APOE antibodies, fusion proteins and uses thereof

Abstract

Methods and compositions for preventing or treating cognitive decline and / or neurodegeneration associated with dementia and / or mild cognitive impairment using antibodies, peptides, fusion proteins or genome editing systems that modulate HSPG / heparin binding affinity of ApoE.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 853,676, filed May 28, 2019, and U.S. Provisional Application No. 62 / 873,019, filed July 11, 2019. The entire contents of the foregoing are incorporated herein by reference.

[0003] Federally funded research or development

[0004] This invention was made with U.S. Government support under Grant No. OD019833 awarded by the National Institutes of Health, Grant Nos. AG054671, AG031581, and AG19610 awarded by the National Institute on Aging, and Grant Nos. NS100121 and NS110048 awarded by the National Institute of Neurological Disorders and Stroke. The U.S. Government has certain rights in this invention. Technical Field

[0005] Described herein are methods and compositions for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment by modulating the heparan sulfate proteoglycan (HSPG) / glycosaminoglycan (GAG) heparin binding affinity of apolipoprotein E (ApoE). Background Art

[0006] Alzheimer's disease (AD) is a chronic neurodegenerative disease that usually starts slowly and gradually worsens over time. It is the cause of 60-70% of dementia cases. The disease process is associated with plaques and neurofibrillary tangles in the brain. There is currently no treatment that can stop or reverse its progression, but some may temporarily improve symptoms. The accumulation, aggregation and deposition of amyloid-β (Aβ) peptides in the brain are at the core of the pathogenesis of Alzheimer's disease (AD). Growing evidence shows that ApoE strongly affects AD pathogenesis by controlling Aβ aggregation and metabolism (Fu et al., Mol Neurodegener 11:37, 2016). APOE affects the production, aggregation and clearance of amyloid protein, is a component of amyloid plaques, and worsens tau-mediated neurodegeneration. ApoE is 299 amino acids long and is polymorphic, with three major alleles (ε2, ε3, and ε4) that differ only by one or two amino acids at positions 112 and 158: ApoE2 (cys112, cys158), ApoE3 (cys112, arg158), and ApoE4 (arg112, arg158). Therefore, there is a need for therapies that target and modulate the function of the ApoE protein for the treatment or prevention of AD and cognitive decline associated with dementia or mild cognitive impairment. Summary of the Invention

[0007] In one aspect, the present disclosure features an isolated monoclonal antibody that specifically binds to one or more (e.g., 1, 2, 3, or 4) HSPG binding sites or one or more (e.g., 1, 2, 3, or 4) HSPG-binding sites of wild-type or mutant apolipoprotein E (ApoE). In some embodiments, the antibody binds to a polypeptide having an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to TEELRVRLASHLRK (SEQ ID NO: 3). In some embodiments, the antibody binds to a polypeptide having an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to TEELRVSLASHLRK (SEQ ID NO: 2). In some embodiments, the antibody binds to one or more (e.g., 1, 2, 3, or 4) HSPG binding sites of wild-type or mutant ApoE2, ApoE3, or ApoE4.

[0008] In some embodiments, the antibody competes for and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL of the reference antibody: (i) comprise the amino acid sequence set forth in SEQ ID NO: 13 and the amino acid sequence set forth in SEQ ID NO: 12, respectively; (ii) comprise the amino acid sequence set forth in SEQ ID NO: 23 and the amino acid sequence set forth in SEQ ID NO: 22, respectively; (iii) comprise the amino acid sequence set forth in SEQ ID NO: 33 and the amino acid sequence set forth in SEQ ID NO: 32, respectively; or (iv) comprise the amino acid sequence set forth in SEQ ID NO: 43 and the amino acid sequence set forth in SEQ ID NO: 42, respectively. In some embodiments of any of the antibodies described herein, the antibody competes for and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain and a light chain, wherein the heavy chain and light chain of the reference antibody comprise the amino acid sequence set forth in SEQ ID NO: 53 and the amino acid sequence set forth in SEQ ID NO: 52.

[0009] In another aspect, provided herein are anti-ApoE antibodies comprising a VH comprising VHCDR1, VHCDR2, and VHCDR3, and a VL comprising VLCDR1, VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3: (i) comprise SEQ ID No: 7, 8, 9, 4, 5, 6, respectively; (ii) comprise SEQ ID No: 17, 18, 19, 14, 15, 16, respectively; (iii) comprise SEQ ID No: 27, 28, 29, 24, 25, 26, respectively; (iv) comprise SEQ ID No: 37, 38, 39, 34, 35, 36, respectively; or (v) SEQ ID No: 47, 48, 49, 44, 45, 46, respectively. In some embodiments of any of the antibodies described herein, (i) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 13 and 12, respectively; (ii) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 23 and 22, respectively; (iii) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 33 and 32, respectively; or (iv) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 43 and 42, respectively. In some embodiments of any of the antibodies described herein, the antibody comprises a heavy chain and a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID Nos: 53 and 52, respectively. In some embodiments, the antibody comprises a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3 heavy chain constant region. In some embodiments, the antibody comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the antibody comprises a human kappa or human lambda light chain constant region. In some embodiments, the antibody is an intact antibody, a single domain antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, an Fv, a scFv, a sc(Fv)2, a diabody, a nanobody, a Fab, or a F(ab')2. In some embodiments, the antibody further comprises a half-life extending moiety. In some embodiments, the antibody further comprises a blood-brain barrier penetrating moiety. In some embodiments, the antibody further comprises a detectable label.In some embodiments, provided herein are pharmaceutical compositions comprising any of the antibodies described herein. In some embodiments, provided herein are one or more polynucleotides encoding any of the antibodies described herein. In some embodiments, provided herein are one or more vectors comprising one or more polynucleotides as described herein. In some embodiments, provided herein are host cells comprising one or more polynucleotides as described herein, or one or more vectors as described herein. On the other hand, provided herein are methods for preparing anti-ApoE antibodies, comprising: (a) culturing any of the host cells described herein under conditions that allow expression of the antibody; and (b) isolating the antibody. In some embodiments, the method further comprises formulating the antibody into a sterile preparation suitable for administration to a human.

[0010] On the other hand, provided herein is an Fc fusion protein comprising: an HSPG binding domain of wild-type ApoE or mutant ApoE, the HSPG binding domain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of: STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60). In some embodiments, the Fc fusion protein comprises the Fc region of a human antibody. In some embodiments, the human antibody is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 molecules. In some embodiments, provided herein are pharmaceutical compositions comprising any of the Fc fusion proteins described herein. In some embodiments, provided herein are one or more polynucleotides encoding the Fc fusion proteins described herein. In some embodiments, provided herein are one or more vectors comprising any of the one or more polynucleotides described herein. In some embodiments, provided herein are host cells comprising one or more polynucleotides described herein, or one or more vectors described herein.

[0011] In another aspect, provided herein is a pharmaceutical composition for eliciting an immune response comprising: (i) a HSPG binding domain of wild-type ApoE or mutant ApoE, wherein the HSPG binding domain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60); and (ii) a pharmaceutically acceptable adjuvant.

[0012] In another aspect, provided herein are pharmaceutical compositions comprising human cells expressing any of the antibodies described herein or any of the Fc fusion proteins described herein.

[0013] In another aspect, provided herein is a method for improving, slowing, delaying the onset, preventing or reversing cognitive decline and / or neurodegeneration associated with dementia and / or mild cognitive impairment in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any of the antibodies, Fc fusion proteins, or pharmaceutical compositions described herein.

[0014] On the other hand, provided herein is a method for improving, slowing, delaying the onset, preventing or reversing cognitive decline and / or neurodegeneration associated with dementia and / or mild cognitive impairment in a human subject in need thereof, the method comprising administering to the subject: (i) a viral vector comprising a nucleotide sequence encoding a gRNA molecule, the gRNA molecule comprising a targeting domain complementary to a target domain from an APOE gene; (ii) a viral vector comprising a nucleotide sequence encoding a Cas9 molecule; and (iii) a viral vector comprising a template nucleic acid, wherein the template nucleic acid comprises an adenine to replace the cytosine at position 19: g.45412013C>A in the APOE gene, wherein the administration results in the production of one or more ApoE R136S alleles in one or more cells of the subject. In some embodiments, the targeting domain of the gRNA molecule comprises a sequence that is identical to or differs from a sequence from Table 5 by no more than 3 nucleotides.

[0015] As used herein, "preventing" or "preventing" refers to reducing the risk of developing a condition.

[0016] In some embodiments, the human subject is diagnosed with Alzheimer's disease or is at risk of developing Alzheimer's disease. In some embodiments, the human subject carries one or more copies of the APOE4 allele. In some embodiments, the human subject carries one or more mutations in at least one gene selected from the group consisting of APP, PSEN1 and PSEN2. In some embodiments, the human subject carries one or more mutations in other genes that cause autosomal dominant Alzheimer's disease (for example, those described in Bateman et al., Alzheimer's Research & Therapy 3(1):1,2011). In some embodiments, the human subject carries all or part of the third copy of chromosome 21. In some embodiments, the human subject is diagnosed with Alzheimer's disease by established biomarkers, such as those obtained by brain imaging or blood or CSF samples. In some embodiments, the human subject is more than (over) 50 years old (for example, more than 55, 60, 65, 70, 75, 80, 85, 90 or 95 years old).

[0017] In some embodiments, the human subject is diagnosed with or is at risk of developing a disorder selected from the group consisting of: vascular cognitive impairment, vascular dementia, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Huntington's disease, neurodegenerative diseases, cerebrovascular diseases, brain injury, chronic traumatic encephalopathy, tauopathies, amyloidopathies, synucleinopathies, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, and aging.

[0018] In yet another aspect, the present invention provides a method for identifying a human subject who is less likely to develop an early-onset neurodegenerative disease, the method comprising: obtaining a biological sample from the subject; detecting the presence of at least one APOE3 mutant allele or the presence of a mutant ApoE3 gene product in the biological sample; and identifying the subject as less likely to develop an early-onset neurodegenerative disease based on the presence of the mutant ApoE3 allele or gene product in the biological sample. In some embodiments, the biological sample is blood, cerebrospinal fluid, saliva, urine, tears, vitreous humor, aqueous humor, or a tissue specimen. In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, retinal degeneration, or glaucoma. In some embodiments, the retinal degeneration is age-related macular degeneration. In some embodiments, the detection comprises determining the sequence of the APOE3 allele in the subject. In some embodiments, the detection comprises determining the presence or absence of an APOE3 sequence encoding an ApoE3 protein having a mutation at R136 compared to the wild-type ApoE3 protein. In some embodiments, the mutation at R136 is R136S, R136H, or R136C. In some embodiments of any of the methods described herein for identifying a human subject less susceptible to developing an early-onset neurodegenerative disease, the method further comprises selecting the subject for inclusion in a clinical trial and optionally administering an experimental treatment, or excluding the subject from the clinical trial if the subject does not have a mutant APOE3 allele. In some embodiments of any of the methods described herein for identifying a human subject less susceptible to developing an early-onset neurodegenerative disease, the method further comprises selecting the subject for inclusion in a clinical trial and optionally administering an experimental treatment, or excluding the subject from the clinical trial if the subject has a mutant APOE3 allele.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use with the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of conflict, the present specification, including definitions, will control.

[0020] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A model showing the structure of the wild-type APOE3 protein. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are shown. The amino acid positions of the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are shown.

[0022] Figure 2 Representative Sanger sequencing results for APOE from control, proband, and offspring samples are shown.

[0023] Figure 3 The pedigrees of the subjects are depicted. Circles represent females, squares represent males, diamonds represent individuals whose sex has been obscured for privacy, arrows depict probands with MCI, and shading denotes individuals with a history of dementia. Deceased individuals are marked with crossed bars. Where necessary, individual APOE and PSEN1 genotypes are indicated to maintain anonymity.

[0024] Figure 4A and 4B Fundus photographs of the right and left eyes, respectively. Figure 4C An infrared image of the right eye is shown, which describes Figure 4D Cross-section of the retina (line) as seen in . Figure 4D Shown are the results of optical coherence tomography (OCT) of the right eye. Figure 4E Displays an infrared image of the left eye. Figure 4F Shown are the results of OCT imaging of the left eye.

[0025] Figure 5 Shown are brain imaging results showing amyloid plaque burden and PHF tau burden in the brains of PSEN1 mutation carriers with late-onset MCI (mild cognitive impairment) and PSEN1 mutation carriers with MCI onset at the typical age for this mutation (44 years).

[0026] Figure 6 Shown are measurements of amyloid burden, tau burden, hippocampal volume, and glucose metabolism in PSEN1 E280A mutation carriers. Red dots represent measurements of carriers with two APOE3ch alleles and unusually late onset of MCI. Black dots represent PSEN1 E280A mutation carriers who developed MCI at a younger age than typical for relatives. Gray dots represent PSEN1 E280A mutation carriers who have not yet developed MCI.

[0027] Figure 7The rate of Aβ42 fibril formation in the presence of wild-type APOE3 or in the absence of APOE is shown, as measured by Thioflavin T fluorescence. Changes in relative fluorescence units (RFU) are plotted against time in minutes (min). (***P < 0.001, ****P < 0.0001)

[0028] Figure 8 Graph showing split-luciferase complementation triggered by amyloid oligomerization (top) and the percentage of luminescence obtained by the split-luciferase complementation assay after 24 hours in culture medium from 293T cells transfected with ApoE3ch or ApoE3 wild-type.

[0029] Figure 9 ELISA results showing heparin binding affinity for APOE2 and APOE4.

[0030] Figure 10 Immunoblot analysis showing the heparin binding affinity of ApoE2, ApoE3, ApoE4, and ApoE3ch.

[0031] Figure 11A and 11B ELISA results showing the heparin binding affinity of ApoE2, ApoE3, ApoE4, and ApoE3ch.

[0032] Figure 12A is a schematic diagram showing the experimental setup used to test the specificity of monoclonal ApoE3 antibodies in blocking ApoE3 / heparin binding. Figure 12B and 12C is a schematic diagram showing the process of passing an ApoE3 protein pre-incubated with a monoclonal antibody through a heparin-binding column, followed by washing and elution.

[0033] Figures 13A-13B Shown are the results of a BCA assay performed on various fractions from the heparin-bound column. Figure 13A The amounts of ApoE3 in various fractions in the absence of ApoE3 antibodies are shown. Figure 13B The amount of ApoE3 in each fraction in the presence of an ApoE3 antibody is shown.

[0034] Figure 14 Shown are immunoblotting results showing the amount of ApoE3 in various fractions pre-incubated with or without monoclonal ApoE3 antibodies.

[0035] Figures 15A-15G ELISA analysis of 19G10-2, 23B2, 2H79-1, 30E1-2, 16H8, 25F1-2, and 29G10-1 antibodies are shown, respectively.

[0036] Figure 16 Immunoblot analysis showing the heparin binding affinity of ApoE3 treated with wild-type ApoE3 peptide and ApoE3ch mutant peptide.

[0037] Figures 17A-17D Modeling showing the interaction of ApoE fragments with heparin. Figure 17A A model of the wild-type ApoE fragment containing amino acids 129-157 that interact with heparin is shown. Figure 17B A model of the ApoE R136S fragment containing amino acids 129-157 that interact with heparin is shown. Figure 17C A model of the wild-type ApoE fragment containing amino acids 114-144 that interact with heparin is shown. Figure 17D A model of the ApoE R136S fragment containing amino acids 114-144 that interact with heparin is shown.

[0038] Figures 18A-18B Heparin affinity chromatography and immunoblot analysis of antibody 1H4 are shown.

[0039] Figure 19 A-19D shows the ELISA results of 1H4-2 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), and ApoE3ch peptide (D).

[0040] Figure 20 Shown are the ELISA results of 1H4-2 serum tested with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, and ApoE3ch peptide.

[0041] Figure 21 Shown are representative ELISA profiles of serial dilutions of antibody 1H4 incubated with human recombinant ApoE3 or mouse recombinant ApoE3.

[0042] Figure 22 ELISA results of monoclonal 1H4 antibodies purified from cloned hybridomas are shown.

[0043] Figure 23A and 23B Heparin affinity chromatography and immunoblot analysis of antibody 7C11 are shown.

[0044] Figure 24 A-24D shows the ELISA results of 7C11-1 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), or ApoE3ch peptide (D).

[0045] Figure 25 Shown are ELISA results from testing 7C11-1 serum with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, or ApoE3ch peptide.

[0046] Figure 26 ELISA results of the monoclonal 7C11-1 antibody purified from the cloned hybridoma are shown.

[0047] Figure 27 Shown are the results of ELISA screening of the 19G10-2 antibody against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins.

[0048] Figure 28A and 28B Heparin affinity chromatography and immunoblot analysis of antibody 19G10-2 are shown.

[0049] Figure 29 Immunoblot of ApoE3 WT incubated with 19G10-2 serum antibody is shown.

[0050] Figure 30 Representative ELISA showing the difference in binding between serum and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3WT or ApoE3ch.

[0051] Figure 31 yes Figure 30 Enlarged view of the Y axis.

[0052] Figure 32 Shown are the ELISA results of the monoclonal 19G10-2 antibody purified from the cloned hybridoma.

[0053] Figure 33 Shown are the results of ELISA screening of the 25F1-2 antibody against the heparin-binding domains of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins.

[0054] Figure 34A and 34B Heparin affinity chromatography and immunoblot analysis of antibody 25F1-2 are shown.

[0055] Figure 35 Immunoblot of ApoE3 WT incubated with 25F1-2 monoclonal antibody is shown.

[0056] Figure 36 Representative ELISA showing the difference in binding between 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3WT or ApoE3ch.

[0057] Figure 37 yes Figure 36 A magnified view of the Y-axis.

[0058] Figure 38 ELISA results of the monoclonal 25F1-2 antibody purified from the cloned hybridoma are shown.

[0059] Figure 39 ELISA screening of 1343 antibodies against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown.

[0060] Figure 40 ELISA screening of 1343 antibodies against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown.

[0061] Figure 41A Shown are immunoblot analyses of ApoE in protein fractions eluted from the heparin column in the presence or absence of 1343 antibody. Figure 41B ELISA analysis of fractions is shown.

[0062] Figure 42A An exemplary experimental outline showing an intraocular model of inducible APOE-dependent Tau hyperphosphorylation. Figure 42B PHF tau in control retinas injected with PBS is shown. Figure 42C A retina injected with recombinant human APOE3 is shown.

[0063] Figures 43A-43I Shown are PHF tau in control retinas compared to retinas injected with mouse 1H4-2 antibody or humanized 1343Ah antibody.

[0064] Figure 44 Shown is a representative binding assay of 1H4 at increasing concentrations (nM) of ApoE3 protein on a Protein A biosensor. DETAILED DESCRIPTION

[0065] The present disclosure discloses that homozygosity for APOE3ch (having two copies of the APOE3 Christchurch (R136S) mutation) is associated with strong resistance to the clinical onset of Alzheimer's disease, and that the R136S mutation significantly reduces the ability of ApoE to bind to heparan sulfate proteoglycans (HSPG) / heparin. Therefore, the present disclosure relates to antibodies that bind to wild-type ApoE and / or ApoE isoforms (one or more) containing the R136S mutation (e.g., antibodies that block the interaction between ApoE and HSPG / GAG / heparin and / or reduce its binding). Fusion proteins comprising peptide fragments (e.g., HSPG / GAG / heparin binding domains) of wild-type and mutant ApoE containing the R136S mutation are also contemplated. These proteins can be administered by human cells expressing such compositions. The present disclosure also relates to small molecules that block the interaction between ApoE and HSPG / heparin, and methods for screening the same small molecules. Compositions and methods for editing the ApoE locus with a genome editing system are also provided. The antibodies, fusion proteins, small molecules and genome editing systems described herein can be used to treat or prevent cognitive decline and / or neurodegenerative diseases associated with dementia and / or mild cognitive impairment (MCI), such as Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease. The antibodies, fusion proteins, small molecules and genome editing systems described herein can also be used to treat or prevent neurodegenerative diseases, cerebrovascular conditions, brain injury, retinal degeneration, optic nerve degeneration or retinal damage.

[0066] Apolipoprotein E (ApoE)

[0067] Apolipoprotein E variants are the main genetic modifiers of AD, promoting susceptibility to late-onset AD. The APOE4 allele causes the cysteine ​​at position 112 to change to arginine and is associated with a 5-fold increase in AD risk in monoallelic carriers, reaching a 20-fold increase in homozygous carriers. APOE2 causes an amino acid change from arginine to cysteine ​​at position 158 and has a protective effect on AD, while the APOE3 allele is considered neutral (Corder et al., Nat Genet (7) 180-184, 1994; Hauser et al., Cure Alzheimer Res (10); 808-817, 2013). APOE affects the production, aggregation and clearance of amyloid proteins, is a component of amyloid plaques, and worsens tau-mediated neurodegenerative diseases. APOE alleles also regulate lipid metabolism and cardiovascular risk. Approximately 5-10% of individuals homozygous for APOE2 develop hyperlipoproteinemia type III (HLP III), while other rare APOE variants are associated with autosomal dominant HLP III. HLP III is characterized by increased plasma cholesterol and triglyceride levels and the presence of nodular or rhabdomyomatous palmaris xanthomas. The mechanisms by which APOE alleles modify AD risk and lead to HLP III are not fully understood. Notable APOE properties affected by specific mutations include differences in: 1) binding affinity for lipids and the LDL receptor; 2) the nature of the interdomain interactions between its N-terminus (amino acids 1 to 199) and C-terminal domains (216 to 299); and, 3) the ability to form homo-oligomers mediated by the C-terminal domain (Frieden et al., PNAS (109): 8913-8918, 2012; Georgiadou et al., PLoS One (6) e27037, 2011; Lalazar et al., J Biol Chem (263) 3542-3545, 1988).

[0068] Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It exists in the form of proteoglycans (HSPGs), in which two or three HS chains are attached to cell surface or extracellular matrix proteins in close proximity. HSPGs are found in hundreds of proteins located in the plasma membrane and extracellular matrix. Protein-protein interactions mediated by HSPGs play a key role in numerous processes related to the pathology of Alzheimer's disease, including amyloid and tau pathology. Heparan sulfate is a member of the glycosaminoglycan family of carbohydrates and is closely related to heparin in structure. Both consist of variably sulfated repeating disaccharide units. Heparan sulfate binds to a large number of extracellular proteins. These are generally referred to as the "heparin interactome" or "heparin-binding proteins" because they are separated by affinity chromatography of the related polysaccharide heparin.

[0069] An exemplary amino acid sequence of human ApoE3 protein (Uniprot Accession No. P02649) is shown below:

[0070]

[0071] At least two HSPG / heparin binding domains have been identified in human ApoE, one located in the N-terminal domain and one located in the C-terminal domain (Weisgraber et al. J Biol Chem, 261(5):2068-76, 1986; Saito et al. J Biol Chem, 278(17):14782-7, 2003). The HSPG / heparin binding domain near arginine 136 (R136) (N-terminal HSPG / heparin binding domain) is functional in full-length lipidated and defatted ApoE, while the HSPG / heparin binding domain in the C-terminal domain is functional only in the absence of the N-terminal domain and in defatted ApoE. The N-terminal HSPG / heparin binding domain is well characterized and comprises amino acid residues 142 to 147 (bold) of SEQ ID NO: 1. The C-terminal HSPG / heparin binding domain is less well characterized and comprises a lysine (K) at position 233 and other charged amino acids nearby, including amino acid residues 211 to 218 and 243 to 272 of SEQ ID NO: 1. The inventors have shown that the arginine at position 136 of ApoE plays a key role in the heparin binding of ApoE. Without wishing to be bound by theory, the underlying mechanism is allosteric regulation of heparin binding mediated by the arginine at position 136. As used herein, allosteric regulation is related to the regulation of ligand binding by an allosteric modulator at one or more allosteric regulatory sites, which are different from the binding site(s) of the ligand. In some embodiments, the one or more allosteric regulatory sites for ApoE and HSPG / heparin binding comprise the arginine at position 136 of ApoE as shown in SEQ ID NO: 1. "HSPG / heparin binding domain(s)", "HSPG / heparin binding site(s)", "HSPG-binding domain(s)" and "HSPG-binding site(s)" are used interchangeably herein.

[0072] anti-ApoE antibodies

[0073] Anti-ApoE antibodies that bind to wild-type or mutant ApoE proteins (e.g., human ApoE proteins) are provided. In some cases, the antibodies described herein bind to wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4), but do not bind to mutant ApoE proteins (e.g., ApoEch). In some cases, the antibodies described herein bind to mutant ApoE proteins (e.g., ApoEch), but do not bind to wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4). In some cases, the antibodies described herein bind to mutant ApoE proteins (e.g., ApoEch) and wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4).

[0074] In some cases, provided herein are antibodies that block the interaction between wild-type ApoE protein (e.g., ApoE2, ApoE3 or ApoE4) and HSPG. Provided herein are antibodies that can reduce or regulate the binding affinity of ApoE protein (e.g., ApoE2, ApoE3 or ApoE4) to HSPG. In some cases, provided herein are antibodies that are bound to the HSPG binding domain of wild-type ApoE protein. In some cases, provided herein are antibodies that are bound to the allosteric regulatory site (e.g., amino acid position 136 of ApoE) that one or more HSPG / ApoE are combined. In some cases, antibodies as described herein reduce fibril formation and / or amyloid oligomerization.

[0075] In some cases, the antibodies provided herein bind to an amino acid sequence in wild-type or mutant ApoE comprising or consisting of TEELRVSLASHLRK (SEQ ID NO: 2). In some cases, the antibodies provided herein bind to an amino acid sequence in wild-type or mutant ApoE comprising or consisting of TEELRVRLASHLRK (SEQ ID NO: 3). In some cases, the amino acid sequence TEELRVSLASHLRK (SEQ ID NO: 2) comprises or consists of an epitope for an antibody provided herein. In some cases, the amino acid sequence TEELRVRLASHLRK (SEQ ID NO: 3) comprises or consists of an epitope for an antibody provided herein. Variants of these sequences can also be used, for example, those that are at least 80%, 85%, 90% or 95% identical to these sequences.

[0076] The calculation of " identity " between two sequences can be carried out as follows. For the purpose of best comparison, the sequences are aligned (for example, for the purpose of best comparison, a room can be introduced in one or both of the first and second nucleic acid sequences, and different sequences can be ignored for the purpose of comparison). The length of the sequence aligned for the purpose of comparison is at least 70% (for example, at least 80%, 90% or 100%) of the reference sequence length. The nucleotides at the corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the nucleotides identical to the corresponding position in the second sequence, the molecules at this position are identical. The percentage identity between the two sequences is a function of the number of identical positions that the sequences share, and considering the number of rooms and the length of each room, it is necessary to introduce these rooms to realize the best comparison of the two sequences.

[0077] The comparison of sequence and the determination of the percent identity between two sequences can be completed using a mathematical algorithm. In some embodiments, the percent identity between two nucleotide sequences is determined using Needleman and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm incorporated into the GAP program in the GCG software package (available at gcg.com), using Blossum 62 matrix, PAM250 matrix, NWSgapdna.CMP matrix. In some embodiments, the percent identity between two amino acid or nucleotide sequences can be determined using E.Meyers and W.Miller ((1989) CABIOS, 4:11-17) algorithm incorporated into the ALIGN program (version 2.0), using PAM120 weight residual table, gap length penalty of 12 and gap penalty of 4.

[0078] The use of the term "antibody" in this disclosure is intended to encompass whole antibodies (as opposed to minibodies, nanobodies, or antibody fragments), bispecific antibodies, tetravalent antibodies, multispecific antibodies, minibodies, nanobodies, and antibody fragments. In some cases, the anti-ApoE antibodies of the present disclosure are whole antibodies. In some cases, the anti-ApoE antibodies of the present disclosure are chimeric antibodies, human antibodies, or humanized antibodies. In certain cases, the heavy chain constant region of the anti-ApoE antibody is a human IgG1, human IgG2, human IgG3, or human IgG4 constant region. In certain cases, the light chain constant region is a human kappa constant region. In other cases, the light chain constant region is a human lambda constant region. In some cases, the antibodies of the present disclosure are designed to have low effector functionality (e.g., through Fc modifications such as N297Q, T299A, etc. See also, Wang, X., Mathieu, M. & Brezski, RJ Protein Cell (2018) 9:63.doi.org / 10.1007 / s13238-017-0473-8 (incorporated herein by reference)). In some cases, the Fc portion of the antibody is a hIgG1 Fc, a hIgG2 Fc, a hIgG3 Fc, a hIgG4 Fc, a hIgG1agly Fc, a hIgG2 SAAFc, a hIgG4(S228P)Fc, or a hIgG4(S228P) / G1 agly Fc (in this format - to minimize effector function - the CH1 and CH2 domains are IgG4 with a "fixed" hinge (S228P) and are aglycosylated. The CH3 domain is hIgG1, or hIgG4(S228P)agly Fc). In one instance, the antibody has one of three scaffolds with reduced effector function: hIgG1 agly(N297Q); hIgG2 SAA (see, Vafa et al. Methods, 65(1): 114-26 (2014); and hIgG4P / G1 agly (see, US 2012 / 0100140A1).

[0079] In some embodiments, the antibodies or ApoE-binding fragments thereof described herein exhibit the binding characteristics and / or biological properties outlined for antibodies 1H4-2, 7C11-1, 19G10-2, 23B2(1343), 2H79-1, 30E1-2, 16H8, 25F1-2, and 29G10-1 as described in the Examples section below.

[0080] In some embodiments, the present disclosure provides antibodies that bind to wild-type human ApoE or a portion thereof and have one or more of the following properties: (i) bind to wild-type human ApoE with high affinity with KD ≤ 20 nM; (ii) compete with wild-type human ApoE for binding to heparin; and (iii) reduce the formation of paired helical fibrils (PHF) Tau in retinal cells.

[0081] In some embodiments, the present disclosure provides antibodies that bind to mutant human ApoE (e.g., those having a mutation at amino acid position 136 of human ApoE, such as ApoEch) or a portion thereof, and have one or more of the following properties: (i) binds to mutant human ApoE (e.g., a mutation at amino acid position 136 of human ApoE, such as ApoEch) with a high affinity of KD ≤ 20 nM; (ii) competes with wild-type human ApoE for binding to heparin; (iii) reduces the formation of paired helical fibrils (PHF) Tau in retinal cells.

[0082] Any of the anti-ApoE antibodies described herein can be used to treat or prevent a condition associated with dementia or mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, or Huntington's disease), a neurodegenerative disease, a cerebrovascular disease, brain injury, retinal degeneration, or retinal injury.

[0083] Exemplary Antibody 1H4-2

[0084] Antibody 1H4-2 was generated by immunization with APOE:KLH-CTEELRVRLASHLRK-CONH2. The amino acid sequences of the complementarity determining regions (CDRs) and heavy and light chain variable regions of 1H4-2 are provided below.

[0085]

[0086] Variable light chain:

[0087] Nucleotide sequence:

[0088] Signal sequence -FR1- -FR2- -FR3- -FR4

[0089]

[0090] Amino acid sequence:

[0091] Signal peptide-FR1- -FR2- -FR3- -FR4

[0092]

[0093] Variable heavy chain:

[0094] Nucleotide sequence

[0095] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0096]

[0097] Amino acid sequence:

[0098] Signal peptide-FR1- -FR2- -FR3- -FR4

[0099]

[0100] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 1H4-2 and a VL comprising three VL CDRs of antibody 1H4-2. The six CDRs can be based on any definition known in the art, such as, but not limited to, the Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0101] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.

[0102] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 13 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0103] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO: 13 and a VL with the amino acid sequence set forth in SEQ ID NO: 12.

[0104] Exemplary antibody 7C11-1

[0105] Antibody 7C11-1 was generated by immunization with APOE:KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54). The amino acid sequences of the complementarity determining regions (CDRs) and heavy and light chain variable regions of 7C11-1 are provided below.

[0106]

[0107] Variable heavy chain:

[0108] Nucleotide sequence

[0109] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0110]

[0111] Amino acid sequence:

[0112] Signal peptide-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0113]

[0114] Variable light chain:

[0115] Nucleotide sequence:

[0116] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0117]

[0118] Amino acid sequence:

[0119] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0120]

[0121] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 7C11-1 and a VL comprising three VL CDRs of antibody 7C11-1. The six CDRs can be based on any definition known in the art, such as, but not limited to, the Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0122] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0123] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 23. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 22. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 23 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 23 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0124] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:23 and a VL with the amino acid sequence set forth in SEQ ID NO:22.

[0125] Exemplary antibody 19G10-2

[0126] Antibody 19G10-2 was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2. The following text provides the amino acid sequences of the complementarity determining regions (CDRs) and the heavy and light chain variable regions of 19G10-2.

[0127]

[0128] Variable light chain:

[0129] Nucleotide sequence:

[0130] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0131]

[0132] Amino acid sequence:

[0133] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0134]

[0135] Variable heavy chain analysis:

[0136] Nucleotide sequence

[0137] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0138]

[0139] Amino acid sequence:

[0140] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0141]

[0142] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 19G10-2 and a VL comprising three VL CDRs of antibody 19G10-2. The six CDRs can be based on any definition known in the art, such as, but not limited to, the Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0143] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0144] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 33. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 32. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 32. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 32. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 32.

[0145] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:33 and a VL with the amino acid sequence set forth in SEQ ID NO:32.

[0146] Exemplary Antibody 25F1-2

[0147] Antibody 25F1-2 was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2. The amino acid sequences of the complementarity determining regions (CDRs) and heavy and light chain variable regions of 25F1-2 are provided below.

[0148]

[0149] Variable light chain analysis:

[0150] Nucleotide sequence:

[0151] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0152]

[0153] Amino acid sequence:

[0154] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0155]

[0156] Variable heavy chain analysis:

[0157] Nucleotide sequence

[0158] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0159]

[0160] Amino acid sequence:

[0161] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0162]

[0163] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 25F1-2 and a VL comprising three VL CDRs of antibody 25F1-2. The six CDRs can be based on any definition known in the art, such as, but not limited to, the Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0164] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0165] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 43. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 42. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 43 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 42. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 43 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 42. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 43 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 42.

[0166] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:43 and a VL with the amino acid sequence set forth in SEQ ID NO:42.

[0167] Exemplary antibody 1343ab

[0168] Antibody 1343ab was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2. The amino acid sequences of the complementarity determining regions (CDRs) and full-length heavy and light chains are provided below.

[0169]

[0170] Full-length heavy chain

[0171]

[0172] Full-length light chain

[0173]

[0174] EC

[0175] N-linked glycosylation was detected on the heavy chain constant region N at position 292. Loss of the C-terminal lysine was observed on the heavy chain.

[0176] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 1343ab and a VL comprising three VL CDRs of antibody 1343ab. The six CDRs can be based on any definition known in the art, such as, but not limited to, the Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0177] In one instance, the anti-ApoE antibody of the present disclosure comprises: (i) a VL comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 48, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46.

[0178] In some cases, the anti-ApoE antibody comprises a heavy chain that is at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 53. In some cases, the anti-ApoE antibody comprises a light chain that is at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 52. In one instance, the anti-ApoE antibody comprises a heavy chain that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 52. In another instance, the anti-ApoE antibody comprises a heavy chain that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 52. In still another instance, the anti-ApoE antibody comprises a heavy chain that is identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain that is identical to the amino acid sequence set forth in SEQ ID NO: 52.

[0179] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a heavy chain having the amino acid sequence set forth in SEQ ID NO:53 and a light chain having the amino acid sequence set forth in SEQ ID NO:52.

[0180] Chimeric, human, or humanized antibodies having the CDR sequences of any of the above antibodies can be produced based on the methods described herein.

[0181] Antibody fragments

[0182] Antibody fragments (e.g., Fab, Fab', F(ab')2, Facb, and Fv) can be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating whole antibodies with enzymes such as papain, pepsin, or plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies produces F(ab')2 or Fab'; and plasmin digestion of whole antibodies produces Facb fragments.

[0183] Alternatively, antibody fragments can be produced recombinantly. For example, a nucleic acid encoding an antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. See, e.g., Co, MS et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, AH, Methods in Enzymology, 178:476-496 (1989); Pluckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymology, (1989) 121:663-669 (1989); and Bird, RE et al., TIBTECH, 9:132-137 (1991)). Antibody fragments can be expressed in Escherichia coli and secreted therefrom, thereby allowing to easily produce a large amount of these fragments. Antibody fragments can be separated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F (ab) 2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another approach, F (ab') 2 fragments can be directly isolated from recombinant host cell cultures. Fab and F (ab') 2 fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent number 5,869,046.

[0184] Conjugated antibodies

[0185] The antibodies disclosed herein may be conjugated antibodies bound to various molecules, including macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive substances (e.g., 90 Y. 131 I), fluorescent substances, luminescent substances, haptens, enzymes, metal chelates and drugs.

[0186] In some embodiments, the antibodies described herein are modified by moieties that improve their stability and / or retention in the circulation, such as in blood, serum, or other tissues, including the brain, for example, by at least 1.5, 2, 5, 10, 15, 20, 25, 30, 40, or 50 times. For example, the antibodies described herein can be combined with (e.g., conjugated to) polymers, such as substantially non-antigenic polymers, such as polyalkylene oxide or polyethylene oxide. Suitable polymers will vary significantly in weight. Polymers with a number average molecular weight of about 200 to about 35,000 daltons (or about 1,000 to about 15,000 and 2,000 to about 12,500) can be used. For example, the antibodies described herein can be conjugated to water-soluble polymers, such as hydrophilic polyethylene polymers, such as polyvinyl alcohol or polyvinyl pyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylated polyols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymer is maintained. Other useful polymers include polyoxyalkylenes, such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides. In some embodiments, the antibodies described herein are modified with moieties that improve their penetration of the blood-brain barrier (e.g., those described in Pardridge, J Cereb Blood Flow Metab 32(11):1959-1972, 2012). Exemplary blood-brain barrier penetrating moieties include, but are not limited to, glucose transporter type 1 (GLUT1), cationic amino acid transporter type 1 (CAT1), monocarboxylate transporter type 1 (MCT1), concentrating nucleoside transporter type 2 (CNT2), active efflux transporters (AETs) (e.g., p-glycoprotein, and those described in Pardridge, J Cereb Blood Flow Metab 32(11):1959-1972, 2012), and additional blood-brain barrier penetrating moieties are known in the art.

[0187] The conjugated antibodies can be prepared by chemically modifying the antibodies described herein or their low molecular weight forms. Methods for modifying antibodies are well known in the art (eg, US 5,057,313 and US 5,156,840).

[0188] Anti-ApoE antibodies can be in the form of full-length (or whole) antibodies, or low molecular weight forms of anti-ApoE antibodies (e.g., biologically active antibody fragments or minibodies), such as Fab, Fab', F(ab')2, Fv, Fd, dAb, scFv, and sc(Fv)2. Other anti-ApoE antibodies encompassed by the present disclosure include single domain antibodies (sdAbs) containing a single variable chain, such as VH or VL, or biologically active fragments thereof. See, for example, Moller et al., J. Biol. Chem., 285(49):38348-38361 (2010); Harmsen et al., Appl. Microbiol. Biotechnol., 77(1):13-22 (2007); US 2005 / 0079574 and Davies et al. (1996) Protein Eng., 9(6):531-7. Like whole antibodies, sdAbs can selectively bind to specific antigens (e.g., ApoE2, ApoE3, ApoE4, or ApoEch). With a molecular weight of only 12-15 kDa, sdAbs are much smaller than ordinary antibodies, even smaller than Fab fragments and single-chain variable fragments.

[0189] In certain embodiments, the anti-ApoE antibody, or its antigen-binding fragment, or its low molecular weight antibody specifically binds to the HSPG / heparin binding domain of ApoE and reduces the severity of symptoms when administered to a human patient or animal model suffering from one or more of the following diseases: dementia and / or mild cognitive impairment (MCI) (e.g., those associated with Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Huntington's disease, or neurodegenerative diseases). In certain embodiments, the anti-ApoE antibody, or its antigen-binding fragment, or its low molecular weight antibody specifically binds to the HSPG / heparin binding domain of ApoE and reduces the severity of symptoms when administered to a human patient or animal model suffering from one or more of the following diseases: neurodegenerative diseases, cerebrovascular diseases (e.g., stroke, carotid stenosis, spinal stenosis, or aneurysm), brain injury (e.g., traumatic brain injury, acquired brain injury), retinal degeneration, glaucoma, or retinal damage. These characteristics of the anti-ApoE antibody or its low molecular weight antibody can be measured according to methods known in the art.

[0190] Nucleic acids, vectors, host cells

[0191] The present disclosure also features nucleic acids encoding the antibodies disclosed herein. Provided herein are nucleic acids encoding the VH CDR1, VH CDR2, and VH CDR3 of the anti-ApoE antibodies described herein. Also provided are nucleic acids encoding the VL CDR1, VL CDR2, and VL CDR3 of the anti-ApoE antibodies described herein. Provided herein are nucleic acids encoding the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the anti-ApoE antibodies described herein. Also provided are nucleic acids encoding the heavy chain variable region (VH) of the anti-ApoE antibodies described herein, and / or nucleic acids encoding the light chain variable region (VL) of the anti-ApoE antibodies described herein. In some cases, provided herein are nucleic acids encoding the VH and / or VL of the anti-ApoE antibodies described herein, linked to a human heavy chain constant region and / or a human light chain constant region, respectively. Also provided herein are nucleic acids encoding both the VH and VL of the anti-ApoE antibodies described herein. In some cases, the nucleic acids described herein include nucleic acids encoding the Fc region of a human antibody (e.g., human IgG1, IgG2, IgG3, or IgG4). In certain cases, the nucleic acids include nucleic acids encoding the Fc region of a human antibody that has been modified to reduce or eliminate effector function (e.g., N297Q or T299A substitutions in the human IgG1 Fc region (numbered according to EU numbering)). In some cases, the nucleic acids include nucleic acids encoding an Fc portion that is hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1agly Fc, hIgG2 SAA Fc, hIgG4(S228P)Fc, or hIgG4(S228P) / G1agly Fc.

[0192] Also disclosed herein are vectors (eg, expression vectors) comprising any of the above-described nucleic acids.

[0193] Furthermore, the present disclosure relates to host cells (eg, bacterial cells, yeast cells, insect cells, or mammalian cells) containing the above-described vector(s) or nucleic acid(s).

[0194] Methods for obtaining anti-ApoE antibodies

[0195] Also provided herein is a method for preparing an anti-ApoE antibody for use in the present method. General methods for preparing antibodies such as monospecific, polyclonal or monoclonal antibodies are known in the art. For monoclonal antibodies, the process involves obtaining antibody-secreting immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) that has been previously immunized in vivo or in vitro with an antigen of interest (e.g., a peptide antigen as described herein). The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells that can replicate indefinitely in cell culture to produce immortal, immunoglobulin-secreting cell lines. The resulting fused cells or hybridomas are cultured and the resulting colonies are screened to produce the desired monoclonal antibodies. Colonies that produce such antibodies are cloned and grown in vivo or in vitro to produce large amounts of antibodies. Kohler and Milstein, Nature 256:495 (1975) describe the theoretical basis and practical methods for fusing such cells.

[0196] Mammalian lymphocytes can be immunized by immunizing an animal (e.g., a mouse) in vivo with a peptide antigen, such as a peptide antigen that is at least 80%, 85%, 90%, or 95% identical to KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) or KLH-CTEELRVSLASHLRK-CONH2 (SEQ ID NO: 55), optionally with one or more substitutions or deletions, for example, of up to 20% of the residues. For example, the method can include immunizing an animal with a peptide comprising a sequence that is at least 80% identical to at least 10 consecutive amino acids from the heparin binding domain of APOE, such as a peptide comprising TEELRVRLASHLRK (SEQ ID NO: 3) or TEELRVSLASHLRK (SEQ ID NO: 2). Such immunizations are repeated at intervals of up to several weeks as needed to obtain sufficient antibody titers. After the last antigen boost, the animal is sacrificed and spleen cells are removed.

[0197] Fusion with mammalian myeloma cells or other fusion partners capable of indefinite replication in cell culture is achieved by known techniques, such as the use of polyethylene glycol ("PEG") or other fusogenic agents (see Milstein and Kohler, Eur. J. Immunol. 6:511 (1976), incorporated herein by reference). Immortalized cell lines are selected that lack enzymes necessary for the utilization of certain nutrients, are capable of rapid growth, and have good fusion capacity, preferably murine, but may also be derived from other mammalian species, including, but not limited to, rat and human. Many such cell lines are known to those skilled in the art, and others are regularly described.

[0198] The procedure for producing polyclonal antibodies is also known. Generally, such antibodies can be produced by subcutaneously administering the protein or polypeptide of the present invention to New Zealand white rabbits, which are first bled to obtain pre-immune serum. For example, the antigen can be injected at six different sites with a total volume of 100 μl per site. Each injection material will contain a synthetic surfactant adjuvant polyether polyol (pluronic polyol), or a crushed acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. Then, the rabbits are bled two weeks after the first injection and regularly reinforced three times with the same antigen every six weeks. Serum samples are then collected 10 days after each reinforcement. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Finally, the rabbits are euthanized with pentobarbital 150 mg / Kg IV. This process and other processes for other polyclonal antibody production are disclosed in E. Harlow, et al., ed., Antibodies: A Laboratory Manual (1988).

[0199] The methods described herein include any one or more steps of producing chimeric antibodies, humanized antibodies, single-chain antibodies, Fab fragments, bispecific antibodies, fusion antibodies, labeled antibodies, or analogs thereof. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor (1988). When derivatives of the antibodies are obtained by phage display technology, surface plasmon resonance employed in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to the same epitope as any of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). For example, the production of chimeric antibodies is described in International Application WO 89 / 09622. Methods for producing humanized antibodies are described, for example, in European application EP-A1 0 239 400 and international application WO 90 / 07861. Other sources of antibodies for use according to the present invention are so-called xenoantibodies. The general principles for producing xenoantibodies, such as human-like antibodies, in mice are described, for example, in international applications WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, and WO 96 / 33735. As described above, the antibodies described herein can exist in a variety of forms in addition to complete antibodies; including, for example, Fv, Fab, and F(ab)2, as well as single chains; see, for example, international application WO 88 / 09344.

[0200] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display technology, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and, for example, those taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd edition. (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, NY, 563-681 (1981), which are incorporated by reference in their entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone including any eukaryotic, prokaryotic or phage clone, rather than a method for producing it. Therefore, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology.

[0201] In the known hybridoma method (Kohler et al., Nature 256 (1975), 495), relatively short-lived or non-immortal lymphocytes from mammals, such as B cells derived from a mouse subject as described herein, are fused with an immortal tumor cell line (e.g., a myeloma cell line) to produce hybrid cells or "hybridomas" that are immortal and capable of producing antibodies encoded by the genes of the B cells. By selection, dilution, and regrowth, the resulting hybrids are separated into individual genetic strains, each containing specific genes for forming a single antibody. They produce antibodies that are homogeneous against the desired antigen and are referred to as "monoclonals" based on their pure genetic lineage.

[0202] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines, and culture media for the formation, selection, and growth of hybridomas are commercially available from many sources and that standardized protocols are well established. Typically, the culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA) as described herein. After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by a limiting dilution process and grown by standard methods; see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp 59-103 (1986). It will further be appreciated that the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0203] In another embodiment, lymphocytes can be selected and variable genes can be separated by micromanipulation. For example, peripheral blood mononuclear cells can be isolated from immune or naturally immune mammals, such as humans, and cultured in vitro for about 7 days. The culture can be screened for specific immunoglobulins that meet the screening criteria. Cells from positive wells can be separated. Single B cells that produce Ig can be separated by FACS or identified in complement-mediated hemolytic plaque assays. B cells that produce Ig can be micromanipulated into tubes, and VH and VL genes can be amplified using, for example, RT-PCR. VH and VL genes can be cloned into antibody expression vectors and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.

[0204] Alternatively, cell lines producing antibodies can be selected and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, techniques suitable for the invention as described below are described in Current Protocols in Immunology, Coligan et al., eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including supplements.

[0205] Antibodies such as those described above can be prepared, for example, by preparing and expressing a synthetic gene encoding the amino acid sequence. Methods for producing variants of any anti-ApoE antibody (e.g., comprising amino acid substitutions) are well known in the art. These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA molecules prepared by encoding the antibody or any portion thereof (e.g., framework region, CDR, constant region). Site-directed mutagenesis is well known in the art (see, e.g., Carter et al., Nucl. Acids Res., 13: 4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82: 488 (1987)). PCR mutagenesis is also applicable to the preparation of amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nucl. Acids Res. 17:723-733 (1989). Another method for preparing sequence variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34:315-323 (1985).

[0206] Antibodies can be produced in bacterial cells or eukaryotic cells. Some antibodies, such as Fab, can be produced in bacterial cells such as Escherichia coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (such as CHO, 293E, COS, Hela). In addition, antibodies (such as scFv) can be expressed in yeast cells such as Pichia pastoris (Pichia) (see, for example, Powers et al., J Immunol Methods.251:123-35 (2001)), Hansenula or Saccharomyces cerevisiae (Saccharomyces). In order to produce an antibody of interest or its antigen-binding fragment, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells and recover antibodies.

[0207] If the antibody is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have features that allow the vector to be amplified in the bacterial cell. In addition, when using E. coli such as JM109, DH5α, HB101 or XL1-Blue as a host, the vector must have a promoter, such as the lacZ promoter (Ward et al., 341:544-546 (1989), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or a T7 promoter that allows efficient expression in E. coli. Examples of such vectors include, for example, M13 series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP and pET (when using this expression vector, the host is preferably a T7 promoter that expresses the T7 promoter). The expression vector may contain a signal sequence for antibody secretion. For production in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) can be used as the signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation.

[0208] If the antibody is to be expressed in animal cells such as CHO, COS and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)) or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or its domain, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in the host cell (e.g., origin of replication) and a selective marker gene. Selective marker genes facilitate selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically, the selective marker gene confers resistance to drugs such as G418, hygromycin or methotrexate to the host cell into which the vector has been introduced. Examples of vectors having a selectable marker include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0209] In one embodiment, the antibody is produced in mammalian cells. Exemplary mammalian host cells for expressing antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr - CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, are used with the DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines, e.g., NSO myeloma cells and SP2 cells, and cells from transgenic animals, e.g., transgenic mammals.

[0210] Antibodies disclosed herein can be separated and purified as substantially pure and homogeneous antibodies from the inside or outside (e.g., culture medium) of a host cell. Separation and purification methods commonly used for antibody purification can be used for the separation and purification of antibodies, and are not limited to any specific method. Antibodies can be separated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be carried out using liquid chromatography such as HPLC and FPLC. The column used for affinity chromatography includes protein A column and protein G column. The example of the column using protein A column includes Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present invention also includes antibodies that have been highly purified using these purification methods.

[0211] Characteristics of antibodies

[0212] The ApoE binding properties of the antibodies described herein can be determined by any standard method, such as one or more of the following methods: Surface plasmon resonance (SPR), BIACORE TManalysis, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and fluorescence resonance energy transfer (FRET).

[0213] Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63: 2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705 and online resources provided by BIAcore International AB. Information from SPR can be used to provide equilibrium dissociation constants (Kd) and K values ​​for binding of a biomolecule to a target. on and K off Accurate and quantitative determination of kinetic parameters.

[0214] The ability of different antibodies to compete with each other for binding to wild-type ApoE or spiked ApoE (e.g., ApoE c h ) can also be evaluated by using BIACORE chromatography techniques (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160: 191-198).

[0215] When using an enzyme immunoassay, a sample containing an antibody, such as culture supernatant of antibody-producing cells or purified antibody, is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, and the plate is incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added, and the absorbance is measured to assess antigen-binding activity.

[0216] Additional general guidance for evaluating antibodies, e.g., immunoblotting and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor press (1988).

[0217] Mutant ApoE proteins, peptides, and fusion proteins thereof

[0218] The present disclosure provides mutant ApoE proteins or fragments thereof containing amino acid substitutions at one or more positions in the HSPG binding domain compared to wild-type ApoE proteins. In some embodiments, mutant ApoE proteins or fragments thereof include amino acids other than arginine at position 136. In some embodiments, mutant ApoE proteins or fragments thereof include serine, histidine or cysteine ​​at position 136. Also provided are nucleic acid (e.g., DNA or RNA) sequences encoding mutant ApoE proteins or fragments thereof, and vectors comprising the nucleic acid sequences. Mutant ApoE proteins or fragments thereof, nucleic acids encoding such proteins or fragments, and vectors comprising nucleic acid sequences can be used to treat or prevent conditions (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease) associated with dementia or mild cognitive impairment (MCI), neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage.

[0219] In some embodiments, the mutant ApoE protein is an ApoEch protein (e.g., ApoE2ch, ApoE3ch, or ApoE4ch protein). Fragments of the ApoEch protein including amino acid position 136 are also contemplated herein. An exemplary sequence of the full-length ApoE3ch protein is shown below. The mutation from arginine to serine is indicated in bold and double underlined.

[0220]

[0221] In some embodiments, the methods disclosed herein allow mutant ApoE proteins or fragments thereof to cross the blood-brain barrier. Mutant ApoE proteins or fragments thereof can be delivered using nanocarriers, including but not limited to polymer nanoparticles, lipid-based nanoparticles, liposomes, micelles, dendrimers, human cells expressing proteins, and nanotubes (see, Dominguez et al. J Nanosci nanotechnol. 14(1):766-79, 2014). In some embodiments, the mutant ApoE protein or fragment thereof is delivered intracarotidally or transmucosally (e.g., intracarotid infusion of a hypertonic solution (arabinose or mannitol); see, Sanchez-Covarrubias et al., Curr Pharm Des. 20(10):1422-49, 2014 and Miyake et al., World J Otorhinolaryngol Head Neck Surg. 1(1):11-16, 2015), or intranasally using chlorotoxin (see, McCall et al., Tissue Barriers 2(4):e944449, 2014). Cryogenic techniques, receptor-mediated transport, cell-penetrating peptides, and cell-mediated delivery can also be used to facilitate passage of the ApoE3ch protein across the blood-brain barrier (see, Pandey et al., Tissue Barriers 4(1):e1129476, 2016). For example, immune cells and stem cells (e.g., neural stem cells, induced pluripotent cells, and mesenchymal stem cells) can be used to carry therapeutic loads across the BBB. Mesenchymal stem cells loaded with nanoparticles can be used for this purpose (see, for example, Roger et al. Biomaterials 31:8393-401, 2010). Genetically modified stem cells (e.g., genetically modified mesenchymal stem cells) can also be used (see, for example, Ebrahimi and Lalvand Hygeia. JDMed. Vol. 5 (1): 90-104, 2013). Chemical drug delivery systems (CDDS) can also be used, such as those described in He et al., Cells, 7 (4): 24, 2018. Other methods of transporting proteins across the blood-brain barrier are known in the art.

[0222] Nucleic acids (e.g., DNA or mRNA) encoding mutant ApoE proteins (e.g., any mutant ApoE protein described herein, such as ApoEch) or fragments thereof are contemplated herein. In some embodiments, the mRNA encoding the ApoEch protein can be modified to increase stability (e.g., those described in Zangi et al., Nat Biotechnol. 31(10):898-907, 2013 and developed by Moderna, Inc.; and those described in Alberer et al., Lancet 390(10101):1511-1520, 2017 and developed by Curevac and BioNTech).

[0223] Viral vectors containing DNA sequences encoding mutant ApoE or fragments thereof are contemplated herein. An exemplary cDNA sequence encoding the full-length ApoE3ch protein (including the signal peptide region) is shown below. Mutations from cytosine to adenine are indicated in bold and double underlined.

[0224]

[0225] Suitable vectors are known in the art. In some embodiments, the viral vector is an AAV vector (such as those described in Rosenberg et al., Hum Gene Ther Clin Dev 29 (1): 24-47, 2018). Also included is a cDNA sequence encoding an ApoE protein containing a mutation at the R136 position in addition to R136S. In some embodiments, the mutation is R136H or R136C.

[0226] Peptides and fusion proteins

[0227] In some embodiments, provided herein is a HSPG / heparin binding domain comprising wild-type or mutant ApoE (e.g., any mutant ApoE protein as described herein) or a peptide consisting thereof. In some cases, provided herein is a peptide amino acid sequence comprising a sequence selected from the group consisting of or consisting of: STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59) and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60). Variants having a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more with these sequences can also be used. Fusion proteins comprising the peptide provided above are also disclosed. In some embodiments, the fusion protein further comprises an Fc region of a human antibody (e.g., human IgG1, IgG2, IgG3, or IgG4). In some cases, the fusion protein comprises an Fc region of a human antibody at the C-terminus of the HSPG / heparin binding domain of wild-type or mutant ApoE. In some cases, the fusion protein comprises an Fc region of a human antibody at the N-terminus of the HSPG / heparin binding domain of wild-type or mutant ApoE.

[0228] In some cases, provided herein is a peptide and fusion protein that competes with wild-type ApoE protein for binding to HSPG / heparin. In some cases, provided herein is a peptide and fusion protein that reduces or regulates the combination between wild-type ApoE protein and HSPG / heparin. In certain embodiments, provided herein is a peptide and fusion protein that inhibits and / or reduces the HSPG / heparin combination of wild-type ApoE protein, and reduces the severity of symptoms when administered to one or more of the following human patients or following animal models: conditions (such as Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Huntington's disease) related to dementia or mild cognitive impairment (MCI), neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage. These features of peptide and fusion protein provided herein can be measured according to methods known in the art.

[0229] Also provided herein are anti-ApoE vaccines that can be used to elicit a protective immune response against ApoE. In some embodiments, the anti-ApoE vaccine comprises one or more ApoE peptides provided herein (e.g., and a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable adjuvants are known in the art.

[0230] Pharmaceutical compositions and methods of administration

[0231] The methods described herein include the use of pharmaceutical compositions comprising as an active ingredient any of the antibodies, peptides, or fusion proteins described herein.

[0232] Pharmaceutical compositions generally include a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration.

[0233] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g., intracranial, intranasal, intracarotid, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), and transmucosal.

[0234] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tonicity, such as sodium chloride or glucose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0235] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be present in a fluid that is easy to inject. It should be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size and by using a surfactant. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid and thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. The extended absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0236] Sterile injectable solutions can be prepared by mixing the required amount of the active compound in an appropriate solvent for one or more of the above-listed ingredients, as needed, and then filtering and sterilizing. Generally, dispersions are prepared by mixing the active compound into a sterile carrier containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0237] Oral compositions typically include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0238] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer. Such methods include those described in US Patent No. 6,468,798.

[0239] Systemic administration of therapeutic compounds as described herein can also be performed transmucosally. For transmucosal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved using nasal sprays or suppositories or injections.

[0240] In one embodiment, the active compound is prepared with a carrier that will protect the therapeutic compound from rapid elimination from the body, such as a controlled release formulation, including an implant and a microencapsulated delivery system. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such preparations can be prepared using standard techniques or, for example, commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent number 4,522,811.

[0241] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0242] CRISPR / Cas9-mediated gene editing of APOE

[0243] Included herein are methods for treating or preventing the following conditions associated with dementia and / or mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease), neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration, or retinal damage. Typically, the method includes administering a therapeutically effective amount of a genome editing system as described herein to a subject in need of such treatment, or to a subject determined to be in need of such treatment. The term "genome editing system" refers to any system with RNA-guided DNA editing activity. The genome editing system disclosed herein includes at least two components adapted from a naturally occurring CRISPR system: gRNA and an RNA-guided nuclease. These two components form a complex that can bind to a specific nucleic acid sequence in a cell and, for example, edit the DNA in or around the nucleic acid sequence by producing one or more single-strand breaks (SSBs or nicks), double-strand breaks (DSBs), and / or base substitutions. For a complete description of the genome editing system, see, for example, WO2018 / 026976. In certain aspects, the present disclosure provides AAV vectors encoding the CRISPR / Cas9 genome editing system, and the use of such vectors in treating or preventing disorders as described herein.

[0244] RNA-guided nuclease / Cas9

[0245] Various RNA-guided nucleases can be used in the present method, for example, as described in WO 2018 / 026976. In some embodiments, the RNA-guided nuclease and composition used in the present method is Staphylococcus aureus (S. aureus) Cas9 or Streptococcus pyogenes (S. pyogenes) Cas9. The exemplary Cas9 proteins of the present disclosure can be isolated or derived from any species, including but not limited to bacteria or archaea. In some embodiments of the present disclosure, the Cas9 sequence is modified to include two nuclear localization sequences (NLS) (e.g., PKKKRKV (SEQ ID NO: 61), and a mini-polyadenylation signal (or Poly-A sequence) at the C- and N-termini of the Cas9 protein. Exemplary NLSs are the SV40 large T antigen NLS (PKKKRRV (SEQ ID NO: 62)) and the nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO: 63)). Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov 15; 1(5):411-415; Freitas and Cunha, Curr Genomics. 2009 Dec; 10(8):550-557. Exemplary polyadenylation signals are

[0246] TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTG ATCAGGCGCG (SEQ ID NO: 64). In some embodiments, the RNA-guided nuclease is a nuclease-dead Cas protein (e.g., dCas9).

[0247] guide RNA

[0248] Provided herein are guide RNAs (gRNAs) designed to target one or more sites in the HSPG binding domain of wild-type ApoE. In some embodiments, gRNAs are designed to introduce a mutation that causes a mutation at amino acid position 136 in wild-type ApoE. In some embodiments, provided herein are guide RNAs designed to introduce R136S mutations in wild-type APOE genes (e.g., APOE2, APOE3, or APOE4), wherein exemplary guide RNAs can be found in Table 5. In some embodiments, a template for repairing double-strand breaks and introducing R136S mutations is also provided. Exemplary template sequences are as follows:

[0249]

[0250] The silent mutation that eliminates the PAM motif is double underlined, the codon corresponding to the R136S mutation is in bold, and the silent mutation that creates a Sad site for cleavage of the PCR product from clones receiving the template is in italics.

[0251] In some embodiments, the guide RNA provided herein is designed to target exon 3 (amino acids 1-61) of a wild-type APOE gene or a variant present in a subject (thus the method can include determining the sequence of the APOE gene in the subject and using the sequence to determine the sequence of a suitable guide RNA for targeting exon 3 in the subject). In some embodiments, double-strand break repair by non-homologous end joining (NHEJ) causes short insertions or deletions that lead to ApoE knockout. Exemplary guide RNA sequences for ApoE knockout are shown in Table 6.

[0252] Base editing

[0253] In some embodiments, the APOE gene is edited using base editing techniques (e.g., Rees and Liu, Nature Reviews Genetics 19, 770-788, 2018; Komor et al., Nature 533, those described in 420-424). In some embodiments, base editing is used to design guide RNA to introduce R136H mutations in wild-type APOE genes (e.g., APOE2, APOE3, or APOE4), where exemplary guide RNAs can be found in Table 6. Base editors that convert C / G to A / T and adenine base editors that convert A / T to G / C can be used to introduce point mutations. Exemplary base editors include those described in Komor et al., Nature 533, 420-424 and Gaudelli et al., Nature 551, 464-471).

[0254] AAV delivery system

[0255] Method includes delivering a CRISPR / Cas9 genome editing system including Cas9 nuclease and one or two guide RNAs to a subject in need. Delivery methods may include, for example, viral delivery, for example, preferably using an adeno-associated virus (AAV) vector comprising a sequence encoding Cas9 and guide RNA (one or more). Adeno-associated virus is a naturally occurring defective virus that requires another virus such as adenovirus or herpes virus as a helper virus to achieve efficient replication and productive life cycle. (Reviewed in Muzyczka et al., Curr. Topics in Micro and Immunol. 158: 97-129 (1992)). AAV vectors can effectively transduce various cell types and can produce long-term expressed transgenes in vivo. AAV vectors have been widely used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, for example, Mingozzi and High, Nature Reviews Genetics 12, 341-355 (2011); Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 Apr; 20(4): 699-708. AAV vectors containing as little as 300 AAV base pairs can be packaged and can produce recombinant protein expression. For example, AAV2, AAV5, AAV2 / 5, AAV2 / 8, and AAV2 / 7 vectors have been used to introduce DNA into photoreceptor cells (see, e.g., Pang et al., Vision Research 2008, 48(3):377-385; Khani et al., Invest Ophthalmol Vis Sci. 2007 Sep;48(9):3954-61; Allocca et al., J. Virol. 2007 81(20):11372-11380). In some embodiments, the AAV vector can include an AAV capsid polypeptide described in PCT / US2014 / 060163 (or a sequence encoding an AAV capsid polypeptide described in PCT / US2014 / 060163); for example, a viral particle comprising an AAV capsid polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 of PCT / US2014 / 060163, and a Cas9 sequence and a guide RNA sequence as described herein. In some embodiments, the AAV capsid polypeptide is an Anc80 polypeptide, such as Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44.In some embodiments, AAV incorporates inverted terminal repeats (ITRs) derived from the AAV2 serotype. Exemplary left and right ITRs are shown in Table 6 of WO 2018 / 026976. However, it should be noted that many modified forms of the AAV2 ITRs are used in the field, and the ITR sequences shown below are exemplary and not intended to be limiting. Modifications of these sequences are known in the art or will be apparent to a skilled artisan and are therefore included within the scope of this disclosure.

[0256] Cas9 expression is driven by promoters known in the art. In some embodiments, expression is driven by one of three promoters: cytomegalovirus (CMV), elongation factor-1 (EFS), or human g-protein receptor coupled kinase-1 (hGRK1), which are specifically expressed in retinal photoreceptor cells. The nucleotide sequences of each of these promoters are provided in Table 5 of WO 2018 / 026976. In certain applications, modification of these sequences is possible or desirable, and such modifications are within the scope of the present disclosure.

[0257] The expression of gRNA in AAV vector is driven by a promoter known in the art. In some embodiments, the polymerase III promoter, such as the human U6 promoter. An exemplary U6 ​​promoter sequence is shown below:

[0258]

[0259] In some embodiments, the nucleic acid or AAV vector shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with one of the nucleic acids or AAV vectors listed above.

[0260] The above-mentioned AAV genome can be packaged into an AAV capsid (e.g., an AAV5 capsid), which can be included in a composition (e.g., a pharmaceutical composition) and / or administered to a subject. Exemplary pharmaceutical compositions comprising an AAV capsid according to the present disclosure can include a pharmaceutically acceptable carrier, such as a balanced saline solution (BSS) and one or more surfactants (e.g., Tween 20) and / or a thermosensitive or reverse-thermosensitive polymer (e.g., Pluronic). Other pharmaceutical formulation ingredients known in the art may also be suitable for use in the compositions described herein.

[0261] Compositions comprising an AAV vector according to the present disclosure can be administered to a subject by any suitable means, including but not limited to injection (e.g., intracranial injection) and intranasal delivery. The concentration of the AAV vector in the composition is selected to ensure that an adequate AAV dose is administered to the subject's brain, taking into account, among other things, the dead volume within the injection device and the relatively limited volume that can be safely administered. Suitable doses may include, for example, 1×10 11 Viral genomes (vg) / mL, 2x10 11 Viral genomes (vg) / mL, 3x10 11 viral genomes (vg) / mL, 4x10 11 Viral genomes (vg) / mL, 5x10 11 viral genomes (vg) / mL, 6x10 11 Viral genomes (vg) / mL, 7x10 11 Viral genomes (vg) / mL, 8xl0 11 Viral genomes (vg) / mL, 9x10 11 Viral genomes (vg) / mL, 1x10 12 vg / mL, 2xl0 12 Viral genomes (vg) / mL, 3x10 12 Viral genomes (vg) / mL, 4x10 12 Viral genomes (vg) / mL, 5x10 12 Viral genomes (vg) / mL, 6x10 12 Viral genomes (vg) / mL, 7x10 12 Viral genomes (vg) / mL, 8xl0 12 Viral genomes (vg) / mL, 9x10 12 Viral genomes (vg) / mL, lx10 13 vg / mL, 2xl0 13 Viral genomes (vg) / mL, 3x10 13 Viral genomes (vg) / mL, 4x10 13 Viral genomes (vg) / mL, 5x10 13 Viral genomes (vg) / mL, 6x10 13 Viral genomes (vg) / mL, 7x10 13 Viral genomes (vg) / mL, 8xl0 13 Viral genomes (vg) / mL or 9x10 13The composition can be delivered to the subretinal or cochlear space in any suitable volume. In some cases, the volume is selected to form a bleb in the subretinal space, such as 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, etc.

[0262] Explants are particularly useful for studying the expression of gRNA and / or Cas9 after viral transduction, and for studying genome editing in relatively short time intervals. These models also allow for higher throughput than is possible in animal models, and can predict expression and genome editing in animal models and subjects. Small (mice, rats) and large animal models (e.g., rabbits, pigs, non-human primates) can be used for pharmacological and / or toxicological studies, and for testing the systems, nucleotides, vectors, and compositions of the present disclosure under conditions and volumes similar to those to be used in the clinic. Since model systems are selected to summarize relevant aspects of human anatomy and / or physiology, the data obtained in these systems will typically (although not necessarily) predict the behavior of AAV vectors and compositions according to the present disclosure in humans and animal subjects.

[0263] Screening method (test compound)

[0264] Included herein are methods for screening test compounds such as polypeptides, polynucleotides, inorganic or organic macromolecules or small molecule test compounds to identify agents for treating or preventing: conditions associated with dementia and / or mild cognitive impairment (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease), neurodegenerative diseases, cerebrovascular diseases, brain injury, retinal degeneration or retinal damage. In some embodiments, the test compound regulates the HSPG / heparin binding properties of ApoE protein (e.g., wild-type ApoE protein). In some embodiments, the test compound reduces the HSPG / heparin binding properties of ApoE protein (e.g., wild-type ApoE protein).

[0265] As used herein, " small molecule " refers to an organic or inorganic small molecule with a molecular weight lower than about 3,000 daltons. Generally, the small molecule that can be used for the present invention has a molecular weight less than 3,000 daltons (Da). Small molecule can be, for example, at least about 100Da to about 3,000Da (for example, about 100 to about 3,000Da, about 100 to about 2500Da, about 100 to about 2,000Da, about 100 to about 1,750Da, about 100 to about 1,500Da, about 100 to about 1,250Da, about 100 to about 1,000Da, about 100 to about 750Da, about 100 to about 500Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000Da or about 100 to about 250Da).

[0266] The test compound can be a member of a natural product or combinatorial chemical library. Various functions, such as charge, aromaticity, hydrogen bond, flexibility, size, length of side chain, hydrophobicity and rigidity, should be covered using a group of different molecules. Combinatorial techniques suitable for synthesizing small molecules are known in the art, for example, as exemplified by Obrecht and Villalgordo, Solid-Supported Combineatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as "separation and merging" or "parallel" synthesis techniques, solid and liquid phase techniques and coding techniques (see, for example, Czarnik, Curr.Opin.Chem.Bio.1:60-6 (1997)). In addition, many small molecule libraries are commercially available. Many suitable small molecule test compounds are listed in U.S. Patent number 6,503,713, which is incorporated herein by reference in its entirety.

[0267] The library screened using the method of the present invention can include various types of test compounds. A given library can include groups of structurally related or unrelated test compounds. In some embodiments, the test compound is a peptide or peptide mimetic molecule. In some embodiments, the test compound is a nucleic acid.

[0268] In some embodiments, for example, using methods known in the art or described herein, and correlating structure with resulting biological activity, such as performing structure-activity relationship studies, test compounds and libraries thereof can be obtained by systematically varying the structure of a first test compound, such as a first test compound that is structurally similar to a known natural binding partner of a target polypeptide, or a first small molecule identified as being capable of binding to a target polypeptide. Those skilled in the art will appreciate that there are various standard methods for generating such structure-activity relationships. Thus, in some cases, the work may be primarily empirical, while in other cases, the three-dimensional structure of an endogenous polypeptide or portion thereof may be used as a starting point for the rational design of one or more small molecule compounds. For example, in one embodiment, a universal library of small molecules is screened, for example, using the methods described herein.

[0269] In some embodiments, the test compound is applied to a test sample, for example, a sample containing one or more ApoE proteins, and one or more effects of the test compound (e.g., HSPG / heparin binding affinity of ApoE protein (one or more)) are evaluated. For example, the ability of the test compound to modify the HSPG / heparin binding affinity of ApoE protein (one or more) can be evaluated using a heparin sepharose column as described herein or an antibody that specifically recognizes the HSPG binding domain of ApoE. In some embodiments, the method for screening a test compound as described herein includes evaluating the ability of the test compound to modify (e.g., inhibit or reduce) the binding of an antibody to one or more HSPG binding sites or one or more HSPG-bound allosteric regulatory sites as described herein that bind to wild-type or mutant ApoE. In some embodiments, the test compound competes with an antibody as described herein for ApoE binding.

[0270] In some embodiments, the test sample is or is derived from (eg, taken from) an in vivo model of a disorder described herein. For example, an animal model, such as a rodent such as a rat, can be used.

[0271] The test compound that has been screened and determined to reduce or modify ApoE and HSPG / heparin combination by the methods described herein can be considered as candidate compounds. Screened in the in vivo model of, for example, illness, such as dementia and / or mild cognitive impairment (such as those related to Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease), neurodegenerative disease, cerebrovascular disease, brain injury, retinal degeneration or retinal damage, and determine that illness, such as one or more symptoms of illness have a desired effect candidate compound, can be considered as candidate therapeutic agent. Once screened in a clinical setting, candidate therapeutic agent is therapeutic agent. Candidate compound, candidate therapeutic agent and therapeutic agent can optionally be optimized and / or derivatized, and formulated to form a pharmaceutical composition together with a physiologically acceptable excipient.

[0272] Thus, test compounds identified as "hits" in a first screen (e.g., test compounds with a desired effect on a condition) can be selected and systematically altered, for example, using rational design, to optimize binding affinity, avidity, specificity, or other parameters. Such optimization can also be screened using the methods described herein. Thus, in one embodiment, the invention comprises screening a first library of compounds using methods known in the art and / or described herein, identifying one or more hits in the library, systematically structurally altering the hits to generate a second library of compounds structurally related to the hits, and screening the second library using the methods described herein.

[0273] Test compounds identified as hits can be considered candidate therapeutic compounds for treating, preventing, or delaying the development and progression of conditions associated with dementia and / or mild cognitive impairment, such as Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease, as described herein, and for treating, preventing, or delaying the development or progression of neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. Various techniques that can be used to determine the structure of "hits" can be used in the methods described herein, such as NMR, mass spectrometry, gas chromatography equipped with an electron capture detector, fluorescence, and absorption spectroscopy. Thus, the present invention also includes compounds identified as "hits" by the methods described herein, as well as methods of administering them and methods for treating, preventing, or delaying the development or progression of the conditions described herein.

[0274] Test compounds identified as candidate therapeutic compounds can be further screened by administration to animal models of conditions associated with any of the conditions described herein. Animals can be monitored for changes in the condition, such as improvement in a parameter of the condition, such as a parameter associated with clinical outcome.

[0275] Treatment

[0276] The methods described herein include methods for treating, preventing or delaying the development or progression of conditions associated with dementia and / or mild cognitive impairment, neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage. In some embodiments, the conditions associated with dementia and / or mild cognitive impairment are Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease. Other non-limiting examples of neurodegenerative diseases include prion disease, motor neuron disease and amyotrophic lateral sclerosis (ALS). Non-limiting examples of cerebrovascular diseases include stroke, carotid stenosis, spinal stenosis and aneurysm. Non-limiting examples of brain damage include traumatic brain injury and acquired brain injury. Retinal degeneration, such as glaucoma and age-related macular degeneration, can involve amyloid beta and neurofibrillary tangle toxicity, establishing a link between retinal degeneration and neurodegeneration (e.g., Alzheimer's disease) (see, e.g., McKinnon, Frontiers in Bioscience 8, s1140-1156, 2003; Johnson et al. PNAS 99(18)11830-11835, 2002; and Sivak, Investigative Ophthalmology & Visual Science, 54(1)871-880, 2013). Therefore, treatments for neurodegenerative diseases can be used to treat retinal or optic nerve degeneration.

[0277] The methods comprise administering to a subject in need or determined to be in need of such treatment a therapeutically effective amount of any of the antibodies, peptides, fusion proteins, or genome editing systems described herein.

[0278] Methods described herein can also be used for subjects at risk of developing any disease as described herein. Subjects at risk of developing Alzheimer's disease can include subjects homozygous or heterozygous for APOE4 alleles, carriers of autosomal dominant Alzheimer's disease pathogenic mutations (such as amyloid beta precursor (APP) gene, PSEN1 gene or PSEN2 gene mutations), trisomy 21 (for example, whose cognitive impairment is only a developmental subject). Subjects at risk of developing Alzheimer's disease can also include subjects with polygenic risk scores associated with the risk of increased development disease, and subjects with brain imaging or other biomarkers (such as biomarkers in body fluids) evidence of Alzheimer's disease. Methods for preventing or delaying the development of disease as described herein can also be used for subjects not at risk of developing the above-mentioned disease, such as subjects over 50 years old (such as 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old or 95 years old).

[0279] As used in this context, "treating" refers to ameliorating at least one symptom of a condition associated with a condition described herein. Typically, Alzheimer's disease results in fibril formation, amyloid aggregation, and cognitive decline; thus, treatment can result in a reduction in fibril formation and / or amyloid aggregation in the brain, a reduction in the formation of tau tangles, improved brain metabolism, improved neurocognitive function, and / or improved cognitive ability.

[0280] Example

[0281] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. In terms of the specific materials mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without exercising inventive ability and without departing from the scope of the invention.

[0282] Example 1: Identification of the APOE3 Christchurch R136S mutation in PSEN1 mutation carriers Materials and methods:

[0283] Clinical evaluation: The institutional review boards of the University of Antioquia, Massachusetts General Hospital, and the Schepens Eye Research Institute of Massachusetts Eye and Ear approved this study. As with all study participants, the proband provided written informed consent. Clinical ratings and neuropsychological testing were performed as described in Table 1. PSEN1 E280A genotyping was performed as previously described. 1 .

[0284] All clinical measures were performed at the University of Antioquia (Medellín, Colombia) and were conducted in Spanish by physicians and psychologists trained in assessment. Neurocognitive testing included a comprehensive multidomain assessment. Some of the tests performed were the Spanish version of the Mini-Mental State Examination (MMSE), the Clinical Dementia Rating (CDR), and the Consortium to Establish a Registry for Alzheimer's disease battery, which have been adapted for this Colombian population. 2 Other tests include the Yesavage Geriatric Depression Scale3 and functional assessment staging tests 4 These tests were completed within six months of the brain imaging.

[0285] A detailed ophthalmologic evaluation was performed. This included assessment of visual acuity, slit lamp, and indirect ophthalmoscopy. Ultra-wide-field fundus and fundus autofluorescence images were obtained using an Optos Panoramic 200Tx imaging system (Optos PLC, Dunfermline, Scotland, UK). In addition, OCT angiography was performed using a Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany) and a Cirrus HD-OCT with AngioPlex (Carl Zeiss Meditec, Dublin, CA).

[0286] Additional studies were conducted after the discovery that carriers of the PSEN1 E280A mutation had two copies of the APOE3ch variant. Fasting serum lipid testing was performed to explore the possibility of type III hyperlipoproteinemia, a condition found in 5-10% of people who are homozygous for the relatively AD-protective APOE2 allele and in most, but not all, APOE3ch carriers. 5 .

[0287] Finally, analysis of data from clinically and neuropathologically validated AD cases and controls from the AD Genetics Consortium was used to elucidate whether homozygosity for the APOE2 allele is associated with a very low risk of late-onset AD dementia.

[0288] Whole exome sequencing: Whole exome capture and sequencing were performed using Illumina chemistry for variant discovery; rare variants with frequencies below 1% in genes previously associated with AD were considered for the search for candidate risk modifiers. Specifically, rare DNA variants (minor allele frequency <1%) within exonic regions and splice site junctions (5 bp into introns) of genes were identified using bioinformatics tools. Whole exome libraries were constructed and sequenced on an Illumina HiSeq 4000 sequencer using 151 bp paired-end reads. A previously described protocol was used with the following modifications: 6Library construction was performed. The genomic DNA input in 10 μL solution was reduced from 3 μg to 50 ng and enzymatic shearing was performed. The dual-index Illumina double-end adapter was replaced with a palindromic forked adapter, in which a unique 8-base index sequence was embedded and added to each end for adapter connection. In-solution hybridization selection was performed using an Illumina Rapid Capture Exome Enrichment Kit with a 38 Mb target region (29 Mb bait). The target region includes 98.3% of the intervals in the Refseq exome database. Prior to hybridization, the dual-index libraries were merged into groups of up to 96 samples. After elution from the streptavidin beads, the enriched library pools were quantified by PicoGreen and then normalized. For cluster amplification and sequencing, quantitative PCR (KAPA Biosystems) is used to quantify the library prepared using bifurcated, indexed joints, standardized to 2 nM using the Hamilton Starlet liquid handling system, and equal volumes are merged using the Hamilton Starlet liquid handling system. The pool is then denatured in 0.1 N NaOH. The denatured sample is diluted into a strip tube using the Hamilton Starlet liquid handling system. Cluster amplification of templates is performed using Illumina cBot according to the manufacturer's protocol (Illumina). Flow cell (flowcell) is sequenced on a HiSeq 4000 sequencing-by-synthesis test kit and then analyzed using RTA2.7.3.

[0289] Exome sequencing data were processed and analyzed using the bioinformatics analysis pipeline of the Center for Personalized Medicine (CPM) Clinical Genomics Laboratory and the Institute for Translational Genomics. In brief, the Dragen Genome Pipeline of EdicoGenome using default parameters was used to perform sequence alignment and variant calling. Open source software samtools and bcftools (samtools.github.io / ) were used together with a set of custom scripts to determine coverage and initial variant filtering based on ExAC (Exome Aggregation Consortium, exac.broadinstitute.org / ) allele frequencies. 7 Sequence alignment was performed against the human hs37d5 bait genome. 8To identify potential modifying variants, a master gene list of 15 genes was generated based on two HPO terms: HP:0002511, Alzheimer's disease; and HP:0003584, late-onset. These genes are AAGAB, ABCC8, AKT2, APOE, APP, BEAN1, GATA1, GCK, HMGA1, HNF1B, HNF4A, LDB3, PAX4, PSEN1, and PSEN2. 9 Rare DNA variants (minor allele frequency <1%) in the exonic regions and splice site junctions (into the intronic 5 bp) of these genes were further annotated and analyzed using a commercial tool (Cartagenia v5.0). Sequence alterations were reported according to the Human Genome Variation Society (HGVS v2.0) nomenclature guidelines.

[0290] Whole-genome sequencing: Whole-genome sequencing (WGS) and Genomizer analysis (v 10.1.0) were used to perform comprehensive and unbiased sequencing of other potential genetic risk modifiers, including those associated with a lower risk of Alzheimer's disease, helping to rule out other potential protective genetic factors. 10 To process the WGS data, the same dragen analysis pipeline as described above was used. The data were aligned to the GRCh37 bait genome (hs37d5). Variants detected at a depth of <10X were filtered out and then annotated using the Variant Effect Predictor (VEP) tool from Ensembl. The VEP version used was v93. The filtered and annotated variant set was then compiled for Genomizer analysis.

[0291] APOE structure display: The image was created using NGL Viewer 12 From the previously published structure of PDB 2L7B 11 The data were obtained and modified from the RCSBPDB (rcsb.org).

[0292] APOE genotyping by Sanger sequencing: The reaction mixture for the amplification process was carried out in a volume of 50 μL and included the following components: 1× PfuUltra II Hostart Master Mix, 1 μL of each primer (10 μmol / L) (forward primer: 5′- A GCCCTTCTCCCCGCCTCCCACTGT-3' (SEQ ID NO: 67) and reverse primer: 5'-CTCCGCCACCTGCTCCTTCACCTCG-3' (SEQ ID NO: 68)), 5% DMSO and 1 μL of genomic DNA (100 ng / μL). 13PCR cycles were run as follows: initial denaturation at 95° C. for 2 minutes, followed by 35 cycles of denaturation at 95° C. for 20 seconds, annealing at 60° C. for 30 seconds, extension at 72° C. for 40 seconds, and a final extension at 72° C. for 5 minutes. PCR products were purified using the QIAquick gel extraction kit from Qiagen and sequenced using the MGHCCIB DNA core using a 3730xl sequencer from Applied Biosystems.

[0293] MRI and PET Imaging: Pittsburgh Compound B (PiB), flortaucipir (FTP) positron emission tomography (PET), and structural magnetic resonance imaging (MRI) measurements were obtained at Massachusetts General Hospital and analyzed at Massachusetts General Hospital and the Banner Alzheimer's Institute as described previously. 14 Fluorodeoxyglucose PET images were acquired and analyzed as previously described at the University of Antioquia, Colombia. 15 Imaging data from cases were compared with data from younger PSEN1 E280A mutation carriers who developed MCI at the expected age of clinical onset in relatives and with data from cognitively intact mutation carriers.

[0294] As mentioned above 16-19 MRI was performed on a 3T Tim Trio (Siemens) and included magnetization-prepared rapid gradient echo (MPRAGE) processed with Freesurfer (FS) to identify gray and white matter and the pial surface to allow segmentation of regions of interest (ROIs) as follows: cerebellar gray matter, hippocampus, and the following Braak stage-related cortices: entorhinal cortex, parahippocampal gyrus, inferior temporal gyrus, fusiform gyrus, and posterior cingulate gyrus.

[0295] 18F-Flortaucipir (FTP) was prepared at MGH with a radiochemical yield of 14 ± 3% and a specific activity of 216 ± 60 GBq / μmol at the end of synthesis (60 minutes) and has been validated for use in humans (Shoup et al., 2013). 11C-Pittsburgh compound B was prepared, and PET images were acquired as previously described. 16All PET images were acquired with a Siemens / CTI (Knoxville, TN) ECAT HR+ scanner (3D mode; 63 image planes; 15.2-cm axial field of view; 5.6-mm transverse resolution and 2.4-mm slice interval). 11C PiB PET was acquired with a bolus injection of 8.5 to 15 mCi, followed by a 60-minute dynamic acquisition of 69 frames (12 x 15 sec, 57 x 60 sec). 18F FTP was acquired with 4 x 5-minute frames over 80–100 minutes after a bolus injection of 9.0 to 11.0 mCi. PET images were reconstructed and attenuation-corrected, and individual frames were evaluated to verify adequate count statistics and the absence of head motion.

[0296] 18F FTP-specific binding in the FS ROI was expressed as the standardized uptake value ratio (SUVR) to the cerebellum, similar to previous reports. 19 , using the FS cerebellar gray matter ROI as a reference. For voxelwise analysis, each subject's MPRAGE was registered to the template MR in SPM8 (SPM), and the spatially transformed SUVR PET data were smoothed using an 8 mm Gaussian kernel to account for individual anatomical differences. 20 To account for possible off-target binding of 18F FTP in the choroid plexus (which could confound hippocampal signaling), we used linear regression to regress the choroid plexus as previously reported. 21 .

[0297] 11C PiB PET data are expressed as distribution volume ratios (DVRs) with the cerebellar gray matter as the reference tissue; regional time-activity curves were used to calculate regional DVRs for each ROI using the Logan graphical method applied to data from 40 to 60 minutes after injection. 16,22 11C PiB retention was assessed using a large cortical ROI aggregate encompassing the previously described frontal, lateral temporal, and retrosplenial cortices (FLR). 23,24

[0298] 8F-fluorodeoxyglucose PET was performed on a 64-section PET / computed tomography system (Biograph mCT; Siemens) while resting in a dark room, after a 30-minute radiotracer uptake period, using 5mCi (185 million Bq) of 18F-fluorodeoxyglucose intravenous administration, followed by 30 minutes of dynamic emission scanning (six 5-minute frames). Images were reconstructed using computed tomography attenuation correction. The ratio of the precuneus to the whole-brain cerebral glucose metabolic rate (CMRgl) was characterized from bilateral regions of interest (ROIs) in each participant's 18F-fluorodeoxyglucose PET images using an automated brain mapping algorithm (SPM8; fil.ion.ucl.ac.uk / spm / software / spm8). The hippocampus to total intracranial volume ratio was characterized from bilateral ROIs in each participant's T1-weighted MR images using FreeSurfer (surfer.nmr.mgh.harvard.edu). All images were visually inspected to verify ROI characterization.

[0299] Amyloid aggregation studies: Human ApoE3 protein fragments (including the carboxyl-terminal domain and histidine tag) with and without the Christchurch variant were synthesized in bacteria, purified (Innovagen), and used for the quantification of amyloid aggregation using Thioflavin T ( ThTβ-amyloid protein (1-42) aggregation kit, catalog number AS-72214) in vitro evaluation of these proteins against Aβ 42 For this assay, 55 μM Aβ 42 Add to a 10 μM solution of wild-type apoE3 protein or mutant 136Arg→Ser ApoE3 protein in a clear, unbound 96-well plate. Then mix the sample with 2 mM Thioflavin T dye and read fluorescence at intervals over 2 hours at Ex / Em = 440 / 484. Maintain the plate at 37°C with 15-second shaking between readings.

[0300] Full-length ApoE3 proteins with and without the Christchurch mutation were also transiently transfected into the Flp-In TM T-REx TM 293 (Thermo Fisher Scientific) mammalian cells to confirm the effects of these proteins on Aβ using a previously published split-luciferase complementation assay. 42 The effect of aggregation. 26 The latter analysis was performed using plasmid #87086 from Addgene. 27Human APOE3 was expressed as WT or APOE3 was introduced into the Christchurch variant by site-directed mutagenesis. Luciferase assay reagents were purchased from Promega.

[0301] result

[0302] Approximately 1,200 carriers of the Columbia presenilin 1 (PSEN1) E280A mutation and 4,600 non-carriers were identified, collectively constituting the world's largest known autosomal dominant Alzheimer's disease (ADAD) family. 28,29 Mutation carriers typically develop mild cognitive impairment (MCI) and dementia at a median age of 44 (95% CI, 43-45) and 49 (95% CI, 49-50) years, respectively. 30,31 Studying autosomal dominant AD (ADAD) mutation carriers who remain cognitively intact until later in life may help identify risk-reducing genetic variants. 32 Characterizing AD biomarkers in these individuals will help understand the potentially targetable mechanisms by which these genes exert their relative protective effects. We identified a carrier of the PSEN1 E280A mutation who did not develop MCI until her 70s, nearly 30 years after the median age of onset.

[0303] This study was conducted in accordance with the guidelines of the Institutional Review Board with the written informed consent of the participant (her exact age and other identifying information have been omitted to protect her anonymity and confidentiality). 42 (Aβ 42 )-overproducing PSEN1 E280A mutation, family informant reports confirmed she was cognitively intact until age 70, then consistent with MCI at annual evaluations at 24-month intervals. 33 criteria. She remained fully independent in basic and instrumental activities of daily living, with no overt signs of deterioration in her ability to perform these activities. At the intake evaluation, her memory deficits were limited to recent events, and her neurologic examination was normal. Her age- and education-adjusted neuropsychological test scores indicated preferential impairment in recall memory, relatively preserved recognition memory, initial learning, naming, visuospatial abilities, and verbal fluency, as well as relatively stable cognitive performance over the 24-month evaluation period (Table 1).

[0304] Table 1: Cognitive test scores and percentiles

[0305]

[0306]

[0307] MMSE: Mini-Mental State Examination

[0308] CERAD: Establishing an Alzheimer's Disease Registry Alliance

[0309] GDS: Geriatric Depression Scale

[0310] EDG: Full Decline Scale

[0311] *Due to her limited literacy skills, the MMSE subtest requiring reading and writing skills was not administered. Her maximum possible score was 23 (not 30).

[0312] **Percentiles are calculated using the standards for this Colombian population.

[0313] A percentile score between the 25th and 75th places her performance within the average range for her age and education.

[0314] A percentile score between 9 and 25 would classify her performance as below average. A percentile score between 2 and 8 would classify her performance as low. A percentile score below 1 would classify her performance as very low.

[0315] #The brain imaging described in this report was obtained three months after initial cognitive testing.

[0316] Whole-exome sequencing confirmed her PSEN1 E280A mutation and revealed that she also had two copies of the rare APOE3 Christchurch R136S (APOEch) mutation. Sanger sequencing confirmed the latter finding. Whole-genome sequencing and Genomizer analysis were used to comprehensively identify and rank all potentially important rare and common variants. 34 Using this approach, the PSEN1 E280A mutation was confirmed to be the participant's major risk factor, and APOE3ch homozygosity was confirmed to be her major resistance factor.

[0317] APOE, the major susceptibility gene for late-onset AD, has three common alleles (APOE2, 3, and 4). Compared with the most common APOE3 / 3 genotype, APOE2 is associated with a lower risk of AD and a greater age of onset of dementia. 35 And each extra copy of APOE4 was associated with higher risk and younger age of onset. 36,37 APOEch variant, arginine-to-serine substitution at amino acid 136 (136Arg→Ser), corresponding to codon 154, 38 Can be located on any common APOE allele, 39 This included the participant's two APOE3 alleles. Figure 1The structural model of the wild-type ApoE3 protein is shown. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are highlighted. The amino acid positions of the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are shown.

[0318] The APOE3ch variant is not present in the AlzAD or ExAC databases, which report approximately 180,000 exomes. R136S was previously identified in APOE2 individuals with HLP III, but its potential role in the progression of AD has not been previously reported. 40 We sequenced DNA samples by whole-genome sequencing from two other PSEN1 E280A carriers with delayed onset at ages 62 and 70. These individuals did not have the APOE3 R136S variant nor APOE2, which had previously been shown to delay disease onset in this family. 41

[0319] To confirm the potential association between the APOE3 R136S mutation and delayed age at onset of AD, we performed whole-genome sequencing and neurological and neuropsychological testing in four offspring of the proband case who were over 50 years of age and predicted to carry APOE3 R136S. Figure 2 Representative Sanger sequencing results for APOE from control, proband, and offspring samples are shown. Top row: Homozygous sequences for C112 are shown in all cases. Middle row: Homozygous sequences for R136 from a control individual are shown in the left panel. The middle panel shows the homozygous mutation that results in the R136S mutation. The right panel shows an example of a heterozygous R136S mutation in an offspring of a proband. Bottom row: Homozygous sequences for R158 are shown in all cases. Figure 3 The pedigrees of the probands are shown, with circles representing females, squares representing males, and diamonds representing individuals whose sex has been obscured for privacy. Arrows depict proband individuals with MCI, and shading indicates individuals with a history of dementia. Deceased individuals are marked with slashes. Individual APOE and PSEN1 genotypes are shown where appropriate to preserve anonymity. Although other unknown genetic or epigenetic factors may contribute to the later age of onset of cognitive impairment in these two related PSEN1 E280A carriers, we propose that the APOE3 R136S variant modifies the AD phenotype by buffering the effects of amyloid-β accumulation in the brain and subsequently delaying the onset of tau pathology, neurodegeneration (i.e., brain atrophy), and symptom onset.

[0320] Carriers of APOEch and other rare mutations in the low-density lipoprotein receptor (LDLR) binding region of APOE typically have type III hyperlipoproteinemia (HLP-III), similar to that observed in 5-10% of APOE2 homozygotes. 43,44 Participants in this report demonstrated HLP-III, including APOEch and elevated triglyceride and total cholesterol levels (see Table 2).

[0321] Table 2: Dyslipidemia examination

[0322]

[0323] HDL-C: high-density lipoprotein cholesterol, VLDL-C: very low-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol. *Normal ranges based on the Merck Manual and laboratory values.

[0324] Detailed laboratory examinations showed abnormal lipid profiles in three of our proband individuals and four offspring carrying APOE3 R136S (Table 3). These four subjects had high levels of total cholesterol and triglycerides. Very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were higher in two offspring and could not be determined in the proband individual and one offspring, whose triglyceride levels were higher than 400 mg / dL (accuracy threshold of indirect method of lipid analysis) (Table 3). Further analysis using direct enzymatic testing showed that the LDL of these two individuals was higher than normal LDL. Despite carrying APOE3R136S and APOE4, the lipid profile of one offspring was within the normal range. Incomplete penetrance of HLP III in APOE2 and R136S mutation carriers has been reported previously. 45 We excluded secondary causes of lipid disorders in these subjects, such as diabetes, obesity, alcoholism, renal disorders, or thyroid disease. None of the mutation carriers had xanthomas, which are diagnostic of HLP III, or cardiovascular disease. The combination of an abnormal lipid profile and the APOE3 R136S mutation in these subjects is consistent with a diagnosis of familial HLP III.

[0325] Table 3: Dementia and dyslipidemia tests in the study population

[0326]

[0327] A detailed ophthalmologic evaluation was performed on a carrier of the PSEN1 E280A mutation with two APOE3ch alleles. The carrier had visual acuity of 20 / 70 in the right eye and 20 / 40 in the left eye. Anterior segment examination noted a dense posterior capsule opacity in the posterior chamber intraocular lens in the right eye. A nuclear sclerotic cataract ( Figure 4A and 4B Posterior segment examination of both eyes was normal, with clear vitreous cavities, normal optic nerve, macula, and peripheral retina. Further testing by optical coherence tomography (OCT) of the right eye was normal, except for a small area of ​​hyper-reflectivity covering the fovea ( Figure 4D ). Figure 4C An infrared image of the right eye is shown, depicting Figure 4D In addition, OCT imaging of the left eye showed a degenerative lamellar hole (caused by Figure 4F * in the figure), with small defects in the external membrane and ellipsoidal layer ( Figure 4F (arrow in the middle).

[0328] Although several mechanisms have been proposed to explain the effects of APOE variants on AD risk, most studies have focused on their effects on Aβ 42 Differential effects of aggregation and plaque burden (APOE2<3<4). 47 In this study, neuroimaging measures were used to elucidate whether participants' resistance to the clinical onset of AD was associated with: a) Aβ 42 overproduction of tau but relatively low Aβ plaque burden or b) relatively high Aβ plaque burden but limited paired helical fibrils (PHFs) tau (neurofibrillary tangle burden) and downstream assays of neurodegeneration.

[0329] Neuroimaging findings among participants included Figure 5 Positron emission tomography (PET) images are superimposed on the medial and lateral surfaces of the left hemisphere. The upper row shows PET measurements of amyloid plaque burden (PiB DVR). The lower row shows PET measurements of paired helical fibril (PHF) tau (i.e., neurofibrillary tangles) burden. The individual with late-onset MCI was in their 70s, compared to the typical age of onset for MCI, which is 44 years.

[0330] like Figure 5As shown, individuals with late-onset MCI have abnormally high PET measurements of Aβ plaque burden, as indicated by a higher mean cortical to cerebellar Pittsburgh compound B (PiB) distribution volume ratio (DVR = 1.96) compared to PSEN1 E280A carriers who develop MCI in their 40s (DVR of 1.49-1.60). Despite her high Aβ plaque burden, the magnitude and / or spatial extent of her PHFtau burden and neurodegeneration were relatively limited: her flortaucipir (tau) PET measurements were limited to the medial temporal region and the occipital region, which is typically less affected, and relatively few other regions that are characteristically affected in the clinical stage of AD ( Figure 5 Her fluorodeoxyglucose PET measurements of brain glucose metabolic rate were preserved in brain regions known to be preferentially affected by AD, including precuneus-to-whole-brain measures that were higher than those in PSEN1 E280A mutation carriers who developed MCI at a younger age and in many younger, cognitively intact mutation carriers.

[0331] Figure 6 Show average cortical amyloid plaque load, entorhinal cortex PHF tau load, hippocampal volume, precuneus glucose metabolism measured value.These measured values ​​are based on brain imaging results, obtained from the PSEN1 E280A mutation carrier (red dots) with two APOE3ch alleles and abnormal late-onset MCI, the PSEN1 E280A mutation carrier (black dots) suffering from MCI under the typical MCI onset age of relatives, and the PSEN1 E280A mutation carrier (grey dots) not yet developed MCI obtained.Amyloid plaque load is expressed as average cortex-to-cerebellum distribution volume ratio (DVR).Paired spiral fiber (PHF) tau load is expressed as entorhinal cortex-to-cerebellum flortaucipir (FTP) standard uptake value ratio (SUVR).The hippocampal volume that can be reduced due to hippocampal atrophy is expressed as hippocampus-to-whole brain volume ratio. Reduced cerebral glucose metabolism in AD-affected brain regions with synaptic dysfunction and loss is reflected in the precuneus-to-whole brain cerebral glucose metabolic rate (CMRgl) ratio. Figure 6As shown, although the PSEN1 E280A mutation carrier with two APOE3ch alleles currently had the highest amyloid plaque burden, she did not have a relatively severe PHF tau burden or hippocampal atrophy, and she had no evidence of precuneus glucose hypometabolism. Her MRI-based hippocampal-to-whole brain volume (a measure of hippocampal atrophy that can be affected by AD and / or normal aging) was within the range of mutation carriers who develop MCI in their 40s. Without wishing to be bound by theory, these results suggest that the resistance of this APOE3ch homozygote to the clinical onset of AD is mediated by a mechanism that limits tau pathology and neurodegeneration even in the face of high Aβ plaque burden.

[0332] To investigate the functional consequences of the APOE3ch variants, Aβ was expressed in the presence of wild-type human ApoE3 protein of bacterial origin, in the presence of mutant ApoE3ch protein, or in the absence of any ApoE protein. 42 In vitro aggregation was compared. Aβ was detected by Thioflavin T fluorescence 42 The rate of fibril formation. 42 Aggregation was highest in the presence of wild-type human ApoE3 protein (C-terminal domain) and lower in the presence of human ApoE3ch (similar to that observed in the presence of ApoE2 48 ), and lowest in the absence of any ApoE ( Figure 7 ).

[0333] This finding was confirmed using a sensitive split-luciferase complementation assay, in which the luciferase signal is reconstituted once the amyloid protein forms oligomers. 48 Oligomers are some of the most toxic amyloid species. 49 Full-length ApoE3ch expression in mammalian cells elicits significantly less Aβ compared to wild-type ApoE3 42 Oligomerization, as luciferase luminescence via oligomer formation was significantly reduced in ApoE3ch compared to wild-type ApoE3 ( Figure 8 These results provide validation for the genetic analysis and suggest that the protective effects of ApoEch may be at least in part due to its ability to promote Aβ 42 Limited aggregation capacity. It remains possible that the study participant could have had larger Aβ plaque deposits, allowing her to survive into her 70s without the APOEch / 3ch genotype, and that the ApoE3ch protein altered the morphology of Aβ aggregation in a way that limited downstream neuroinflammation, tau pathology, neurodegeneration, and cognitive decline.

[0334] A small number of Colombian relatives were found to carry one copy of the APOE3ch mutation. 50Four PSEN1 E280A mutation carriers developed MCI at a median age of 45 years. Therefore, it is speculated that APOE3ch homozygosity may be required to significantly reduce risk and delay the clinical onset of autosomal dominant AD. Due to the small sample size, APOEch heterozygous individuals may still have partial protection against cognitive decline associated with autosomal dominant AD and substantial protection against sporadic late-onset AD and / or neurodegeneration.

[0335] These results suggest that APOE variants have different degrees of pathogenic function (APOEch and APOE2<3<4), and that APOE3ch / 3ch and APOE2 / 2 are associated with the greatest loss of function. Interventions that safely and adequately edit APOE, reduce its expression, or inhibit its pathogenic function could have profound implications for the treatment and prevention of AD. Interestingly, inhibition of APOE expression in the brain using antisense oligonucleotides in mice with Aβ overproduction altered Aβ plaque morphology and reduced dystrophic neurites. 51 This approach may be feasible because loss of APOE expression is tolerated in middle-aged men who are homozygous for the frameshift variant. 52 Furthermore, the availability of statins for the treatment of HPL-III supports the potential tolerability of ApoE-lowering therapy. See, for example, Reiman et al. Nat Commun 1191):667, 2020.

[0336] Without wishing to be bound by theory, these results further suggest that homozygosity for APOE3ch and APOE2 is associated with strong resistance to the clinical onset of AD; these genotypes exert their beneficial effects by directly or indirectly limiting downstream tau pathology and neurodegeneration; these effects are not solely based on the size of Aβ plaque burden, despite the relative reduction in Aβ aggregation mediated by APOE. These findings suggest a role for APOE in the understanding, treatment, and prevention of AD and may stimulate interest in developing APOE-modifying genes and drug therapies for this condition.

[0337] Example 2: Heparin Binding Properties of APOE3ch Mutant Protein

[0338] Materials and methods

[0339] Heparin column protocol: A 1 ml heparin column (BioVision-6554-1) was used to compare the heparin binding affinity of ApoE2, ApoE3, ApoE3ch, and ApoE4 protein isoforms. The column was acclimatized to room temperature for 1 hour before use. The column was washed with 5 mL of 20 mM TRIS-HCL (pH 7.5). A 1 mL sample containing 50 μg / mL of APOE recombinant protein in 20 mM TRIS-HCL (pH 7.5) was then circulated through the column 5 times. The column was then washed 5 times with 20 mM TRIS-HCL (pH 7.5). An increasing NaCl gradient (0.025-1 M) in 20 mM TRIS-HCL was passed through the column, and 1 mL fractions were collected and subsequently prepared for immunoblotting.

[0340] Immunoblotting: Immunoblotting confirmed the elution of ApoE isoforms in fractions collected from the heparin-bound column. Fractions were diluted to a final volume of 40 μl in 10 μl RIPA buffer (Cell Signaling Technology), 4 μl DTT (1 M) and 10 μl Laemmli buffer. TGX TM Samples were separated on precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), and probed with mouse anti-his tag (Novus biologicals.) and IRDye800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey infrared imaging system and visualized in Image Studio version 2.1 (LI-COR Biosciences). Individual gels were combined to generate Figure 10 .

[0341] Heparin plate ELISA protocol: ELISA was performed using heparin microplates (Bioworld; 50-197-531). They were blocked for one hour using sample preparation reagent (DY008). The heparin plates were incubated with 0.1 μg / well of each recombinant ApoE protein isoform (ApoE2, ApoE3, ApoE3ch, and ApoE4) for 2 hours, then washed five times with PBS containing a NaCl gradient (0-0.5 M), followed by three washes in wash buffer (DY008). Anti-His tag antibody was incubated overnight at 1:10,000 (Novusbiologicals; NBP2-61482). The plates were then washed five times to ensure removal of unbound primary antibody, incubated with donkey anti-rabbit-HRP (1:10000) for 45 minutes, and then washed five times to ensure removal of the secondary antibody. Before adding 100 μl of tetramethylbenzidine (Millipore) to start the detection phase of the reaction, sulfuric acid from the ELISA kit (DY008) was heated to 37°C. After incubation for 5 minutes, sulfuric acid was added to terminate the reaction. The plate was then read using a SPECTRAmax plus 384 (Molecular Devices). The reading wavelength was 450 nm. To calculate the amount of antigen present in the sample, a standard curve was drawn based on serially diluted recombinant Notch3 protein using Prism 6 (GraphPad Software).

[0342] result

[0343] Heparan sulfate proteoglycans (HSPGs) are glycosaminoglycans found in hundreds of proteins in the plasma membrane and extracellular matrix. Protein-protein interactions mediated by HSPGs play a key role in numerous processes associated with Alzheimer's pathology, including amyloid and tau pathology, as well as neurodegenerative disorders. The ability of various ApoE isoforms, including ApoEch, to bind heparin, a glycosaminoglycan commonly used to mimic HSPG-protein interactions, was investigated. Briefly, fractions containing the ApoE isoforms ApoE2 and ApoE4 eluted from a heparin column under an increasing NaCl gradient (0-0.65 M) were analyzed using ELISA. Figure 9 As shown, the ApoE variant ApoE4, which is associated with a higher risk of Alzheimer's disease, has a higher affinity for heparin than the ApoE2 variant, which is known to be protective. Next, fractions containing His-tagged ApoE2, ApoE3, ApoE4, and ApoE3ch eluted from the heparin column under an increasing NaCl gradient (0-0.65M) were analyzed by immunoblotting. Figure 10As shown, heparin binding of ApoE3ch was impaired and much lower than that of ApoE2. The affinity of ApoE isoforms for heparin was also analyzed using the heparin plate ELISA protocol as described in Materials and Methods. Figures 11A-11B As shown, ApoE3ch exhibited significantly lower levels of heparin binding, as ApoE3ch was released from the heparin column at much lower NaCl concentrations than those required for ApoE4 release.

[0344] Example 3: Generation of antibodies against wild-type ApoE and ApoEch mutant proteins

[0345] Materials and methods

[0346] Antibody competition assay: Antibodies were incubated with ApoE3 recombinant protein (50 μg / ml in 20 mM Tris-HCl) at a ratio of 1:10 and incubated at room temperature for 3 hours. A negative control containing only culture medium and a positive control containing only recombinant protein ApoE3 were used. The antibody / ApoE3 recombinant protein solution and control were passed through a heparin column and exposed to an increasing NaCl gradient (as described in Example 2 above). Fractions were collected and evaluated by ELISA and immunoblotting.

[0347] BCA Assay: First, fractions collected from the heparin column were screened using the bicinchoninic acid assay (BCA assay) (Pierce BCA Protein Assay Kit). The assay was performed using 200 μl of a mixture of reagents A and B and 25 μl of each fraction. A 96-well plate was incubated at 37°C for 30 minutes and read at 562 nm. The plate was read using a Synery2 microplate reader (BioTek Instrument, Inc.) and Gen5 version 1.11 software.

[0348] Immunoblotting: Immunoblotting confirmed the elution of ApoE3 recombinant protein in the fractions collected from the heparin-bound column. 10 μl of the fractions were diluted to a final volume of 40 μl using RIPA buffer (Cell Signaling Technology), 10x (DTT 1M) and 4x Laemmli buffer. TGX TMSamples were separated on precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), and probed with mouse anti-his tag (Novus bios) and IRDye 800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences).

[0349] ELISA: ELISA was used to test the affinity of antibodies designed against the heparin-binding domain of ApoE for ApoE3 and ApoEch mutant recombinant proteins. Ni-NTA HisSorb plates (Qiagen) were washed three times with wash buffer 1 (DY008). ApoE recombinant protein was suspended in buffer (DY008) to produce a final concentration of 0.5 ug / ml. The plates were incubated with 200 μl of ApoE recombinant protein for 2 hours and washed five times with 1x wash buffer (DY008). The plates were then incubated overnight at 4°C with serially diluted antibodies from 1:1,000 to 1:32,000. The plates were then washed five times in 1X wash buffer (DY008) and incubated with anti-mouse HRP (Abcam; ab97046, 1:10,000) for 45 minutes, followed by five washes with 1x wash buffer to ensure complete removal of unbound secondary antibody. Sulfuric acid from an ELISA kit (DY008) was heated to 37°C. 100 μl of tetramethylbenzidine (Millipore) was added to initiate the detection phase of the reaction. After a 5-minute incubation, sulphuric acid was added to terminate the reaction. The plate was then read using a Synery2 microplate reader (BioTek Instrument, Inc.) and Gen5 version 1.11 software.

[0350] result

[0351] Monoclonal antibodies were generated against amino acids 130 to 143 of ApoE and tested for their effect on binding between full-length ApoE3 protein and heparin. Briefly, full-length wild-type ApoE3 protein or those pre-incubated with monoclonal antibodies were passed through a heparin column and cycled five times to ensure maximum ApoE3 binding. The column was then washed five times with 20 mM Tris-HCl (pH = 7.5) and exposed to an increasing NaCl (0 to 1 M) gradient in 20 mM Tris-HCl (pH = 7.5). The eluates from the Tris-HCl washes and from the different NaCl concentrations were collected. Figures 12A-12C ).

[0352] The fractions collected from the column were first screened by the bicinchoninic acid (BCA) assay. Figure 13A As shown in FIG, the protein signal of wild-type ApoE3 was detected in the fraction on the right side of the curve, indicating strong binding of ApoE3 to heparin. In contrast, when ApoE3 was pre-incubated with a monoclonal antibody, a strong signal was observed in the early fraction with low ionic strength ( Figure 13B To verify the results, immunoblotting was used to analyze the column washes and NaCl gradient fractions collected from the heparin column. Figure 14 As shown, pre-incubation of wild-type ApoE3 with a monoclonal antibody (A3Ab) (designated 1343 in Arboleda-Velasquez et al., Nature Medicine, 25, pages 1680-1683 (2019)) reduced its ability to bind heparin to a level similar to that of the ApoE3ch mutant protein. The individual gels were combined to generate Figure 14 These results suggest that antibodies could be used to alter the binding properties of ApoE to heparin, thereby preventing or treating Alzheimer's disease or related dementias or neurodegenerative disorders.

[0353] To generate monoclonal antibodies against the ApoE heparin binding domain, mice were immunized with wild-type ApoE peptide: KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) and ApoEch peptide: KLH-CTEELRVSLASHLRK-CONH2 (SEQ ID NO: 55). Cysteine ​​residues were added to the N-terminus to promote the binding of the peptide. Cell fusions were obtained from positive clones and cell suspensions tested for activity against wild-type and mutant peptides and proteins. As described in the Materials and Methods section, seven antibodies generated were analyzed by ELISA as examples. The 19G10-2 antibody serum showed specificity for both the full length and C-terminus of the APOE3ch mutant protein and some interactions with the wild-type APOE3 protein ( Figure 15A ). The 23B2 antibody showed reactivity to both the wild-type ApoE3 and ApoE3ch mutant C-terminal and full-length recombinant proteins ( Figure 15B ). The 2H79-1 antibody showed nonspecific binding to bovine serum albumin (BSA) and showed no affinity for wild-type ApoE3 or ApoE3ch mutant ( Figure 15C Both 30E1-2 and 16H8 antibodies showed reactivity to full-length and C-terminal ApoE3ch mutant proteins and the C-terminal form of wild-type ApoE3, but did not react with full-length wild-type APOE3 protein ( Figure 15D and 15EThe 25F1-2 antibody serum showed high affinity for both full-length and C-terminal ApoE3ch mutant proteins, appeared to have variable binding to the C-terminus of the wild-type ApoE3 protein, and had some interaction with full-length wild-type ApoE3 ( Figure 15F ). The 29G10-2 antibody showed high affinity to the full-length and C-terminal ApoE3ch mutant protein, and also showed reactivity to C-terminal wild-type ApoE3 and BSA. Finally, the 29G10-2 antibody did not interact with the full-length wild-type ApoE3 protein ( Figure 15G ).

[0354] The variable heavy chain (VH), variable light chain (VL), and complementarity determining region (CDR) sequences of 25F1-2 and 19G10-2 are described herein.

[0355] In addition, the following parental clones specific for wild-type ApoE were generated and tested for specificity to wild-type ApoE peptide (WT peptide), wild-type ApoE protein (WT protein), mutant ApoE peptide (ApoE ch; Mut peptide) and mutant ApoE 3ch protein (Mut protein) as shown in Table 4. These values ​​represent the absorbance levels detected by ELISA. The bold clones show specificity to wild-type ApoE peptide (KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) and wild-type ApoE protein).

[0356] Table 4

[0357] WT peptide WT protein Mut protein Mut protein 1D5 1.742 1.086 0.060 0.062 1H4 2.578 2.113 0.070 0.056 3A6 2.412 0.733 0.059 0.056 7C3 1.698 1.245 0.064 0.051 7C4 2.097 0.586 0.057 0.056 7C11 1.282 0.689 0.058 0.055 16G6 0.739 0.449 0.076 0.067 Positive control 1.980 1.171 0.254 0.144 Negative control 0.068 0.051 0.056 0.048

[0358] Example 4: Production of fusion proteins containing the APOE heparin binding domain

[0359] Materials and methods

[0360] Peptide competition assay: Wild-type ApoE3 and ApoE3ch mutant peptides (50 μg / ml) were incubated with ApoE3 recombinant protein (50 μg / ml, prepared in 20 mM Tris-HCl) at room temperature for 3 hours. The peptide / ApoE3 recombinant protein solution was then passed through a heparin column and exposed to an increasing NaCl gradient (as described in Examples 2 and 3 above). Fractions were collected and evaluated by immunoblotting.

[0361] Immunoblotting: Immunoblotting confirmed the elution of ApoE3 in fractions collected from the heparin-bound column. Fractions were diluted to a final volume of 40 μl in 10 μl RIPA buffer (Cell Signaling Technology), 10X (DTT 1M) and 4X Laemmli buffer. TGXTM Samples were separated on precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), and probed with mouse anti-his tag (Novus biologicals) and IRDye800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences).

[0362] result

[0363] Peptides containing amino acids 130-143 of wild-type ApoE protein and ApoEch mutant protein, respectively, were generated. To examine the effect of these peptides on the binding between wild-type ApoE3 recombinant protein and heparin agarose, peptide competition assays were performed as described in Materials and Methods. Figure 16 As shown, the wild-type ApoE peptide caused a one-fraction shift in the binding of wild-type ApoE3 recombinant protein, indicating that the peptide can compete with wild-type full-length ApoE3 for heparin binding. These results suggest that ApoE fragments containing amino acids 130-143 of the wild-type ApoE protein can be used to alter the binding properties of ApoE to heparin, thereby preventing or treating Alzheimer's disease or related dementias or neurodegenerative disorders.

[0364] In order to increase the protein stability of the peptide, the backbone Fc IgG2 constructs pfuse-hfc1 and pfcn-hg2 (Invivogen) were used to generate C-terminal and N-terminal fusion proteins containing the heparin binding domain or the allosteric regulatory site of the heparin binding domain of human ApoE (wild type and R136S mutant forms). The human ApoE fragment excludes the sites of APOE2 and APOE4 variants. The administration of ApoE fragments with R at position 136 can compete with endogenous ApoE for interactions with binding partners including HSPG, leading to protection against neurodegeneration. The administration of ApoE fragments with S at position 136 can bind to molecules that do not bind to wild-type ApoE, leading to protection against neurodegeneration. The amino acid sequences of the fragments from wild-type and R136S mutant ApoE used to generate fusion proteins are shown below (the R136 position is marked in bold and double underlined).

[0365] Downstream of the R136 fragment

[0366] WT

[0367]

[0368] mutant

[0369]

[0370] Upstream of the R136 fragment

[0371] WT

[0372]

[0373] mutant

[0374]

[0375] Figures 17A-17D Model showing the interaction of ApoE fragments with heparin. Figure 17A Model showing the wild-type ApoE fragment (downstream of the R136 fragment, helix) interacting with heparin. Figure 17B A model of the ApoE R136S fragment (downstream of the R136 fragment, helix) interacting with heparin is shown. Figure 17C Model showing the wild-type ApoE fragment (upstream of the R136 fragment, helix) interacting with heparin. Figure 17D A model of the ApoE R136S fragment (upstream of the R136 fragment, helix) interacting with heparin is shown.

[0376] The nucleic acid and amino acid sequences of exemplary fusion protein constructs containing the downstream of the R136 fragment or the upstream of the R136 fragment are shown below.

[0377] 184Q pfcn-hg2 ApoE 114-144

[0378] Nucleic acid sequence (SEQ ID NO:81)

[0379]

[0380]

[0381] Amino acid sequence (SEQ ID NO: 82)

[0382]

[0383] 184R pfcn-hg2 ApoE 114-144R136S nucleotide sequence (SEQ ID NO: 83)

[0384]

[0385]

[0386]

[0387] Amino acid sequence (SEQ ID NO: 84)

[0388]

[0389] 197F pfuse hfc2 ApoE 114-144

[0390] Nucleic acid sequence (SEQ ID NO:85)

[0391]

[0392]

[0393] Amino acid sequence (SEQ ID NO: 86)

[0394]

[0395] 197G pfuse-hfc2 ApoE 114-144R136S nucleic acid sequence (SEQ ID NO: 87)

[0396]

[0397] Amino acid sequence (SEQ ID NO: 88)

[0398]

[0399] 184U pfcn hg2 ApoE 129-157 nucleic acid sequence (SEQ ID NO:89)

[0400]

[0401]

[0402] Amino acid sequence (SEQ ID NO:90)

[0403]

[0404] 184V pfcn hg2 129-157R136S nucleic acid sequence (SEQ ID NO:91)

[0405]

[0406]

[0407]

[0408] Amino acid sequence (SEQ ID NO:92)

[0409]

[0410] 197H pfuse hfc2 ApoE 129-157 nucleic acid sequence (SEQ ID NO:93)

[0411]

[0412]

[0413] Amino acid sequence (SEQ ID NO:94)

[0414]

[0415] 197I pfuse hfc2 ApoE 129-157R136S nucleic acid sequence (SEQ ID NO:95)

[0416]

[0417]

[0418] Amino acid sequence (SEQ ID NO: 96)

[0419]

[0420] Example 5: CRISPR-Cas9-mediated editing and base editing of APOE

[0421] In order to introduce the R136S mutation in APOE using CRISPR-Cas9, a gRNA sequence was designed (Table 5). In one embodiment, the gRNA sequence was cloned into lentiCRISPR v2 using two oligonucleotides to form a linker containing a 20-base sequence, which was cloned into the BsmB1 site downstream of the U6 promoter. To support repair, a template with two additional silent mutations was designed. The template has 50 bases located on both sides of the mutation region. Exemplary template sequences are as follows:

[0422]

[0423] The silent mutation that eliminates the PAM motif is double underlined, the codon corresponding to the R136S mutation is in bold, and the silent mutation that creates a Sad site for cleavage of the PCR product in the clone receiving the template is in italics.

[0424] Table 5: gRNA design for introducing the R136S mutation

[0425]

[0426] Without wishing to be bound by theory, the mechanism proposed for APOE3ch mutation is the loss of function (e.g., in combination with HSPG). Therefore, CRISPR-Cas9 is used to design gRNA sequences to "knockout" APOE (Table 6). Design gRNA to target exon 3 (amino acids 1-61) of ApoE. In one example, by using 2 oligonucleotides, the gRNA sequence is cloned into lentiCRISPRv2 to form a joint containing 20 base sequences, which is cloned into the BsmB1 site downstream of the U6 promoter. Repair is accomplished by non-homologous end joining (NHEJ), which is an error-prone process and generally results in short insertions or deletions, thereby causing APOE to be knocked out.

[0427] Table 6: gRNA design for APOE knockout

[0428]

[0429] The R136H mutation in APOE is expected to have a similar effect to the R136S mutation. Therefore, in order to introduce the R136H mutation in APOE using base editing technology, the following gRNA was designed (Table 7). The gRNA sequence GAGGCGCACCCGCAGCTCCT was cloned into pLenti sgRNA (addgene 71409), and two oligonucleotides were used to form a linker containing a 20-base sequence, which was cloned into the BsmB1 site downstream of the U6 promoter. Plasmid Addgene base editor plasmid pCMV-BE3 (#73021) was used to generate base editors.

[0430] Table 7: gRNA design for APOE base editing

[0431]

[0432] Example 6: High-throughput screening of molecules that modify ApoE and heparin binding

[0433] In order to screen molecules that affect the binding of ApoE to heparin, ApoE protein is pre-incubated with candidate polypeptides, small molecules, nucleic acids, lipids or carbohydrates. The pre-incubated ApoE protein is introduced into a heparin-coated surface (such as a plate or column) so that it binds to heparin / HSPG / GAG and washes away unbound ApoE and candidate molecules. Alternatively, before applying the ApoE protein, the heparin-coated surface (such as a plate) can be pre-incubated with a candidate molecule. The level of ApoE binding to heparin is detected using antibodies, protein assays or fluorescence, and the effect of the candidate molecule on ApoE / heparin binding is evaluated. Candidate molecules that reduce ApoE / heparin binding can represent new therapies for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment in human subjects with such treatment needs. Examples of such molecules include EZ-482 (see, for example, Mondal et al. Biochemistry 55(18):2613-21, 2016. The structure of EZ-482 is shown below).

[0434]

[0435] Example 7: High-throughput screening of molecules that modify ApoE and bind to anti-ApoE antibodies

[0436] In order to screen the molecules that affect the binding of ApoE and anti-ApoE antibodies, the ApoE protein is pre-incubated with candidate polypeptides, small molecules, nucleic acids, lipids or carbohydrates. The pre-incubated ApoE protein is introduced into a surface (e.g., plate or column) coated with an antibody (such as any anti-ApoE antibody as described herein) that binds to the HSPG binding site of ApoE, so that it binds the antibody, and unbound ApoE and candidate molecules are washed away. Alternatively, before applying the ApoE protein, the surface (e.g., plate) coated with heparin can be pre-incubated with the available candidate molecules. Antibodies, protein assays or fluorescence detection are used to detect the level of anti-ApoE binding to anti-ApoE antibodies, and the impact of the candidate molecules on binding is evaluated. The candidate molecules that increase or decrease the binding of ApoE / heparin can represent a new therapy for preventing or treating the cognitive decline associated with dementia and / or mild cognitive impairment in human subjects with such treatment needs.

[0437] Example 8: Antibody Characterization

[0438] Antibodies binding to wild-type ApoE and ApoEch mutants were further evaluated using heparin affinity chromatography, immunoblotting, subclone analysis, monoclonal antibody screening, screening for selectivity between huApoE3 and msApoE, and in vivo subretinal injection.

[0439] method

[0440] Chromatographic experiments

[0441] At room temperature (RT), His-tagged recombinant ApoE peptide (50 μg / mL) was incubated for 3 hours with each antibody diluted 1:10 in 20mM Tris HCl buffer (pH 7.5). Heparin binding of the sample was tested using a heparin agarose column. In short, the column was brought to room temperature and washed 5 times with 20mM Tris-HCL. After collecting 10 μL for WB experiments, the protein input was loaded onto the column. The input cycle was passed through the column 5 times. The effluent was collected and the column was subsequently washed 5 times, and the recovered fractions were labeled "washings". For each 0.05M step of a NaCl salt gradient from 0 to 1M, 1mL fractions were recovered. The 5M fraction was also tested to ensure that the protein was completely released from the column. WB or ELISA was used to test the changes in ApoE binding to heparin.

[0442] Immunoblotting

[0443] Samples for Western blotting analysis were prepared by diluting 10 μL of each fraction in 4X sample buffer (Laemmli's SDS-Sample buffer, BP-110R, Boston bioproducts), 4 μL DTT (Sigma Aldrich), and 16 μL 1X RIPA buffer. Electrophoresis was performed under denaturing conditions using a vertical electrophoresis chamber (Biorad). Bands were separated on a 4-20% precast gel (Biorad) using a constant voltage (70 V for 15 min, 100 V for 1 h). Proteins were transferred to a nitrocellulose membrane (Millipore) for 1 hour at a constant voltage of 70 V. Before incubation with primary antibody (Anti-His, rb, 1:5000, Novus biological) and secondary antibody (Donkey anti-rb-800, 1:10000, Licor), membrane was blocked at room temperature for 1 hour using Odyssey blocking buffer (Licor) and washed with TBS-0.05% Tween 20 (Thermo fisher) for 3X 10 '. Image acquisition was completed using Odyssey scanner. Image J was used to analyze data. Data were normalized by input and expressed as the standardized intensity relative to fraction number (0=input, 0.05M NaCl step gradient increased in 1-27=20mM Tris HCl pH 7.5, 5M NaCl in 20mM Tris HCl pH 7.5).

[0444] ELISA

[0445] To test the selectivity of the antibodies for huApoE3WT, huApoE3ch, or msApoE, anti-his ELISA-coated plates were incubated with 0.0025 μg / μL target protein for 2 hours at room temperature with slow shaking. The wells were washed 5 times with 1X wash buffer (R&D) and then incubated overnight at 4°C on a shaker (100 μL / well) with serial dilutions of the Innovagen antibody of interest. The next day, the wells were washed 5 times with sample buffer and incubated with rabbit anti-mouse HRP conjugate buffer (1:10,000; 100 μL / well; Abcam) for 45 minutes. After washing 5 more times, the plates were incubated with chromogen A+B 1:1 to initiate the colorimetric reaction (100 μL / well). The reaction was stopped with 2N sulfuric acid (R&D stop solution, 50 μL / well), and the absorbance at 450 nm was detected spectroscopically.

[0446] ELISA was used to test the affinity of antibodies designed for the heparin binding domain of APOE to APOE3 and APOEch mutant recombinant proteins. The Ni-NTA HisSorb Plates (Qiagen) plates were washed 3 times with wash buffer 1 (DY008) and the APOE recombinant protein was suspended in buffer (DY008) to produce a final concentration of 0.5ug / ml. The plates were incubated with 200ul for 2 hours. The plates were then washed 5 times with 1x wash buffer (DY008) and then incubated overnight at 4°C with a serial dilution series of antibodies from 1:1000 to 1:32000. The plates were then washed 5 times in 1X wash buffer (DY008). The plates were then incubated for 45 minutes with anti-mouse HRP (Abcam; ab97046) (1:10000). The plates were then washed 5 times in 1x wash buffer to ensure complete removal of unbound secondary antibodies. Sulfuric acid from the ELISA kit (DY008) was heated to 37°C before adding 100 μl of tetramethylbenzidine (Millipore) to initiate the detection phase of the reaction. After a 5-minute incubation, sulphuric acid was added to terminate the reaction. The plate was then read using a Synery 2 microplate reader (BioTek Instrument. Inc) and Gen5 version 1.11 software.

[0447] result

[0448] Antibody 1H4 was evaluated using heparin affinity chromatography and immunoblotting. ApoE3 was incubated with a negative control (vehicle, top blot) or 1H4 (bottom blot), and fractions were subjected to heparin affinity chromatography and immunoblotting ( Figure 18A The ApoE3-positive band is indicated by an arrow and was detected using an anti-his tag antibody (rb, 1:5,000) that specifically detects the his tag of recombinant human APOE. Figure 18B Display by Figure 18A Quantification of WB blot bands detected with the anti-his tag antibody shown in . Intensity normalized to input. These results demonstrate that ApoE3 binding to heparin is reduced in the presence of antibody and that antibody 1H4 competes with ApoE for heparin binding. N = 2 independent experiments.

[0449] Next, subclonal analysis of 1H4 serum was performed. Figure 19 A-19D shows the ELISA results of 1H4-2 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), and ApoE3ch peptide (D). The results are expressed as the optical density at 450 nm versus the dilution factor of the serum tested. Figure 20 It is a comparison Figure 19 A-19D shows a graph of the results.

[0450] Figure 21 Representative ELISA profiles of serial dilutions of antibody 1H4 incubated with human recombinant ApoE3 or mouse recombinant ApoE3 are shown. The results indicate that the antibody preferentially binds to the human protein over the mouse protein and that antibody 1H4 is selective for human ApoE. Results are shown as mean optical density detected at 450 nm ± sem (n = 2).

[0451] Next, monoclonal 1H4 antibodies were purified from the cloned hybridomas and evaluated by ELISA. Figure 22 Results of ELISA experiments are shown.

[0452] Antibody 7C11 was evaluated using heparin affinity chromatography, immunoblotting, quantitative ELISA of chromatography fractions, and competitive ELISA for binding analysis.

[0453] Heparin affinity chromatography fractions of ApoE3 were incubated with negative control (vehicle, top blot) or antibody 7C11 (bottom blot) and immunoblotted ( Figure 23A The ApoE3-positive band is indicated by an arrow and was detected using an anti-his tag antibody (rb, 1:5,000) that specifically detects the his tag of the recombinant peptide. Figure 23B Display by Figure 23A Quantification of WB blot bands detected with the anti-his tag antibody shown in . Intensity was normalized to input. These results indicate that ApoE3 binding to heparin is reduced in the presence of the antibody and that 7C11 competes with ApoE for heparin binding.

[0454] Next, subclonal analysis of 7C11-1 serum was performed. Figure 24A-24D shows the ELISA results of 7C11-1 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), or ApoE3ch peptide (D). The results are shown as the optical density at 450 nm versus the dilution factor of the serum tested. Figure 25 It is a comparison Figure 19 A-19D shows the results in a graph. Next, the monoclonal 7C11-1 antibody was purified from the cloned hybridoma and evaluated by ELISA. Figure 26 Results of ELISA experiments are shown.

[0455] The 19G10-2 antibody was further evaluated using heparin affinity chromatography, immunoblotting, quantitative ELISA of chromatographic fractions, and ELISA for binding analysis. ELISA screening of the 19G10-2 antibody was performed against the heparin binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins. Figure 27 As shown, the 19G10-2 antibody exhibited specificity for both the full-length and C-terminal domains of the APOE3ch (amino acids 125 to 299) mutant recombinant protein, and some interaction with APOE3 WT.

[0456] ELISA analysis was performed on heparin affinity chromatography fractions of ApoE3 incubated with negative control (vehicle, top blot) or antibody 19G10-2 (bottom blot) ( Figure 28A The ApoE3-positive band is indicated by an arrow and was detected using an anti-his tag antibody (rb, 1:5,000) that specifically detects the his tag of the recombinant peptide. Figure 28B Display by Figure 28A Quantification of WB blots detected by the anti-his tag antibody shown in . Intensity is normalized to input. These results show that, although designed for the ApoE3ch-HSPG domain, 19G10-2 competes with wild-type ApoE for heparin binding and causes ApoE3 to bind to heparin reduced. Without wishing to be bound by theory, antibody 19G10-2 may recognize and / or stabilize conformational specific features of APOE (e.g., APOE polymers or aggregates) that are unlikely to bind heparin / HSPG / GAG.

[0457] Figure 29 Immunoblots of ApoE3 WT incubated with 19G10-2 serum antibody are shown. Top blot: The membrane was labeled with a secondary mouse probe to detect the 19G10-2 antibody. The bottom membrane was incubated with an anti-his tag as described above to detect the ApoE3-positive fraction. This analysis shows that the antibody-APOE complex (left blot; top and bottom) does not bind heparin, while free APOE binds heparin with high affinity (right blot; bottom).

[0458] Figure 30 Representative ELISA showing differential binding of serum and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3WT or ApoE3ch. The data demonstrates the superior selectivity of this antibody for the ApoE3ch variant. Figure 31 The Y-axis is an enlarged graph showing some limited binding of antibody 19G10-2 in the presence of ApoE3 WT (serum, gray; monoclonal, black). Next, monoclonal 19G10-2 antibodies were purified from cloned hybridomas and evaluated by ELISA. Figure 32 Results from ELISA experiments are shown. The signal of recognition of full-length WT APOE is higher than that of WT ApoE peptide, indicating that conformation-specific features are involved.

[0459] The 25F1-2 antibody was further evaluated using heparin affinity chromatography, immunoblotting, quantitative ELISA of chromatography fractions, and ELISA for binding analysis. Figure 33 ELISA screening of 25F1-2 antibodies against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown. Figure 33 As shown, the 25F1-2 antibody showed high affinity for the full-length and C-terminal APOE3 mutant proteins, however, it did not appear to have strong binding to the C-terminus of the APOE3WT protein and showed limited interaction with the APOE3WT full-length protein. The results are shown as optical density at 450 nm versus the dilution factor of the serum tested.

[0460] Immunoblotting was performed on heparin affinity chromatography fractions of ApoE3 incubated with negative control (vehicle, top blot) or antibody 25F1-2 (bottom blot). Figure 34A The ApoE3-positive band is indicated by an arrow and was detected using an anti-His tag antibody (rb, 1:5,000) that specifically detects the his tag of the recombinant peptide. Figure 34B Display by Figure 34A Quantification of WB blot bands detected with anti-his tag antibodies as shown. Intensities were normalized to input. These results indicate that, despite being designed against the ApoE3ch-HSPG domain, 25F1-2 competes with wild-type ApoE for heparin binding and results in decreased ApoE3 binding to heparin.

[0461] Figure 35Immunoblots of ApoE3 WT incubated with the 25F1-2 monoclonal antibody are shown. Top blot: The membrane was labeled with a secondary mouse antibody probe to detect 25F1-2. The bottom membrane was incubated with an anti-his tag as described above to detect the ApoE3-positive fraction. This analysis shows that the antibody-APOE complex (left blot; top and bottom) does not bind heparin, while free ApoE binds heparin with high affinity (right blot, bottom).

[0462] Figure 36 Representative ELISA showing the difference in binding between 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3WT or ApoE3ch. These results demonstrate the superior selectivity of this antibody for the ApoE3ch variant. Figure 37 yes Figure 36 The Y-axis of the graph shows the binding profile of antibody 25F1-2 in the presence of ApoE3 WT. Next, monoclonal 25F1-2 antibodies were purified from cloned hybridomas and evaluated by ELISA. Figure 38 Results from ELISA experiments are shown. The signals for some recognitions of full-length WT APOE were higher than those for WT ApoE peptide, indicating conformation-specific binding.

[0463] Mouse antibody 1343ab (renamed from 23B2) was evaluated using heparin affinity chromatography and immunoblotting, and quantitative ELISA of chromatography fractions. Figure 39 and 40 ELISA screening of the 1343 antibody against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown. 1343 exhibits reactivity to both the C-termini of APOE3 WT and APOE3ch mutants, as well as full-length recombinant APOE protein (mutant refers to the Christchurch mutant).

[0464] ApoE was immunoblotted in protein fractions eluted from the heparin column using an increasing NaCl gradient in the presence or absence of 1343 ( Figure 41A Individual blots were cropped between 25 and 50 kDa. Blank spaces separate individual blots. FT = flow through. ELISA was performed to quantify the differences in NaCl elution patterns of different ApoEs in the presence and absence of 1343 ( Figure 41B ). N = 3 columns per isomer were analyzed twice side by side on different days in independent experiments to quantify differences. Error bars depict the standard error of the mean.

[0465] Validated CDR sequences from mouse antibodies 1H4-2, 7C11-1, 19G10-2, 25F1-2, and 1343ab were grafted into human IgG frameworks (IgG2 or IgG4) to generate humanized counterparts.

[0466] Example 9: In vivo validation of ApoE antibodies

[0467] An intraocular model of inducible APOE-dependent Tau hyperphosphorylation (paired-helical fibril formation) was generated and used to test the ability of ApoE antibodies to inhibit Tau pathology, a hallmark of neurodegeneration. Figure 42A An exemplary experimental summary is shown.

[0468] Briefly, his-tagged recombinant human APOE3 was injected into B6;C3-Tg(Prnp-MAPT*P301S)PS19Vle / J mice (Jackson Laboratory 008169) (Yoshiyama et al. 53(3):337-51, 2007). This mouse model contains the human tau P301S mutation and is a validated animal model for Alzheimer's disease and other tauopathies such as frontotemporal dementia (see, for example, Bugiani et al. 58(6):667-77, 1999). Mice injected with PBS were used as controls.

[0469] like Figure 42B As shown, in the control retina of 6-week-old mice, paired helical fibers of phosphorylated tau (PHF) are absent in ganglion cells and their axonal fibers (the signal in blood vessels labeled with isolectin B4 is background signal). In contrast, administration of recombinant human APOE3 (his tag) triggered strong formation of PHFs detected with AT8 antibody. PHFs were strong in ganglion cell axons (arrows) and ganglion cell bodies. Human APOE was detected around ganglion cell bodies using anti-His antibody.

[0470] Mouse 1H4-2 antibody and humanized 1343Ah antibody were injected intravitreally into the eyes of mice from the above mouse model (final volume 2 μL). The animals were sacrificed on day 3 after injection, and the retinas were dissected and immunolabeled. The retinas were stained with DAPI, isolectin, and AT8 (pTAU), which can identify phosphorylated paired helical fibril tau (PHF tau). The retinas were imaged using an SP8 confocal microscope. Figure 43B 、 43CAs shown in Figures 43F and 43G, APOE3 WT resulted in a significant increase in PHF tau, which was significantly reduced by 1H4-2 (***p < 0.001). Similarly, administration of the humanized 1H4-2 IgG2 / κ recombinant monoclonal antibody effectively reduced the induction of APOE-dependent PHF tau pathology in vivo ( Figure 43H ; **p<0.01, ***p<0.001). Figure 43D and 43H As shown, humanized 1343Ah significantly reduced PHF Tau levels (**p<0.01, ***p<0.001).

[0471] The similarity in efficacy between the mouse monoclonal antibody and the corresponding humanized antibody confirmed the affinity of the binding domain for ApoE or ApoEch, and that the binding properties were retained during the humanization process. This suggests that the binding properties of the CDRs can be transferred from the original mouse IgG1 to other proteins, including human IgG2.

[0472] The binding affinity between ApoE3 and monoclonal antibody 1H4 (mAb 1H4) was determined using the advanced kinetics module (Blitz Pro, FB-609928, version 1.3.1.3) of the BLItz system. Briefly, the protein A biosensor was loaded with mAb 1H4 and the association and dissociation constants were determined at concentrations of the full-length ApoE3 protein (Innovagen) increasing from 0 to 571.4 nM. The following run settings were used: an initial baseline of 30 s in the experimental medium, 120 s loading of the ligand (mAb 1H4) on the biosensor, and a new baseline of 30 s before the binding (120 s) and dissociation (120 s) steps. A total of 6 runs of 420 s were performed at 2200 rpm and room temperature to determine the binding parameters of ApoE3. Global fitting and step correction were performed for the dissociation experiments using BLItz software (version 1.1.0.7). Figure 44 Representative binding measurements of ApoE3 protein and 1H4 at increasing concentrations (nM) on a Protein A biosensor are shown. KD, Ka, and Kd were calculated using the BLItz system (Table 8). The upper graph represents the association step, and the lower graph represents the dissociation step of the binding kinetics. The X-axis represents time in seconds and the Y-axis represents binding in nM, respectively.

[0473] Table 8

[0474]

[0475] Example 10: In vivo validation of APOE fragment fusion protein

[0476] Next, a similar in vivo model was used to test a fusion protein containing an APOE fragment that includes the HSPG binding domain and the Fc region of human IgG. Briefly, 0.78 μg of recombinant full-length APOE was used to induce tau pathology. 0.14 μg of the fusion protein was injected intravitreally into mice. Figure 43E and 43I As shown, the fusion protein reduced APOE-dependent tau pathology in neurons.

[0477] Example 11: Sequence of chimeric antibody

[0478] The chimeric antibody sequences with CDRs transferred from mouse to human IgG2 or IgG4 are shown below:

[0479] Normal font = carrier

[0480] Italics = signal peptide

[0481] Underline = VL / VH

[0482] Double underline = constant part (human κ / IgG4 / IgG2)

[0483] Sequence of the expression vector for mAb 1H4 IgG2 / κ:

[0484] >p1H4.VL.hk

[0485]

[0486]

[0487]

[0488] >p1H4.VH.hIgG2

[0489]

[0490]

[0491]

[0492] Sequence of the expression vector for mAb 1343Ah IgG2 / κ:

[0493] >p1343Ah.VL.hk

[0494]

[0495]

[0496] >p1343Ah.VH.hIqG2

[0497]

[0498]

[0499]

[0500] Sequence of the expression vector for mAb 19G10 IgG4 / κ:

[0501] >p19G10.VL.hk

[0502]

[0503]

[0504]

[0505] >p19G10.VH.hIgG4

[0506]

[0507]

[0508]

[0509] Sequence of the expression vector for mAb 25F1 IgG4 / κ:

[0510] >p25F1.VL.hk

[0511]

[0512]

[0513]

[0514] >p20F1.VH.hIgG4

[0515]

[0516]

[0517]

[0518] Sequence of the expression vector for mAb 7C11.1 IgG2 / κ:

[0519] >p7C11.1.VL.hk

[0520]

[0521]

[0522]

[0523] > p7C11.1.VH.hIgG2

[0524]

[0525]

[0526]

[0527]

[0528] References

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[0540] 12.Rose,A.S.,et al.NGL viewer:web-based molecular graphics for largecomplexes.Bioinformatics 34,3755-3758(2018).

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[0581] Other implementation plans

[0582] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. An anti-ApoE antibody, wherein the antibody comprises a heavy chain variable region (VH) comprising VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region (VL) comprising VLCDR1, VLCDR2 and VLCDR3, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of the antibody: (i) consisting of the amino acid sequences described in SEQ ID No: 7, 8, 9, 4, 5, 6, respectively; or (ii) consisting of the amino acid sequences described in SEQ ID Nos: 17, 18, 19, 14, 15, and 16, respectively.

2. The antibody according to claim 1, wherein: The VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of the antibody are composed of the amino acid sequences recorded in SEQ ID Nos: 17, 18, 19, 14, 15, and 16, respectively, and wherein the VH and the VL of the antibody comprise amino acid sequences that are at least 80% identical to the amino acid sequences recorded in SEQ ID NOs: 22 and 23, respectively.

3. The antibody according to claim 1, wherein The VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of the antibody are composed of the amino acid sequences recorded in SEQ ID Nos: 7, 8, 9, 4, 5, and 6, respectively, and wherein the VH and the VL of the antibody comprise amino acid sequences that are at least 80% identical to the amino acid sequences recorded in SEQ ID NOs: 13 and 12, respectively.

4. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

5. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a human kappa or human lambda light chain constant region.

6. The antibody according to any one of claims 1 to 3, wherein the antibody is a whole antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, Fv, scFv, sc(Fv)2, Fab or F(ab')2.

7. The antibody of any one of claims 1 to 3, further comprising a half-life extending moiety.

8. The antibody according to any one of claims 1 to 3, further comprising a blood-brain barrier penetrating portion.

9. The antibody according to any one of claims 1 to 3, further comprising a detectable label.

10. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9.

11. One or more polynucleotides encoding the antibody according to any one of claims 1 to 6.

12. One or more vectors comprising one or more polynucleotides according to claim 11.

13. A host cell comprising one or more polynucleotides according to claim 11, or one or more vectors according to claim 12.

14. A method for preparing an anti-ApoE antibody, the method comprising: (a) culturing the host cell according to claim 13 under conditions that allow expression of the antibody; and (b) isolating the antibody.

15. The method of claim 14, further comprising formulating the antibody into a sterile formulation suitable for administration to humans.

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