavy chain variable region is SEQ ID NO:

2.

60. An isolated ApoE antibody, wherein the light chain variable region comprises SEQ ID NO:74; and / or the heavy chain variable region comprises SEQ ID NO:

77.

61. The isolated antibody of claim 60, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:72 or SEQ ID NO:73; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:75 or SEQ ID NO:

76.

62. 61. The isolated antibody of claim 60, wherein the light chain variable region comprises SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74; and / or the heavy chain variable region comprises SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:

77.

63. 63. The isolated antibody of any of claims 60 to 62, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:21; and / or the amino acid sequence of the heavy chain variable region is SEQ ID NO:

22.

64. 63. The isolated antibody of any of claims 60-62, wherein the light chain variable region is SEQ ID NO:21 and the heavy chain variable region is SEQ ID NO:

22.

65. An isolated ApoE antibody, wherein the light chain variable region comprises SEQ ID NO:101; and / or the heavy chain variable region comprises SEQ ID NO:

104.

66. 66. The isolated antibody of claim 65, wherein the light chain variable region further comprises one or more sequences selected from SEQ ID NO:99 or SEQ ID NO:100; and / or the heavy chain variable region further comprises one or more sequences selected from SEQ ID NO:102 or SEQ ID NO:

103.

67. 66. The isolated antibody of claim 65, wherein the light chain variable region comprises SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101; and / or the heavy chain variable region comprises SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:

104.

68. 68. The isolated antibody of any of claims 65 to 67, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:97 and / or the amino acid sequence of the heavy chain variable region is SEQ ID NO:

98.

69. 69. The isolated antibody of any of claims 1 to 68, wherein the framework regions of each variable region have at least 75% sequence identity with a human framework region sequence.

70. 70. The isolated antibody of any of claims 1 to 69, wherein the isolated antibody further comprises one or more constant regions, or portions of constant regions, that have at least 90% sequence identity to a human constant region sequence.

71. 71. The isolated antibody of any of claims 1-70, wherein the antibody is a monoclonal antibody or an antibody fragment.

72. 72. The isolated antibody of any one of claims 1 to 71, wherein the antibody is a monoclonal antibody.

73. (a) an isolated anti-ApoE antibody comprising: a light chain variable region comprising L3 of SEQ ID NO:25; and / or a heavy chain variable region comprising H3 of SEQ ID NO:

84.

74. 74. The isolated antibody of claim 73, wherein H3 of the heavy chain is sequence number 85.

75. The isolated antibody of claim 73 or 74, wherein the light chain variable region further comprises L1 of SEQ ID NO: 78, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 80, H2 of SEQ ID NO: 82, or any combination thereof.

76. The isolated antibody of claim 75, wherein the light chain variable region comprises L1 of SEQ ID NO:78, L2 of SEQ ID NO:24, and L3 of SEQ ID NO:25; and / or the heavy chain variable region comprises H1 of SEQ ID NO:80, H2 of SEQ ID NO:82, and H3 of SEQ ID NO:

84.

77. The isolated antibody of claim 73 or 74, wherein the light chain variable region further comprises L1 of SEQ ID NO: 79, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 81, H2 of SEQ ID NO: 83, or any combination thereof.

78. 78. The isolated antibody of claim 77, wherein the light chain variable region comprises L1 of SEQ ID NO:79, L2 of SEQ ID NO:24, and L3 of SEQ ID NO:25; and / or the heavy chain variable region comprises H1 of SEQ ID NO:81, H2 of SEQ ID NO:83, and H3 of SEQ ID NO:

85.

79. 79. The isolated antibody of any of claims 73 to 78, wherein the amino acid sequence of the light chain variable region is selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:

19.

80. 80. The isolated antibody of any of claims 73 to 79, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:

20.

81. 81. The isolated antibody of any of claims 73 to 80, wherein: (a) the amino acid sequence of the light chain variable region is SEQ ID NO: 9 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 10; (b) the amino acid sequence of the light chain variable region is SEQ ID NO: 11 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 12; (c) the amino acid sequence of the light chain variable region is SEQ ID NO: 13 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 14; (d) the amino acid sequence of the light chain variable region is SEQ ID NO: 15 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 16; or (e) the amino acid sequence of the light chain variable region is SEQ ID NO: 19 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

20.

82. 75. The isolated antibody of claim 73 or 74, wherein H3 of the heavy chain variable region is sequence number 50.

83. 83. The isolated antibody of claim 82, wherein the light chain variable region further comprises L1 of SEQ ID NO: 47, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 48, H2 of SEQ ID NO: 49, or any combination thereof.

84. 83. The isolated antibody of claim 82, wherein the light chain variable region comprises L1 of SEQ ID NO: 47, L2 of SEQ ID NO: 24, and L3 of SEQ ID NO: 25; and / or the heavy chain variable region comprises L1 of SEQ ID NO: 48, L2 of SEQ ID NO: 49, and L3 of SEQ ID NO:

50.

85. 75. The isolated antibody of claim 73 or 74, wherein H3 of the heavy chain variable region is sequence number 54.

86. 86. The isolated antibody of claim 85, wherein the light chain variable region further comprises L1 of SEQ ID NO: 51, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 52, H2 of SEQ ID NO: 53, or any combination thereof.

87. 87. The isolated antibody of claim 86, wherein the light chain variable region comprises L1 of SEQ ID NO:51, L2 of SEQ ID NO:24, and L3 of SEQ ID NO:25; and / or the heavy chain variable region comprises H1 of SEQ ID NO:52, H2 of SEQ ID NO:53, and H3 of SEQ ID NO:

54.

88. 75. The isolated antibody of claim 73 or 74, wherein H3 of the heavy chain variable region is sequence number 58.

89. 89. The isolated antibody of claim 88, wherein the light chain variable region further comprises L1 of SEQ ID NO: 55, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 56, H2 of SEQ ID NO: 57, or any combination thereof.

90. The isolated antibody of claim 89, wherein the anti-ApoE antibody comprises L1 of SEQ ID NO:55, L2 of SEQ ID NO:24, and L3 of SEQ ID NO:25; and / or H1 of SEQ ID NO:56, H2 of SEQ ID NO:57, and H3 of SEQ ID NO:

58.

91. 75. The isolated antibody of claim 73 or 74, wherein H3 of the heavy chain variable region is sequence number 62.

92. 92. The isolated antibody of claim 91, wherein the light chain variable region further comprises L1 of SEQ ID NO:59, L2 of SEQ ID NO:24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO:60, H2 of SEQ ID NO:61, or any combination thereof.

93. 93. The isolated antibody of claim 92, wherein the light chain variable region comprises L1 of SEQ ID NO:59, L2 of SEQ ID NO:24, and L3 of SEQ ID NO:25; and / or the heavy chain variable region comprises H1 of SEQ ID NO:60, H2 of SEQ ID NO:61, and H3 of SEQ ID NO:

62.

94. 75. The isolated antibody of claim 73 or 74, wherein H3 of the heavy chain variable region is sequence number 71.

95. The isolated antibody of claim 94, wherein the light chain variable region further comprises L1 of SEQ ID NO: 68, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 69, H2 of SEQ ID NO: 70, or any combination thereof.

96. The isolated antibody of claim 94, wherein the light chain variable region comprises L1 of SEQ ID NO:68, L2 of SEQ ID NO:24, and L3 of SEQ ID NO:25; and / or the heavy chain variable region comprises H1 of SEQ ID NO:69, H2 of SEQ ID NO:70, and H3 of SEQ ID NO:

71.

97. An isolated anti-ApoE antibody comprising a light chain variable region comprising L3 of SEQ ID NO:88; and / or a heavy chain variable region comprising H3 of SEQ ID NO:

94.

98. 98. The isolated antibody of claim 97, wherein the L3 of the light chain is SEQ ID NO: 89 and / or the H3 of the heavy chain is SEQ ID NO:

95.

99. The isolated antibody of claim 97 or 98, wherein the light chain variable region further comprises L1 of SEQ ID NO: 86, L2 of SEQ ID NO: 30, or any combination thereof, and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 90, H2 of SEQ ID NO: 92, or any combination thereof.

100. The isolated antibody of claim 99, wherein the light chain variable region comprises L1 of SEQ ID NO: 86, L2 of SEQ ID NO: 30, and L3 of SEQ ID NO: 88; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 90, H2 of SEQ ID NO: 92, and H3 of SEQ ID NO:

94.

101. The isolated antibody of claim 97 or 98, wherein the light chain variable region further comprises L1 of SEQ ID NO: 87, L2 of SEQ ID NO: 30, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 91, H2 of SEQ ID NO: 93, or any combination thereof.

102. The isolated antibody of claim 101, wherein the light chain variable region comprises L1 of SEQ ID NO:87, L2 of SEQ ID NO:30, and L3 of SEQ ID NO:89; and / or the heavy chain variable region comprises H1 of SEQ ID NO:91, H2 of SEQ ID NO:93, and H3 of SEQ ID NO:

95.

103. The isolated antibody of any of claims 97 to 102, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 3 or SEQ ID NO:

17.

104. The isolated antibody of any of claims 97 to 103, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:4 or SEQ ID NO:

18.

105. 105. An isolated antibody according to any one of claims 97 to 104, wherein: (a) the amino acid sequence of the light chain variable region is SEQ ID NO: 3 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4; or (b) the amino acid sequence of the light chain variable region is SEQ ID NO: 17 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

18.

106. 99. The isolated antibody of claim 97 or 98, wherein L3 of the light chain variable region is SEQ ID NO: 31 and / or H3 of the heavy chain variable region is SEQ ID NO:

34.

107. The isolated antibody of claim 106, wherein the light chain variable region further comprises L1 of SEQ ID NO:29, L2 of SEQ ID NO:30, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO:32, H2 of SEQ ID NO:33, or any combination thereof.

108. The isolated antibody of claim 107, wherein the light chain variable region comprises L1 of SEQ ID NO:29, L2 of SEQ ID NO:30, and L3 of SEQ ID NO:31; and / or the heavy chain variable region comprises H1 of SEQ ID NO:32, H2 of SEQ ID NO:33, and H3 of SEQ ID NO:

34.

109. 99. The isolated antibody of claim 97 or 98, wherein L3 of the light chain variable region is SEQ ID NO: 64 and / or H3 of the heavy chain variable region is SEQ ID NO:

67.

110. The isolated antibody of claim 109, wherein the light chain variable region further comprises L1 of SEQ ID NO: 63, L2 of SEQ ID NO: 30, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 65, H2 of SEQ ID NO: 66, or any combination thereof.

111. The isolated antibody of claim 110, wherein the light chain variable region comprises L1 of SEQ ID NO: 63, L2 of SEQ ID NO: 30, and L3 of SEQ ID NO: 64; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 65, H2 of SEQ ID NO: 66, and H3 of SEQ ID NO:

67.

112. An isolated ApoE antibody comprising a light chain variable region comprising L3 of SEQ ID NO:96; and / or a heavy chain variable region comprising H3 of SEQ ID NO:

28.

113. The isolated antibody of claim 112, wherein the light chain variable region further comprises L1 of SEQ ID NO: 23, L2 of SEQ ID NO: 24, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 26, H2 of SEQ ID NO: 27, or any combination thereof.

114. The isolated antibody of claim 113, wherein the light chain variable region comprises L1 of SEQ ID NO: 23, L2 of SEQ ID NO: 24, and L3 of SEQ ID NO: 96; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 26, H2 of SEQ ID NO: 27, and H3 of SEQ ID NO:

28.

115. 15. The isolated antibody of any of claims 112 to 114, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 1; and / or the amino acid sequence of the heavy chain variable region is SEQ ID NO:

12.

116. 115. The isolated antibody of any of claims 112-114, wherein the light chain variable region is SEQ ID NO:1 and the heavy chain variable region is SEQ ID NO:

2.

117. An isolated ApoE antibody comprising a light chain variable region comprising L3 of SEQ ID NO:74; and / or a heavy chain variable region comprising H3 of SEQ ID NO:

77.

118. The isolated antibody of claim 117, wherein the light chain variable region further comprises L1 of SEQ ID NO: 72, L2 of SEQ ID NO: 73, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 75, H2 of SEQ ID NO: 76, or any combination thereof.

119. The isolated antibody of claim 118, wherein the light chain variable region comprises L1 of SEQ ID NO: 72, L2 of SEQ ID NO: 73, and L3 of SEQ ID NO: 74; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 75, H2 of SEQ ID NO: 76, and H3 of SEQ ID NO:

77.

120. 120. The isolated antibody of any of claims 117 to 119, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:21; and / or the amino acid sequence of the heavy chain variable region is SEQ ID NO:

22.

121. 121. The isolated antibody of any of claims 117-120, wherein the light chain variable region is SEQ ID NO:21 and the heavy chain variable region is SEQ ID NO:

22.

122. An isolated ApoE antibody comprising a light chain variable region comprising L3 of SEQ ID NO:101; and / or a heavy chain variable region comprising H3 of SEQ ID NO:

104.

123. The isolated antibody of claim 122, wherein the light chain variable region further comprises L1 of SEQ ID NO: 99, L2 of SEQ ID NO: 100, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 102, H2 of SEQ ID NO: 103, or any combination thereof.

124. The isolated antibody of claim 123, wherein the light chain variable region comprises L1 of SEQ ID NO:99, L2 of SEQ ID NO:100, and L3 of SEQ ID NO:101; and / or the heavy chain variable region comprises H1 of SEQ ID NO:102, H2 of SEQ ID NO:103, and H3 of SEQ ID NO:

104.

125. 125. The isolated antibody of any of claims 122 to 124, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:97; and / or the amino acid sequence of the heavy chain variable region is SEQ ID NO:

98.

126. 126. The isolated antibody of any of claims 122-125, wherein the light chain variable region is SEQ ID NO:97 and the heavy chain variable region is SEQ ID NO:

98.

127. An isolated antibody comprising a light chain variable region comprising L3 of SEQ ID NO:107; and / or a heavy chain variable region comprising H3 of SEQ ID NO:

113.

128. 128. The isolated antibody of claim 127, wherein L3 of the light chain is SEQ ID NO: 108; and / or H3 of the heavy chain is SEQ ID NO:

114.

129. The isolated antibody of claim 127 or 128, wherein the light chain variable region further comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 109, H2 of SEQ ID NO: 111, or any combination thereof.

130. The isolated antibody of claim 129, wherein the light chain variable region comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, and L3 of SEQ ID NO: 107; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 109, H2 of SEQ ID NO: 111, and H3 of SEQ ID NO:

113.

131. The isolated antibody of claim 127 or 128, wherein the light chain variable region further comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 110, H2 of SEQ ID NO: 112, or any combination thereof.

132. The isolated antibody of claim 131, wherein the light chain variable region comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, and L3 of SEQ ID NO: 108; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 110, H2 of SEQ ID NO: 112, and H3 of SEQ ID NO:

114.

133. The isolated antibody of any of claims 127 to 132, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 115 or SEQ ID NO:

121.

134. The isolated antibody of any of claims 127 to 133, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:116 or SEQ ID NO:

122.

135. 135. The isolated antibody of any of claims 127-134, wherein: (a) the amino acid sequence of the light chain variable region is SEQ ID NO: 115 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 116; or (b) the amino acid sequence of the light chain variable region is SEQ ID NO: 121 and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

122.

136. 129. The isolated antibody of claim 127 or 128, wherein L3 of the light chain is SEQ ID NO: 117; and / or H3 of the heavy chain is SEQ ID NO:

120.

137. The isolated antibody of claim 136, wherein the light chain variable region further comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 118, H2 of SEQ ID NO: 119, or any combination thereof.

138. The isolated antibody of claim 137, wherein the light chain variable region comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, and L3 of SEQ ID NO: 117; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 118, H2 of SEQ ID NO: 119, and H3 of SEQ ID NO:

120.

139. 129. The isolated antibody of claim 127 or 128, wherein L3 of the light chain variable region is SEQ ID NO: 123; and / or H3 of the heavy chain variable region is SEQ ID NO:

126.

140. The isolated antibody of claim 139, wherein the light chain variable region further comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, or any combination thereof; and / or the heavy chain variable region further comprises H1 of SEQ ID NO: 124, H2 of SEQ ID NO: 125, or any combination thereof.

141. The isolated antibody of claim 140, wherein the light chain variable region comprises L1 of SEQ ID NO: 105, L2 of SEQ ID NO: 106, and L3 of SEQ ID NO: 123; and / or the heavy chain variable region comprises H1 of SEQ ID NO: 124, H2 of SEQ ID NO: 125, and H3 of SEQ ID NO:

126.

142. 142. The isolated antibody of any of claims 73 to 141, wherein the framework regions of each variable region have at least 75% sequence identity with human framework region sequences.

143. The isolated antibody of any of claims 73 to 142, further comprising one or more constant regions, or portions of constant regions, that have at least 90% sequence identity to a human constant region sequence.

144. The isolated antibody of any of claims 73-143, wherein the antibody is a monoclonal antibody or an antibody fragment.

145. A method for reducing amyloid plaque deposition and / or CAA deposition in the brain of a subject in need thereof, the method comprising administering a therapeutically effective amount of at least one anti-ApoE antibody, wherein the antibody (a) binds to human ApoE4 with a KD of about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, (b) preferentially binds to recombinant alipidated human ApoE4 compared to ApoE4 derived from human plasma or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4, and (c) binds to human ApoE in amyloid plaques in unfixed brain tissue.

146. The method of claim 145, wherein the antibody is administered peripherally.

147. The method of claim 146, wherein the antibody is administered locally, optionally directly into the central nervous system.

148. The method of any of claims 145 to 147, wherein amyloid plaque deposition is reduced in the hippocampus.

149. The method of any of claims 145 to 147, wherein amyloid plaque deposition is reduced in the cerebral cortex.

150. 148. The method of any of claims 145-147, wherein deposition of CAA is reduced in perforator arterioles on the surface of the cerebral cortex and / or in leptomeningeal arterioles on the surface of the cerebral cortex.

151. The method of any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 1, 3, 4, 73, 75 or 76.

152. The method according to any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 2, 5, 6, 74, 77 or 78.

153. The method of any of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any of claims 10 to 12 or claims 82 to 84; (b) claims 13 to 15 or claims 85 to 87; (c) claims 16 to 18 or claims 88 to 89; (d) claims 19 to 21 or claims 91 to 93; or (e) claims 22 to 24 or claims 94 to 96.

154. The method of any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 25, 27, 28, 97, 99 or 100.

155. The method of any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 26, 29, 30, 98, 101 or 102.

156. The method according to any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of (a) claims 34 to 36 or claims 106 to 108; or (b) claims 37 to 39 or claims 109 to 111.

157. The method of any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 40, 42, 43, 127, 129 or 130.

158. The method of any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 41, 44, 45, 128, 131 or 132.

159. The method according to any of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any of (a) claims 49 to 51 or claims 137 to 139; or (b) claims 52 to 54 or claims 140 to 141.

160. The method according to any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 55 to 57 or claims 112 to 114.

161. The method according to any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 65 to 67 or claims 122 to 124.

162. The method according to any one of claims 145 to 150, wherein the antibody is an anti-ApoE antibody according to any one of claims 60 to 62 or claims 117 to 119.

163. The method of any of claims 145 to 162, wherein each variable region of the anti-ApoE antibody comprises a framework region that has at least 75% sequence identity with a human framework region sequence.

164. The method of any of claims 73-142, wherein the anti-ApoE antibody further comprises one or more constant regions, or portions of constant regions, having at least 90% sequence identity with a human constant region sequence.

165. The method of any one of claims 145 to 164, wherein the anti-ApoE antibody is a monoclonal antibody or an antibody fragment.

166. 1. A method of treating at least one Aβ plaque-associated condition or at least one CAA-associated condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of at least one anti-ApoE antibody, wherein the antibody (a) binds to human ApoE4 with a KD of about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, (b) preferentially binds to recombinant, non-lipidated human ApoE4 compared to ApoE4 derived from human plasma or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4, and (c) binds to human ApoE in amyloid plaques in unfixed brain tissue.

167. The method of claim 166, wherein the treatment comprises preventing, attenuating, ameliorating or ameliorating at least one symptom or sign of Aβ plaques or at least one CAA-associated symptom in the subject.

168. The method of claim 166 or 167, wherein the Aβ plaque-associated condition or CAA-associated condition is selected from neurodegeneration, cognitive impairment, behavioral changes, language abnormalities, impaired emotional regulation, seizures, nervous system structural impairment, nervous system dysfunction, increased risk of developing Alzheimer's disease, or increased risk of developing cerebral amyloid angiopathy.

169. The method of any of claims 166 to 168, wherein the antibody is administered locally, optionally directly into the central nervous system.

170. The method of any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 1, 3, 4, 73, 75 or 76.

171. The method of any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 2, 5, 6, 74, 77 or 78.

172. The method of any of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any of the following: (a) claims 10 to 12 or claims 82 to 84; (b) claims 13 to 15 or claims 85 to 87; (c) claims 16 to 18 or claims 88 to 89; (d) claims 19 to 21 or claims 91 to 93; or (e) claims 22 to 24 or claims 94 to 96.

173. The method of any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 25, 27, 28, 97, 99 or 100.

174. The method of any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 26, 29, 30, 98, 101 or 102.

175. The method according to any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of (a) claims 34 to 36 or claims 106 to 108; or (b) claims 37 to 39 or claims 109 to 111.

176. The method of any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 40, 42, 43, 127, 129 or 130.

177. The method of any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 41, 44, 45, 128, 131 or 132.

178. The method according to any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of (a) claims 49 to 51 or claims 137 to 139; or (b) claims 52 to 54 or claims 140 to 141.

179. The method according to any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 55 to 57 or claims 112 to 114.

180. The method according to any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 65 to 67 or claims 122 to 124.

181. The method according to any one of claims 166 to 168, wherein the antibody is an anti-ApoE antibody according to any one of claims 60 to 62 or claims 117 to 119.

182. The method of any of claims 166 to 168, wherein each variable region of the anti-ApoE antibody comprises a framework region that has at least 75% sequence identity with a human framework region sequence.

183. The method of any of claims 166-168, wherein the anti-ApoE antibody further comprises one or more constant regions, or portions of constant regions, having at least 90% sequence identity with a human constant region sequence.

184. The method of any one of claims 155 to 184, wherein the anti-ApoE antibody is a monoclonal antibody or an antibody fragment.

185. A method for measuring the binding affinity of an anti-ApoE antibody to human plasma ApoE, comprising: (a) contacting human plasma ApoE with a blocking protein; (b) exposing the plasma ApoE blocked with said protein to a test anti-ApoE antibody; and (c) detecting the presence of anti-ApoE antibody bound to said plasma ApoE.

186. The method of claim 186, further comprising exposing the protein-blocked plasma ApoE in step (b) to various concentrations of anti-ApoE antibody, and (d) calculating the IC50 binding affinity of the antibody.

187. An isolated anti-ApoE antibody that does not specifically bind to plasma-derived ApoE, which, when administered to an animal, has a pharmacokinetic clearance value in plasma that is less than 25-fold the clearance value of an isotype control antibody.

188. The isolated antibody of claim 188, having a plasma clearance value that is less than 20, 15, 10, 5 or 3 times that of an isotype control.

189. The isolated antibody of claim 189, having a plasma clearance value less than three-fold that of an isotype control.

190. The isolated antibody of claim 188, wherein the isotype control binds to an antigen that is not present in the animal.

191. The isolated antibody of claim 188, wherein the isotype control binds to a viral or bacterial antigen.

192. The isolated antibody of claim 188, wherein the isotype control is an IgG antibody.

193. The isolated antibody of claim 188, wherein the isotype control is an IgG2a antibody.

194. The isolated antibody of claim 188, wherein the animal is a human.

195. The isolated antibody of claim 188, wherein the animal is a mouse modified to express human ApoE.

196. 189. The isolated antibody of claim 188, wherein the antibody binds to non-lipidated ApoE.

197. 200. The isolated antibody of claim 198, wherein the delipidated ApoE is recombinant ApoE.

198. The isolated antibody of claim 198, wherein non-lipidated ApoE is present in amyloid plaques.

199. 200. The isolated antibody of claim 198, wherein the non-lipidated ApoE is in an aggregated form.

200. 200. The isolated antibody of claim 198, wherein the antibody preferentially binds aggregated ApoE over non-aggregated ApoE.

201. 202. The isolated antibody of claim 200 or 201, wherein aggregated ApoE is prepared by oxidation or heat treatment of ApoE and isolated by ultracentrifugation.

202. 203. The isolated antibody of any of claims 188-202, wherein the ApoE is ApoE4.

203. 204. The isolated antibody of any of claims 188-203, wherein the antibody binds to non-lipidated ApoE4.

204. 204. The isolated antibody of any of claims 188-203, wherein the antibody binds preferentially to non-lipidated ApoE3 and / or ApoE4 over other non-lipidated ApoE isoforms.

205. The isolated antibody of claim 188, wherein the antibody (a) binds to human ApoE4 with a KD of about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, (b) preferentially binds to recombinant, non-lipidated human ApoE4 compared to ApoE4 derived from human plasma or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4, and (c) binds to human ApoE in amyloid plaques in unfixed brain tissue.

206. The antibody, a light chain variable region comprising SEQ ID NO:25, and / or a heavy chain variable region comprising SEQ ID NO:84; a light chain variable region comprising SEQ ID NO:88, and / or a heavy chain variable region comprising SEQ ID NO:94; a light chain variable region comprising SEQ ID NO:74, and / or a heavy chain variable region comprising SEQ ID NO:77; a light chain variable region comprising L3 of SEQ ID NO:25, and / or a heavy chain variable region comprising H3 of SEQ ID NO:84; a light chain variable region comprising L3 of SEQ ID NO:88, and / or a heavy chain variable region comprising H3 of SEQ ID NO:94; or A light chain variable region comprising L3 of SEQ ID NO:74 and / or a heavy chain variable region comprising H3 of SEQ ID NO:77 The isolated antibody of claim 188, comprising:

207. 203. An assay for detecting anti-ApoE antibodies that bind to aggregated ApoE, including the aggregated ApoE of claim 202.

208. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 144 or claims 188 to 207, and a pharma- ceutically acceptable carrier or excipient.

209. 210. The pharmaceutical composition of claim 209, further comprising a dispersing agent, buffer, surfactant, preservative, solubilizer, isotonicity agent or stabilizer.

210. The pharmaceutical composition of claim 209, wherein the carrier comprises saline, an ion exchanger, alumina, aluminum stearate, lecithin, serum proteins, human serum albumin, a buffer substance, phosphate, glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, a salt or electrolyte, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, a zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, wool fat, or a combination thereof.

211. An antibody according to any of claims 1 to 144 or claims 188 to 207 for use as a medicament.

212. The antibody of any of claims 1 to 144 or claims 188 to 207 for use in the treatment of reducing amyloid plaque deposits and / or CAA deposits in the CNS.

213. The antibody of any of claims 1 to 154 for use in the treatment of at least one Aβ plaque-related condition or at least one CAA-related condition.

214. The antibody of any of claims 1 to 144 or claims 188 to 207 for use in the treatment of Alzheimer's disease.

215. 208. The antibody of any of claims 1 to 144 or claims 188 to 207 for use in the manufacture of a medicament for reducing amyloid plaque deposition and / or CAA deposition in the CNS.

216. The antibody of any of claims 1 to 144 or claims 188 to 207 for use in the manufacture of a medicament for treating at least one Aβ plaque-related condition or at least one CAA-related condition.

217. 208. The antibody of any of claims 1 to 144 or claims 188 to 207 for use in the manufacture of a medicament for the treatment of Alzheimer's disease.

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