Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0085—Brain, e.g. brain implants; Spinal cord
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the invention relates to anti-ApoE antibodies and compositions thereof.
• the invention also relates to compositions and methods for delaying and/or preventing ⁇ amyloidosis.
• the invention also relates to compositions and methods for delaying and/or preventing ⁇ plaque associated symptoms and/or cerebral amyloid angiopathy (CAA) associated symptoms, such as those associated with Alzheimer's disease (AD) or CAA in a subject.
• CAA cerebral amyloid angiopathy
• the invention relates to modulating the concentration of amyloid- ⁇ ( ⁇ ) in the brain of a subject.
• AD Alzheimer's disease
• AD Alzheimer's disease
• It is currently estimated to afflict over 5 million people in the United States, with an expected increase to 13 million by the year 2050.
• Alzheimer's disease leads to loss of memory, cognitive function, and ultimately loss of independence. It takes a heavy personal and financial toll on the subject and the family. Because of the severity and increasing prevalence of the disease in the population, it is urgent that better treatments be developed.
• Cerebral amyloid angiopathy occurs in about 90% of individuals who develop AD, as well as in some individuals independently of AD. CAA can lead to ischemic and hemorrhagic strokes causing severe disability or death. There are no current treatments for CAA.
• AD amyloid- ⁇
• the neuropathologic and neurochemical hallmarks of AD include synaptic loss and selective neuronal death, a decrease in certain neurotransmitters, and the presence of abnormal proteinaceous deposits within neurons (neurofibrillary tangles) and in the extracellular space (cerebrovascular, diffuse, and neuritic plaques).
• the characteristic features of CAA include the buildup of fibrillar forms of ⁇ in penetrating and leptomeningeal arterioles on the surface of the cerebral cortex. CAA can lead to ischemic or
• the main constituent of the plaques seen in AD and CAA is ⁇ , a 38-43 amino acid sequence peptide cleaved from the amyloid precursor protein (APP).
• APP amyloid precursor protein
• soluble ⁇ is secreted primarily by neurons, but also other cell types. Excessive ⁇ deposition may result from increased ⁇ synthesis, e.g. as occurs in familial early-onset AD and in some cases of familial early onset CAA, decreased ⁇ clearance in the brain, or increased ⁇ fibrillogenesis.
• ⁇ over-production occurs in the more common late-onset forms of AD suggests that insufficient ⁇ clearance may drive ⁇ deposition and amyloid plaque formation and CAA as well.
• the apolipoprotein E gene (ApoE) remains the most widely replicated genetic risk factor for late-onset AD and CAA, with carriers of the ⁇ 4 allele having a 3-15-fold greater risk as well as an earlier age of disease onset.
• ApoE4 carriers represent over 60% of the AD population whereas the ⁇ 2 allele is least represented in AD and may be protective in some populations.
• the human ApoE isoforms differ at amino acid position 1 12 or 158 ( ⁇ 2 has Cys1 12, Cys158; ⁇ 3 has Cys1 12, Arg158; ⁇ 4 has Arg1 12, Arg158).
• Mouse ApoE and human ApoE share ⁇ 70% amino acid homology.
• ApoE4 has a greater propensity towards an unstructured, molten globule state and is more likely to form aggregates, as compared to ApoE2 and ApoE3.
• ApoE is mainly produced by glia and predominantly functions to distribute cholesterol and lipids to neurons.
• the majority of ApoE in the CNS is found on HDL-like lipoprotein particles, and iipidation of ApoE is regulated by the ABCA1 and ABCG1 transporters.
• ABCA1 knockout mice crossed to transgenic AD mice produce ApoE that is poor!y-lipidated and these mice have increased ⁇ plaques.
• the brains of AD transgenic mice overexpressing ABCA1 contain well- iipidated apoE lipoprotein particles and have reduced ⁇ plaque deposition. Binding of ApoE to LDLR, LRP1 , apoER2, and VLDLR, which are expressed on various neural cell types, enables the uptake of lipoprotein particles through receptor-mediated
• the amino acid difference at position 158 for ApoE2 confers decreased receptor binding to the LDLR and humans with the ⁇ 2/ ⁇ 2 genotype have increased risk for type III hyperlipoproteinemia.
• One aspect of the invention encompasses an isolated anti-ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 86, an L2 of SEQ ID NO: 30, an L3 comprising SEQ ID NO: 88, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 90, an H2 comprising SEQ ID NO: 92, an H3 comprising SEQ ID NO: 94, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 87, an L2 of SEQ ID NO: 30, an L3 comprising SEQ ID NO: 89, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 91 , an H2 comprising SEQ ID NO: 93, an H3 comprising SEQ ID NO: 95, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 78, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 80, an H2 comprising SEQ ID NO: 82, an H3 comprising SEQ ID NO: 84, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 79, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 81 , an H2 comprising SEQ ID NO: 83, an H3 comprising SEQ ID NO: 86, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 comprising SEQ ID NO: 107, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 109, an H2 comprising SEQ ID NO: 1 1 1 , an H3 comprising SEQ ID NO: 1 13, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 comprising SEQ ID NO: 108, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 1 10, an H2 comprising SEQ ID NO: 1 12, an H3 comprising SEQ ID NO: 1 14, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 23, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 26, an H2 comprising SEQ ID NO: 27, an H3 comprising SEQ ID NO: 28, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 29, an L2 of SEQ ID NO: 30, an L3 comprising SEQ ID NO: 31 , or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 32, an H2 comprising SEQ ID NO: 33, an H3 comprising SEQ ID NO: 34, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 47, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 48, an H2 comprising SEQ ID NO: 49, an H3 comprising SEQ ID NO: 50, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 51 , an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 52, an H2 comprising SEQ ID NO: 53, an H3 comprising SEQ ID NO: 54, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 55, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 56, an H2 comprising SEQ ID NO: 57, an H3 comprising SEQ ID NO: 58, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 59, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 60, an H2 comprising SEQ ID NO: 61 , an H3 comprising SEQ ID NO: 62, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 63, an L2 of SEQ ID NO: 30, an L3 comprising SEQ ID NO: 64, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 65, an H2 comprising SEQ ID NO: 66, an H3 comprising SEQ ID NO: 67, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 68, an L2 of SEQ ID NO: 24, an L3 comprising SEQ ID NO: 25, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 69, an H2 comprising SEQ ID NO: 70, an H3 comprising SEQ ID NO: 71 , or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 72, an L2 of SEQ ID NO: 73, an L3 comprising SEQ ID NO: 74, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 75, an H2 comprising SEQ ID NO: 76, an H3 comprising SEQ ID NO: 77, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 comprising SEQ ID NO: 123, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 124, an H2 comprising SEQ ID NO: 125, an H3 comprising SEQ ID NO: 126, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 99, an L2 of SEQ ID NO: 100, an L3 comprising SEQ ID NO: 101 , or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 102, an H2 comprising SEQ ID NO: 103, an H3 comprising SEQ ID NO: 104, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses an isolated anti- ApoE antibody comprising (a) a light chain variable region comprising an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 comprising SEQ ID NO: 1 17, or any combination thereof; and/or (b) a heavy chain variable region comprising an H1 comprising SEQ ID NO: 1 18, an H2 comprising SEQ ID NO: 1 19, an H3 comprising SEQ ID NO: 120, or any combination thereof.
• Compositions comprising the antibody including but not limited to pharmaceutical compositions, are contemplated herein.
• the antibody is a humanized antibody.
• Another aspect of the invention encompasses a method of improving a clinical sign of ⁇ amyloidosis which comprises administering an effective amount of an anti-ApoE antibody to a living human subject.
• the invention encompasses a method of effectively treating at least one clinically detectable ⁇ plaque associated symptom and/or CAA associated symptom which comprises administering an effective amount of an anti-ApoE antibody to a living human subject.
• the invention encompasses a method of treating Alzheimer's disease which comprises administering an effective amount of an anti-ApoE antibody to a living human subject.
• the invention encompasses a method of treating CAA which comprises administering an effective amount of an anti-ApoE antibody to a living human subject.
• the anti-ApoE antibody may be an antibody described above.
• compositions useful to treat at least one clinically detectable ⁇ plaque associated symptom comprising a pharmaceutically effective amount of an anti- ApoE antibody adapted for administration to a living human subject.
• the anti-ApoE antibody may be an antibody described above.
• the medicinal composition is effectively administered to a living subject system ically..
• compositions useful to treat at least one clinically detectable CAA associated symptom comprising a pharmaceutically effective amount of an anti-ApoE antibody adapted for administration to a living human subject.
• the anti-ApoE antibody may be an antibody described above.
• the pharmaceutical composition is effectively administered to a living subject system ically..
• compositions useful to treat Alzheimer's disease comprise a pharmaceutically effective amount of an anti-ApoE antibody adapted for administration to a living human subject.
• the anti-ApoE antibody may be an antibody described above.
• the pharmaceutical composition is effectively administered to a living subject system ically.
• compositions useful to treat CAA comprise a pharmaceutically effective amount of an anti-ApoE antibody adapted for administration to a living human subject.
• the anti-ApoE antibody may be an antibody described above.
• the pharmaceutical composition is effectively administered to a living subject
• kits comprising a container containing a pharmaceutical composition comprising a pharmaceutically effective amount of an anti-ApoE antibody adapted for administration to a living human subject and any medical devices to be used for said administration.
• the anti-ApoE antibody may be an antibody described above.
• FIG. 1 depicts an image of a Western blot.
• the brain lysates from ApoE KO mice or mice expressing ApoE3, ApoE4 or murine ApoE were immunoblotted with GA-50, HJ151 , or HJ152.
• the Western blot shows that HJ151 is ApoE4 specific and HJ152 recognizes both ApoE3 and ApoE4.
• GA-50 is a positive control.
• FIG. 2 depicts an image of a Western blot.
• the brain lysates from ApoE KO mice or mice expressing ApoE3, ApoE4 or murine ApoE were immunoblotted with HJ153 or HJ154.
• the Western blot shows that HJ153 and HJ154 recognize both ApoE3 and ApoE4.
• FIG. 3 depicts images of brain tissue from 5XFAD APP transgenic mice expressing different human ApoE isoforms and stained using biotinylated HJ153 or HJ154 antibodies. The figure shows these two antibodies stain ApoE in the neuropil, in astrocytes, and in amyloid plaques if plaques are present. Brain tissue from ApoE KO mouse was used as negative control.
• FIG. 4 depicts images of several immunoblots.
• Samples containing ApoE2, ApoE3 or ApoE4 were immunoprecipitated using HJ152, HJ153 or HJ154.
• Materials immunoprecipitated with anti-ApoE antibodies (labeled as IP) and the solution after immunoprecipitation (labeled as post IP) were immunoblotted after running an SDS-PAGE gel and transfer to nitrocellulose membrane using GA-50, a commercial anti-ApoE antibody.
• WUE4 a monoclonal antibody against human ApoE, was used as a positive control.
• the results showed that HJ152 was able to immunoprecipitate some of the ApoE from the samples while there was still some ApoE remaining in the post IP product.
• HJ153 and HJ154 were able to immunoprecipitate all ApoE from the samples.
• FIG. 5A-H depicts graphs showing aspects of various antibodies. Specifically, FIG. 5A-D graphically shows the results of an ELISA using various antibodies. ELISA plates were coated with 0.5 pg/ml of recombinant apoE2 (square), apoE3 (open circle), or apoE4 (closed circle). Then different concentrations of (A)
• HJ151 , (B) HJ153, (C) HJ154, or (D) HJ156 were loaded on the plates. Horse-radish peroxidase labeled goat anti-mouse secondary antibodies were used to detect binding. The results show that HJ153 and HJ154 bind ApoE2, ApoE3 and ApoE4 as detected in the ELISA. HJ151 is specific for ApoE4 and HJ156 binds only ApoE3 and ApoE4.
• FIG. 5E-H are graphs depicting surface plasmon resonance profiles for various antibodies.
• Anti-ApoE antibodies were serially diluted 3-fold (starting at 100 nM for HJ153 (E), and 1000 nM for HJ151 (F), and HJ1 156 (G)) for detection of binding to biotinylated- recombinant apoE4 captured on a streptavidin chip. Samples were injected at a flow rate of 30 ⁇ /minute. (H) Apparent KD values of HJ151 , HJ153 and HJ156 were calculated based on the SPR experiment.
• FIG. 6 depicts a graph showing some anti-ApoE antibodies decrease ⁇ plaques in APP/PS1 -21 E4/E4 mice after ICV infusion.
• APP/PS1 -21 E4/E4 mice received continuous intracerebroventricular (ICV) infusion of PBS (negative control), mouse lgG2ab (negative control), HJ5.1 (anti- ⁇ antibody, positive control), or anti-apoE antibody (HJ151 , HJ154, and HJ156) beginning at 2 months of age.
• ICV intracerebroventricular
• Anti- ApoE antibody or control antibody (2mg/ml) was filled into a subcutaneous osmotic minipump (Alzet, model 2006) and infused through a surgically implanted catheter into the left lateral cerebral ventricle (bregma -0.4 mm, 1 .0 mm lateral to midline, 2.5 mm below the skull), infusing fluid at the speed of 1 .2 ⁇ /min for 6 weeks.
• the mice were perfused and the sections were stained for ⁇ plaques using anti- ⁇ antibody HJ3.4B.
• the percent of area covered by plaques in the cerebral cortex dorsal to hippocampus was quantified.
• FIG. 7 depicts a graph showing some anti-ApoE antibodies decrease fibrillar plaque load after ICV infusion in APP/PS1 -21 E4/E4 mice.
• APP/PS1 - 21 E4/E4 mice received continuous intracerebroventricular (ICV) infusion of PBS (negative control), mouse lgG2ab (negative control), HJ5.1 (anti- ⁇ antibody, positive control), or anti-apoE antibody (HJ151 , HJ154, and HJ156) from 2 months of age.
• ICV intracerebroventricular
• Anti- apoE antibody or control antibody (2mg/ml) was filled into a subcutaneous osmotic minipump (Alzet, model 2006) and infused through a surgically implanted catheter into the left lateral cerebral ventricle (bregma -0.4 mm, 1 .0 mm lateral to midline, 2.5 mm below the skull) at the speed of 1 .2 ⁇ /min for 6 weeks.
• the mice were perfused and the brain sections were stained for fibrillar plaques using Thioflavine S. The area covered by plaques in the cerebral cortex dorsal to
• hippocampus was quantified.
• FIG. 8A-F depicts graphs showing that anti-ApoE antibodies decrease insoluble ⁇ 42 in APP/PS1 -21 E4/E4 mice after ICV infusion.
• APP/PS1 -21 E4/E4 mice received continuous intracerebroventricular (ICV) infusion of PBS (negative control), mouse lgG2ab (negative control), HJ5.1 (anti- ⁇ antibody, positive control), or anti-apoE antibody (HJ151 , HJ154, and HJ 156) from 2 months of age.
• ICV intracerebroventricular
• the mice were perfused and the cortical tissue was homogenized in PBS, Triton and 5M Guanidine sequentially.
• FIG. 9 depicts a graph showing the body weight of APP/PS1 -21 E4/E4 mice intraperitoneally injected with anti-ApoE antibodies (1 injection/week for 7 weeks).
• FIG. 10 depicts a graph showing that HJ151 , HJ155, and HJ156 do not significantly decrease soluble ⁇ 40 in the PBS fraction of brain tissue homogenate in APP/PS1 -21 E4/E4 mice intraperitoneally injected with anti-ApoE antibodies (1 injection/week for 7 weeks).
• FIG. 11 depicts a graph showing that HJ151 , HJ155, and HJ156 do not significantly decrease soluble ⁇ 42 in the PBS fraction of brain tissue homogenate of APP/PS1 -21 E4/E4 mice intraperitoneally injected with anti-ApoE antibodies (1 injection/week for 7 weeks).
• FIG. 12 depicts a graph showing that HJ156 significantly decreases insoluble ⁇ 40 in the 5M guandine fraction of brain tissue homogenate of APP/PS1 -21 E4/E4 mice intraperitoneally injected with anti-ApoE antibodies (1 injection/week for 7 weeks). ( * p ⁇ 0.05)
• FIG. 13 depicts a graph showing that HJ156 significantly decreases insoluble ⁇ 42 in the 5M guandine fraction of brain tissue homogenate of APP/PS1 -21 E4/E4 mice intraperitoneally injected with anti-ApoE antibodies (1 injection/week for 7 weeks).
• FIG. 14 is a graph depicting relative antibody concentration in the cortex of APPPS1 -21/APOE4 mice expressing recombinant (r) HJ151 , r HJ151 with D265A mutation ( ⁇ ), rHJ156, and rHJ156A.
• APPPS1 -21/APOE4 mice were injected at P0 with AAV2/8 that express full length rHJ151 and rHJ156 with or without the D265A mutation.
• antibody concentration in the PBS soluble fraction of cortex was measured by ELISA.
• the relative level of each antibody was calculated by using its hybridoma-derived, purified antibody as a standard.
• FIG. 15 depicts a graph showing that HJ151 and HJ156
• FIG. 16 depicts a graph showing that HJ151 and HJ156 reduce insoluble ⁇ 40 in the guanidine fraction of brain tissue homogenate of APP/PS1 -21 E4/E4 mice injected with anti-ApoE antibodies expressed in the brain with the use of an adenoassociated virus (AAV) 2/8 vector.
• AAV adenoassociated virus
• 17A-B depict graphs showing that HJ151 and HJ156 significantly reduce amyloid ⁇ (A) and fibrillar plaque area (B) in brain sections from APP/PS1 -21 E4/E4 mice injected with anti-ApoE antibodies expressed in the brain with the use of an adenoassociated virus (AAV) 2/8 vector.
• HJ151 and HJ156 with a D265A mutation have no effect.
• FIG. 18A-G depicts unfixed tissue sections (20 pm thickness) from APPPS1 -21/apoE 4 4 (APPPS1 -21/E4), 5XFAD/apoE knockout (5XFAD/EKO) mice and human apoE 4 4 (human E4) brains that were stained using biotinylated (“B") antibodies HJ3.4B (A), HJ151 B (B), HJ152B (C), HJ153B (D), HJ154B (E), HJ155B (F), and HJ156B (G).
• FIG. 19A-D shows antibody detection of ApoE in human plasma lipoprotein particles.
• A shows a schematic of a plasma binding assay used to demonstrate the detection of ApoE in lipoprotein particles from human plasma coated onto an ELISA plate, while
• B-D each show a graph of the results from the binding assay for various antibodies. Detection of ApoE is observed with HJ153, HJ154, HJ1510, HJ151 1 , HJ1517, HJ1530, and HJ1534.
• FIG. 20A-C shows antibody detection of ApoE in a competition ELISA.
• A shows a schematic of a plasma competition assay used in assessing preference for antibody binding to coated recombinant alipidated ApoE4 (recApoE; 0.5 ug/mL) after pre-incubation in various dilutions of human plasma, while
• B-C each show a graph of the results from competition ELISA for various antibodies, "x ApoE” is an abbreviation for "anti-ApoE antibody.”
• FIG. 21A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E2/E2 mice that were immunostained for ApoE with anti-ApoE antibody HJ156B at 20ug/ml (A,C) or 50ug/ml (B,D) at two different magnifications. Scale bars, 400 ⁇ .
• FIG. 22A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E3/E3 mice that were immunostained for ApoE with anti-ApoE antibody HJ156B at 20ug/ml (A,C) or 50ug/ml (B,D) at two different magnifications. Scale bars, 400 ⁇ .
• FIG. 23A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E4/E4 mice that were immunostained for ApoE with anti-ApoE antibody HJ156B at 20ug/ml (A,C) or 50ug/ml (B,D) at two different magnifications. Scale bars, 400 ⁇ .
• FIG. 24A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E4/E4 mice that were immunostained for ApoE with anti-ApoE
• FIG. 25A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E4/E4 mice that were immunostained for ApoE with anti-ApoE
• FIG. 26A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E4/E4 mice that were immunostained for ApoE with anti-ApoE
• FIG. 27A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E4/E4 mice that were immunostained for ApoE with anti-ApoE
• FIG. 28A-D shows images of unfixed frozen brain sections from APP/PS1 -21 E4/E4 mice that were immunostained for ApoE with anti-ApoE
• FIG. 33A-F shows images of brain sections immunostained for ⁇ with anti- ⁇ antibody (HJ3.4-biotin).
• A,D Brain sections from non-treated, 2 month old mice before the development of ⁇ plaques.
• B,E Brain sections from 3.5 month old mouse treated with PBS.
• FIG. 34A-F shows images of brain sections immunostained for ⁇ with anti- ⁇ antibody (HJ3.4-biotin).
• A,D Brain sections from 3.5 month old mouse treated with HJ151 at 50 mg/kg.
• B,E Brain sections from 3.5 month old mouse treated with HJ155 at 50 mg/kg.
• FIG. 35A-F shows images of brain sections stained with X-34 dye that recognizes fibrillar plaques.
• A,D Brain sections from non-treated, 2 month old mice before the development of ⁇ plaques.
• B,E Brain sections from 3.5 month old mouse treated with PBS.
• FIG. 36A-F shows images of brain sections stained with X-34 dye that recognizes fibrillar plaques.
• A,D Brain sections from 3.5 month old mouse treated with HJ151 at 50 mg/kg.
• B,E Brain sections from 3.5 month old mouse treated with HJ155 at 50 mg/kg.
• FIG. 37A-F are graphs depicting the binding profile of HJ151 , HJ153 and HJ156 with lipidated apoE.
• A,B ELISA following coating of plasma on plates. Plasma from apoE KO, APOE2, APOE3 and APOE4 mice was coated onto the plates. Chi153 and chi156 of different concentrations were loaded. The captured antibodies were detected with HRP-goat anti-human IgG antibody.
• C,D ELISA following coating of antibodies on plates. Plates were coated with HJ153 and HJ156. Plasma from mice with different genotypes was loaded. The captured ApoE was detected with HRP-goat-polyclonal anti-ApoE.
• Plasma competition experiment was performed via ELISA format. Recombinant ApoE4 was coated onto the plates. HJ151 (50 nM), HJ153 (4 nM), and HJ156 (50 nM) pre-incubated with serially diluted plasma from APOE4 Kl mice was loaded to the plates. The HJ15 antibodies bound to the plates were detected with HRP-Goat anti-mouse IgG antibodies.
• F Plasma antibody concentrations of HJ153, HJ156, or control IgG following IP injection into APOE4 Kl or EKO mice. HJ156 was dosed at 2, 10 and 50 mg/kg and plasma samples collected by submandibular puncture. HJ153 was dosed at 10 mg/kg.
• Control murine lgG2a (mslgG2a) was anti-Her2 and dosed at 10 mg/kg. Quantification of dosed antibodies in plasma was by antigen capture ELISA using coated recombinant apoE4 to detect HJ153 or HJ156 with recombinant Her2 used to detect the control IgG. Peripheral clearance of HJ156 is similar to Control IgG with target mediated clearance observed by day 14. HJ153 exhibited high clearance and reached lower limits by 48 hours.
• FIG. 38A-E are graphs depicting the binding of HJ151 and HJ156 with ApoE4 in the amyloid plaques in unfixed mouse brain sections and specificity for heat-induced aggregates of ApoE4.
• B Binding of HJ 151 , HJ153 and HJ156 to untreated recombinant ApoE4 (untreated) and ApoE4 that has been incubated at 40°C for 24 hr (40C).
• C Incubation of ApoE4 at 40°C for 24 hour results in the formation of aggregates recovered in the pellet fraction after
• FIG. 39A-D are images of HJ151 , HJ156 and control antibody binding to human ApoE4 in living mouse brain.
• Negative control lgG2ab HJ151 and (C) HJ156 conjugated with Alexa 594 were applied directly onto the surface of the brain in living APPPS1 -21/APOE4 mice that were 6 months of age and the binding of antibodies was observed under 2-photon microscopy. The amyloid was labeled using methoxy-X04.
• the middle panel is the higher power images of the area in the white frame of the top panel. Arrows indicate plaques.
• the bottom panel is the higher power images of the area in the yellow frame of the top panel. Arrows indicate CAA.
• D D
• Control hulgG or chi156 at 50 mg/kg body weight (i.p.) were injected for one (0 hour) or two doses (0 and 48 hour) and APPPS1 -21/APOE4 mice sacrificed at 48 hours after final injection.
• FIG. 40 depicts a graph showing that HJ156 activates microglia to facilitate plaque clearance.
• 4-month old APPPS1 -21/APOE4 mice that already had existing plaques were administered a short-term treatment of HJ151 and HJ156 antibodies (4 doses by IP injection every 3 days). Stained sections were analyzed for CD45+ microglia area relative to the amount of fibrillar plaques. HJ151 had no effect on the amount of activated microglia. * p ⁇ 0.05.
• FIG. 41 depicts staining of microglia and plaques after acute immunization of HJ156.
• FIG. 42A-B graphically shows the results of a chronic dose-range efficacy study.
• the mice were perfused with ice-cold PBS containing 0.3% heparin.
• the cerebral cortices were sequentially homogenized with cold PBS and 5 M guanidine buffer in the presence of 1 ⁇ protease inhibitor mixture.
• (A) ⁇ 40 and (B) ⁇ 42 in the guanidine fraction were determined by ELISA.
• One-way ANOVA followed by Tukey post-test was performed to compare different groups. Data were expressed as mean ⁇ SEM. * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 .
• FIG. 43A-C graphically shows the binding of (A) HJ151 , (B) HJ154, and (C) HJ156 antibodies to untreated recombinant and heat-induced aggregates of ApoE2, ApoE3, and ApoE4.
• the aggregated ApoE was induced by incubating ApoE at 1 mg/ml concentration at 40°C for 24 hours. The aggregates then were recovered in the pellet fraction following ultracentrifugation at 186,000 g for 1 hour.
• the untreated ApoE or heat-induced aggregates of ApoE were coated directly to ELISA plates at 0.5 g/ml overnight at 4°C.
• the method comprises effectively administering to a living subject a therapeutically effective amount of an anti- ApoE antibody that (a) binds to human ApoE4 with a KD between about 0.1 pM to about 10 ⁇ , or between about 0.1 pM to about 1 ⁇ , (b) preferentially binds recombinant alipidated human ApoE4 as compared to ApoE4 derived from human plasma or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4, and (c) binds to human ApoE in amyloid plaque in unfixed brain tissue.
• the present invention encompasses the discovery that anti-ApoE antibodies with these characteristics provide a treatment for subjects with ⁇ amyloidosis including, but not limited to, subjects diagnosed with a disease characterized by brain ⁇ plaques, subjects diagnosed with a disease characterized by vascular ⁇ plaques in the brain, subjects diagnosed with ⁇ plaque-associated symptoms, subjects diagnosed with CAA- associated symptoms, subjects with clinical signs of ⁇ amyloidosis that may or may not have ⁇ plaque associated symptoms and/or CAA associated symptoms, subjects diagnosed with Alzheimer's disease, and subjects diagnosed with CAA (collectively referred to, herein, as "subjects in need of treatment").
• Methods for identifying clinical signs of ⁇ amyloidosis in asymptomatic patients are known in the art and discussed below. I. Definitions
• subject refers to a human, or to a non-human animal expressing human ApoE.
• treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease/disorder.
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
• Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
• Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented.
• ⁇ refers to peptides derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP).
• APP amyloid precursor protein
• the gene encoding APP is located on chromosome 21 .
• ⁇ peptides are typically 37-43 amino acid sequences long, though they can have truncations and modifications changing their overall size. They can be found in soluble and insoluble compartments, in monomeric, oligomeric and aggregated forms, intracellular ⁇ or extracellularly, and may be complexed with other proteins or molecules.
• the adverse or toxic effects of ⁇ may be attributable to any or all of the above noted forms, as well as to others not described specifically.
• two such ⁇ isoforms include ⁇ 40 and ⁇ 42; with the ⁇ 42 isoform being particularly fibrillogenic or insoluble and associated with disease states.
• ⁇ amyloidosis is clinically defined as evidence of ⁇ deposition in the brain or blood vessels of the brain, typically in the form of amyloid plaques or CAA.
• Diseases associated with ⁇ amyloidosis include, but are not limited to, preclinical Alzheimer's disease, Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia, and inclusion body myositis.
• An "increased risk of developing a disease associated with ⁇ amyloidosis” refers to a risk that is elevated over the expected risk given the subject's age, family history, genetic status and other known risk factors.
• a "clinical sign of ⁇ amyloidosis” refers to a measure of ⁇ deposition known in the art.
• Clinical signs of ⁇ amyloidosis may include, but are not limited to, ⁇ deposition identified by amyloid imaging (e.g. PiB PET, fluorbetapir, or other imaging methods known in the art) or by decreased cerebrospinal fluid (CSF) ⁇ 42 or ⁇ 42/40 ratio.
• amyloid imaging e.g. PiB PET, fluorbetapir, or other imaging methods known in the art
• CSF cerebrospinal fluid
• Clinical signs of ⁇ amyloidosis may also include measurements of the metabolism of ⁇ , in particular measurements of ⁇ 42 metabolism alone or in comparison to measurements of the metabolism of other ⁇ variants (e.g. ⁇ 37, ⁇ 38, ⁇ 39, ⁇ 40, and/or total ⁇ ), as described in U.S. Patent Serial Nos. 14/366,831 , 14/523, 148 and 14/747,453, each hereby incorporated by reference in its entirety. Additional methods are described in Albert et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 170-179; McKhann et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 263-269; and Sperling et al.
• a subject with clinical signs of ⁇ amyloidosis may or may not have symptoms associated with ⁇ deposition.
• subjects with clinical signs of ⁇ amyloidosis are at an increased risk of developing a disease associated with ⁇ amyloidosis.
• An " ⁇ plaque associated symptom” or a “CAA associated symptom” refers to any symptom caused by or associated with the formation of amyloid plaques or CAA, respectively, being composed of regularly ordered fibrillar aggregates called amyloid fibrils.
• Exemplary ⁇ plaque associated symptoms may include, but are not limited to, neuronal degeneration, impaired cognitive function, impaired memory, altered behavior, emotional dysregulation, seizures, impaired nervous system structure or function, and an increased risk of development or worsening of Alzheimer's disease or CAA.
• Neuronal degeneration may include a change in structure of a neuron
• Impaired cognitive function may include but is not limited to difficulties with memory, attention, concentration, language, abstract thought, creativity, executive function, planning, and organization.
• Altered behavior may include, but is not limited to, physical or verbal aggression, impulsivity, decreased inhibition, apathy, decreased initiation, changes in personality, abuse of alcohol, tobacco or drugs, and other addiction-related behaviors.
• Emotional dysregulation may include, but is not limited to, depression, anxiety, mania, irritability, and emotional incontinence.
• Seizures may include but are not limited to generalized tonic-clonic seizures, complex partial seizures, and non-epileptic, psychogenic seizures.
• Impaired nervous system structure or function may include, but is not limited to, hydrocephalus, Parkinsonism, sleep disorders, psychosis, impairment of balance and coordination. This may include motor impairments such as monoparesis, hemiparesis, tetraparesis, ataxia, ballismus and tremor. This also may include sensory loss or dysfunction including olfactory, tactile, gustatory, visual and auditory sensation. Furthermore, this may include autonomic nervous system impairments such as bowel and bladder dysfunction, sexual
• this may include hormonal impairments attributable to dysfunction of the hypothalamus and pituitary gland such as deficiencies and dysregulation of growth hormone, thyroid stimulating hormone, lutenizing hormone, follicle stimulating hormone, gonadotropin releasing hormone, prolactin, and numerous other hormones and modulators.
• ApoE (NP_000032.1 , UniProtKB Identifier P02649) is an apolipoprotein expressed from the APOE gene mapped to chromosome 19 (for example, the nucleotide sequence identified as GenBank Accession Number
• NM_000041 or NCBI Reference Sequence: NC_000019.10
• ApoE2 Cys1 12, Cys158
• ApoE3 Cys1 12, Arg158
• ApoE4 Arg1 12, Arg158
• ApoE refers to "human ApoE”
• Recombinant ApoE refers to ApoE encoded by a nucleic acid that has been introduced into a system (e.g. a prokaryotic cell, a eukaryotic cell, or a cell-free expression system) that supports expression of the nucleic acid and its translation into a protein.
• lipidated ApoE refers to ApoE recombinantly produced in a prokaryotic cell.
• antibody as used herein, is used in the broadest sense and encompasses various antibody and antibody-like structures, including but not limited to full-length monoclonal, polyclonal, and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as heavy chain antibodies and antibody fragments provided they exhibit the desired antigen-binding activity.
• the domain(s) of an antibody that is involved in binding an antigen is referred to as a "variable region" or “variable domain,” and is described in further detail below.
• a single variable domain may be sufficient to confer antigen-binding specificity.
• Antibodies may or may not be glycosylated, though glycosylated antibodies may be preferred.
• An "isolated" antibody is one which has been separated from a component of its natural environment.
• an antibody is purified to greater than 95% or 99% purity as determined by methods known in the art.
• an antibody mimetic refers to a polypeptide or a protein that can specifically bind to an antigen but is not structurally related to an antibody.
• Antibody mimetics have a mass of about 3 kDa to about 20 kDa.
• Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies.
• Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA
• oligonucleotides and have high specificity and affinity for their targets.
• Aptamers interact with and bind to their targets through structural recognition, a process similar to that of an antigen-antibody reaction.
• Aptamers have a lower molecular weight than antibodies, typically about 8-25 kDa.
• full length antibody and intact antibody may be used interchangeably, and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
• the basic structural unit of a native antibody comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
• Light chains are classified as gamma, mu, alpha, and lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
• the amino-terminal portion of each light and heavy chain includes a variable region of about 100 to 1 10 or more amino acid sequences primarily responsible for antigen recognition (VL and VH, respectively).
• each chain defines a constant region primarily responsible for effector function.
• the variable and constant regions are joined by a "J" region of about 12 or more amino acid sequences, with the heavy chain also including a "D” region of about 10 more amino acid sequences.
• Intact antibodies are properly cross-linked via disulfide bonds, as is known in the art.
• variable domains of the heavy chain and light chain of an antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
• FRs conserved framework regions
• HVRs hypervariable regions
• a single VH or VL domain may be sufficient to confer antigen-binding specificity.
• antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991 ).
• FR Framework region
• the FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence: FR1 -HVR1 -FR2-HVR2-FR3- HVR3-FR4.
• the FR domains of a heavy chain and a light chain may differ, as is known in the art.
• hypervariable region refers to each of the regions of a variable domain which are hypervariable in sequence (also commonly referred to as “complementarity determining regions” or “CDR") and/or form structurally defined loops ("hypervariable loops") and/or contain the antigen-contacting residues ("antigen contacts").
• CDR complementarity determining regions
• antibodies comprise six HVRs: three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3).
• an HVR derived from a variable region refers to an HVR that has no more than two amino acid substitutions, as compared to the corresponding HVR from the original variable region.
• Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1 ), 50-52 (L2), 91 -96 (L3), 26-32 (H1 ), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 -917 (1987)); (b) CDRs occurring at amino acid residues 24- 34 (L1 ), 50-56 (L2), 89-97 (L3), 31 -35b (H1 ), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
• HVR residues and other residues in the variable domain are numbered herein according to Kabat et al., supra.
• Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
• the term includes native sequence Fc regions and variant Fc regions.
• a human IgG heavy chain Fc region extends from Cys226, or from
• the C-terminal lysine (Lys447) of the Fc region may or may not be present.
• numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 .
• a "variant Fc region” comprises an amino acid sequence that can differ from that of a native Fc region by virtue of one or more amino acid substitution(s) and/or by virtue of a modified glycosylation pattern, as compared to a native Fc region or to the Fc region of a parent polypeptide.
• a variant Fc region can have from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
• the variant Fc region herein may possess at least about 80% homology, at least about 90% homology, or at least about 95% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide.
• an "antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
• Non-limiting examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; single-chain forms of antibodies and higher order variants thereof; single-domain antibodies, and multispecific antibodies formed from antibody fragments.
• Single-chain forms of antibodies may include, but are not limited to, single-domain antibodies, single chain variant fragments (scFvs), divalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra- scFvs), diabodies, and triabodies and tetrabodies.
• ScFv's are comprised of heavy and light chain variable regions connected by a linker. In most instances, but not all, the linker may be a peptide.
• a linker peptide is preferably from about 5 to 30 amino acids in length, or from about 10 to 25 amino acids in length.
• the linker allows for stabilization of the variable domains without interfering with the proper folding and creation of an active binding site.
• a linker peptide is rich in glycine, as well as serine or threonine.
• ScFvs can be used to facilitate phage display or can be used for flow cytometry, immunohistochemistry, or as targeting domains.
• ScFvs may also be conjugated to a human constant domain (e.g. a heavy constant domain is derived from an IgG domain, such as lgG1 , lgG2, lgG3, or lgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE).
• a human constant domain e.g. a heavy constant domain is derived from an IgG domain, such as lgG1 , lgG2, lgG3, or lgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE.
• Diabodies, triabodies, and tetrabodies and higher order variants are typically created by varying the length of the linker peptide from zero to several amino acids.
• multivalent binding antibody variants can be generated using self-assembling units linked to the variable domain.
• single-domain antibody refers to an antibody fragment consisting of a single, monomeric variable antibody domain.
• Multispecific antibodies include bi-specific antibodies, tri-specific, or antibodies of four or more specificities. Multispecific antibodies may be created by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains can be covalently linked.
• “Monoclonal antibody” refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone.
• Monoclonal antibody is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be produced using hybridoma techniques well known in the art, as well as recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies and other technologies readily known in the art. Furthermore, the monoclonal antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.
• a heterologous compound e.g., an enzyme or toxin
• a “heavy chain antibody” refers to an antibody that consists of two heavy chains.
• a heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from a cartiliaginous fish.
• a “humanized antibody” refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response in humans following administration but retains similar binding specificity and affinity as the starting non-human antibody.
• a humanized antibody binds to the same or similar epitope as the non-human antibody.
• the term "humanized antibody” includes an antibody that is composed partially or fully of amino acid sequences derived from a human antibody germline by altering the sequence of an antibody having non-human hypervariable regions ("HVR"). The simplest such alteration may consist simply of substituting the constant region of a human antibody for the murine constant region, thus resulting in a human/murine chimera which may have sufficiently low
• variable region of the antibody is also humanized by techniques that are by now well known in the art.
• the framework regions of a variable region can be substituted by the corresponding human framework regions, while retaining one, several, or all six non-human HVRs.
• Some framework residues can be substituted with corresponding residues from a non-human VL domain or VH domain (e.g., the non-human antibody from which the HVR residues are derived), e.g., to restore or improve specificity or affinity of the humanized antibody.
• Substantially human framework regions have at least about 75% homology with a known human framework sequence (i.e.
• HVRs may also be randomly mutated such that binding activity and affinity for the antigen is maintained or enhanced in the context of fully human germline framework regions or framework regions that are substantially human. As mentioned above, it is sufficient for use in the methods of this discovery to employ an antibody fragment.
• humanized antibody refers to an antibody comprising a substantially human framework region, at least one HVR from a nonhuman antibody, and in which any constant region present is
• substantially human substantially human.
• Substantially human constant regions have at least about 90% with a known human constant sequence (i.e. about 90%, about 95%, or about 99% sequence identity).
• all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences.
• humanized immunoglobulins may be carried out as follows, or using similar methods familiar to those with skill in the art (for example, see Almagro, et al. Front. Biosci. 2008, 13(5): 1619-33).
• a murine antibody variable region is aligned to the most similar human germline sequences (e.g. by using BLAST or similar algorithm).
• the CDR residues from the murine antibody sequence are grafted into the similar human "acceptor" germline.
• one or more positions near the CDRs or within the framework e.g., Vernier positions
• several versions of humanized antibodies with different reversion mutations are generated and empirically tested for activity.
• the humanized antibody variant with properties most similar to the parent murine antibody and the fewest murine framework reversions is selected as the final humanized antibody candidate.
• Anti-ApoE antibodies disclosed herein can be described or specified in terms of the epitope(s) that they recognize or bind.
• the portion of a target polypeptide that specifically interacts with the antigen binding domain of an antibody is an "epitope.”
• ApoE can comprise any number of epitopes, depending on the source of the protein (e.g. mouse, rat, cynomolgus monkey, human, etc.), isoform (e.g. ApoE2, ApoE3, ApoE4), conformational state of the isoform (e.g.
• an "epitope" on ApoE can be a linear epitope or a conformational epitope, and in both instances can include non-polypeptide elements, e.g., an epitope can include a carbohydrate or lipid side chain.
• affinity refers to a measure of the strength of the binding of an individual epitope with an antibody's antigen binding site.
• an "anti-ApoE antibody,” as used herein, refers to an isolated antibody that binds to recombinant human ApoE4 or ApoE4 isolated from human brain with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 ⁇ , preferably about 0.1 pM to about 1 ⁇ , more preferably about 0.1 pM to about 100 nM.
• KD affinity constant or affinity of interaction
• Anti-ApoE antibodies disclosed herein can also be described or specified in terms of their cross-reactivity.
• the term "cross-reactivity" refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
• an antibody is cross- reactive if it binds to an epitope other than the one that induced its formation.
• the cross- reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.
• certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least about 85%, at least about 90%, or at least about 95% identity (as calculated using methods known in the art) to a reference epitope.
• An antibody can be said to have little or no cross- reactivity if it does not bind epitopes with less than about 95%, less than about 90%, or less than about 85% identity to a reference epitope.
• An antibody can be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.
• the epitope(s) to which anti-ApoE antibodies of this disclosure bind may be unique to ApoE4, may be common to ApoE4 and another ApoE isoform (e.g. ApoE2 and/or ApoE3), or may be an ApoE4 epitope that is related, but not identical, to an epitope in another isoform.
• an anti-ApoE antibody does not preferentially bind to ApoE2, ApoE3, or ApoE4.
• an anti-ApoE antibody preferentially binds to ApoE4, or preferentially binds to ApoE3 and ApoE4.
• an antibody that preferentially binds to an ApoE isoform binds to that isoform more readily than it would a different ApoE isoform.
• preferentially binds it is meant that the antibody specifically binds to an epitope of a first antigen more readily than it would bind to another epitope of the first antigen or another epitope of a second antigen.
• an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope.
• an antibody can be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (KD) that is less than the antibody's KD for the second epitope.
• KD dissociation constant
• an antibody can be considered to bind an ApoE isoform preferentially if the binding half maximal concentration (EC 50 ) of the antibody for that isoform is at least about 10-fold, 50-fold, or 100-fold less than EC 50 for the other isoforms as measured in an ELISA or similar assay.
• an antibody can be described as not preferentially binding ApoE2, ApoE3, or ApoE4 if the EC50 for the antibody for each of the isoforms varies by less than 10-fold.
• Another aspect of isolated, anti-ApoE antibodies of this disclosure is that they bind to human ApoE in amyloid plaque in unfixed brain tissue.
• Unfixed brain tissue refers to brain tissue that is not fixed with paraformaldehyde or other fixatives.
• An anti-ApoE antibody is described as "binding to human ApoE in amyloid plaques in unfixed brain tissue" when the staining pattern is consistent with binding to either ⁇ in parenchymal brain amyloid plaques or ⁇ in deposits around blood vessels in the brain in the form of CAA.
• Another aspect of isolated, anti-ApoE antibodies of this disclosure is that they preferentially bind recombinant alipidated human ApoE4 as compared to ApoE4 derived from human plasma or human ApoE4 derived from plasma of a transgenic mouse expressing human ApoE4.
• this can be measured by (a) coating recombinant alipidated human ApoE4 on an ELISA plate, (b) incubating an anti- ApoE antibody in the presence of varying dilutions of human plasma or plasma from a transgenic mouse expressing human ApoE4 (e.g., 20-fold to 1000-fold dilutions of plasma comprising about 1 -5 ⁇ ApoE) to produce a pre-incubated antibody-plasma mixture for each dilution, (c) adding the a pre-incubated antibody-plasma mixtures to the ELISA plate from (a) and allowing the mixtures to equilibrate with the coated
• an antibody that preferentially binds recombinant alipidated ApoE4 will demonstrate similar binding to the plate at plasma dilutions between about 1000-fold up to about 20-fold (i.e., loss of binding signal no greater than 20%, or preferably no greater than 10%). Further details are provided in Example 13. In certain embodiments, an isolated, anti- ApoE antibody does not specifically bind to ApoE derived from plasma.
• Clearance is a pharmacokinetic parameter that describes the efficiency of irreversible elimination of a drug from systemic circulation, expressed as volume of blood/plasma/serum cleared of drug per unit time.
• An anti-ApoE antibody does not specifically bind to ApoE derived from plasma, for example, when it is administered to an animal expressing human ApoE and the clearance value of the antibody is less than 25 times that of an isotype control antibody (the amounts administered to the animal being similar for both antibodies). For example, see FIG. 37F and Example 19.
• Another aspect of isolated, anti-ApoE antibodies of this disclosure is that they may or may not have a variant Fc region.
• an Fc region can be modified to have increased or decreased affinity for an Fc receptor on a microglial cell and/or an altered glycosylation pattern.
• an anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 94.
• the heavy chain variable region further comprises SEQ ID NO: 90 and/or SEQ ID NO: 92.
• the heavy chain variable region further comprises SEQ ID NO: 91 and/or SEQ ID NO: 93.
• the antibody has a light chain variable region comprising SEQ ID NO: 88 or SEQ ID NO: 89.
• the light chain variable region can further comprise (a) SEQ ID NO: 86 or SEQ ID NO: 87; and/or (b) SEQ ID NO: 30.
• an anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 95.
• the heavy chain variable region further comprises SEQ ID NO: 90 and/or SEQ ID NO: 92.
• the heavy chain variable region further comprises SEQ ID NO: 91 and/or SEQ ID NO: 93.
• the antibody has a light chain variable region comprising SEQ ID NO: 88 or SEQ ID NO: 89.
• the light chain variable region can further comprise (a) SEQ ID NO: 86 or SEQ ID NO: 87; and/or (b) SEQ ID NO: 30.
• an anti-ApoE antibody is selected from Table A.
• SEQ ID NO: 90 SEQ ID NO: 92
• SEQ ID NO: 90 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 90 SEQ ID NO: 92
• SEQ ID NO:86 SEQ ID NO: 90 SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 90 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 30 SEQ ID NO: 90
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 90 SEQ ID NO: 92
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 90 SEQ ID NO: 92
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 30 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 90 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 90
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 90
• SEQ ID NO: 92 SEQ ID NO: 92
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 90
• SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 92
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 92
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 90
• SEQ ID NO:86 SEQ ID NO: 30
• SEQ ID NO: 88 SEQ ID NO: 94
• SEQ ID NO: 30 SEQ ID NO: 90 SEQ ID NO: 92
• SEQ ID NO: 30 SEQ ID NO: 90 SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO: 30 SEQ ID NO: 90 SEQ ID NO: 94
• SEQ ID NO: 30 SEQ ID NO: 88 SEQ ID NO: 90 SEQ ID NO: 30 SEQ ID NO: 88 SEQ ID NO: 90 SEQ ID NO: 92
• SEQ ID NO: 30 SEQ ID NO: 88 SEQ ID NO: 90 SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO: 30 SEQ ID NO: 88 SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO: 30 SEQ ID NO: 88 SEQ ID NO: 90 SEQ ID NO: 94
• SEQ ID NO: 88 SEQ ID NO: 90 SEQ ID NO: 92 SEQ ID NO: 94
• SEQ ID NO:86 SEQ ID NO: 88 SEQ ID NO: 90 SEQ ID NO: 92 SEQ ID NO: 94
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 3 or a VH that has one or more HVRs derived from SEQ ID NO: 4.
• the HVR derived from SEQ ID NO: 3 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 29, an L2 of SEQ ID NO: 30, an L3 of SEQ ID NO: 31 , or any combination thereof (e.g. antibodies 1 -7 in Table B).
• the HVR derived from SEQ ID NO: 4 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 32, an H2 of SEQ ID NO: 33, an H3 of SEQ ID NO: 34, or any combination thereof (e.g. antibodies 8-14 in Table B).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 4 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 3.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 29, an L2 of SEQ ID NO: 30, an L3 of SEQ ID NO: 31 , or any combination thereof (e.g. antibodies 15-63 in Table B).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 3 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 4.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 3, 4, 29, 30, 31 , 32, 33, and 34, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 17 or a VH that has one or more HVRs derived from SEQ ID NO: 18.
• the HVR derived from SEQ ID NO: 17 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 63, an L2 of SEQ ID NO: 30, an L3 of SEQ ID NO: 64, or any combination thereof (e.g. antibodies 64-70 in Table B).
• the HVR derived from SEQ ID NO: 18 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 65, an H2 of SEQ ID NO: 66, an H3 of SEQ ID NO: 67, or any combination thereof (e.g. antibodies 71 -77 in Table B).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 18 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 17.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 63, an L2 of SEQ ID NO: 30, an L3 of SEQ ID NO: 64, or any combination thereof (e.g.
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 17 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 18.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 17, 18, 63, 30, 64, 65, 66, and 67, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• each of the exemplary antibodies described above may also contain a variant Fc region, including but not limited to a variant Fc region that is modified to alter the natural interaction with the microglia FcR.
• an isolated antibody of Group I recognizes an epitope listed in Tables 4-7, for example, as described for the exemplary antibody HJ152 or HJ1514.
• TABLE B Exemplary Group I Antibodies
• SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 30 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 34
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 33
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 34
• SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 34
• SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 29 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33
• SEQ ID NO: 29 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 29 SEQ ID NO: 31 SEQ ID NO: 33 SEQ ID NO: 34
• SEQ ID NO: 29 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 34
• SEQ ID NO: 65 SEQ ID NO: 67 78 SEQ ID NO: 63 SEQ ID NO: 65
• an anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 84.
• the heavy chain variable region further comprises SEQ ID NO: 80 and/or SEQ ID NO: 82.
• the heavy chain variable region further comprises SEQ ID NO: 81 and/or SEQ ID NO: 83.
• the antibody has a light chain variable region comprising SEQ ID NO: 25.
• the light chain variable region can further comprise (a) SEQ ID NO: 78 or SEQ ID NO: 79; and/or (b) SEQ ID NO: 24.
• an anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 85.
• the heavy chain variable region further comprises SEQ ID NO: 80 and/or SEQ ID NO: 82.
• the heavy chain variable region further comprises SEQ ID NO: 81 and/or SEQ ID NO: 83.
• the antibody has a light chain variable region comprising SEQ ID NO: 25.
• the light chain variable region can further comprise (a) SEQ ID NO: 78 or SEQ ID NO: 79; and/or (b) SEQ ID NO: 24.
• an anti-ApoE antibody is selected from Table C.
• SEQ ID NO: 80 SEQ ID NO: 82
• SEQ ID NO: 80 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 80
• SEQ ID NO:78 SEQ ID NO: 80 SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 80
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 80 SEQ ID NO: 82
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 80
• SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 80 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 80 SEQ ID NO: 82
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 82
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 82
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 80 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 84
• SEQ ID NO: 24 SEQ ID NO: 80
• SEQ ID NO: 24 SEQ ID NO: 80 SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 80 SEQ ID NO: 82 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 80 SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO:78 SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO:78 SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO: 82 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 25 SEQ ID NO: 82
• SEQ ID NO:78 SEQ ID NO: 25 SEQ ID NO: 82
• SEQ ID NO: 84 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 25 SEQ ID NO: 84
• SEQ ID NO:78 SEQ ID NO: 25 SEQ ID NO: 80
• SEQ ID NO: 84 SEQ ID NO: 84
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 9 or a VH that has one or more HVRs derived from SEQ ID NO: 10.
• the HVR derived from SEQ ID NO: 9 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 47, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 1 -7 in Table D).
• the HVR derived from SEQ ID NO: 10 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 48, an H2 of SEQ ID NO: 49, an H3 of SEQ ID NO: 50, or any combination thereof (e.g. antibodies 8-14 in Table D).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 10 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 9.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 47, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 15-63 in Table D).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 9 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 10.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 85%, 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 9, 10, 47, 24, 25, 48, 49, and 50, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 1 1 or a VH that has one or more HVRs derived from SEQ ID NO: 12.
• the HVR derived from SEQ ID NO: 1 1 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 51 , an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 64-70 in Table D).
• the HVR derived from SEQ ID NO: 12 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 52, an H2 of SEQ ID NO: 53, an H3 of SEQ ID NO: 54, or any combination thereof (e.g. antibodies 71 -77 in Table D).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 12 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 1 1 .
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 51 , an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g.
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 1 1 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 12.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 85%, 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 1 1 , 12, 51 , 24, 25, 52, 53, and 54, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 13 or a VH that has one or more HVRs derived from SEQ ID NO: 14.
• the HVR derived from SEQ ID NO: 13 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 55, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 127-133 in Table D).
• the HVR derived from SEQ ID NO: 14 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 56, an H2 of SEQ ID NO: 57, an H3 of SEQ ID NO: 58, or any combination thereof (e.g. antibodies 134-140 in Table D).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 14 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 13.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 55, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 141 -189 in Table D).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 13 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 14.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 13, 14, 55, 24, 25, 56, 57, and 58, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 15 or a VH that has one or more HVRs derived from SEQ ID NO: 16.
• the HVR derived from SEQ ID NO: 15 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 59, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 190-196 in Table D).
• the HVR derived from SEQ ID NO: 16 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 60, an H2 of SEQ ID NO: 61 , an H3 of SEQ ID NO: 62, or any combination thereof (e.g. antibodies 197-203 in Table D).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 16 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 15.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 59, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 204-252 in Table D).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 15 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 16.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 15, 16, 59, 24, 25, 60, 61 , and 62, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 19 or a VH that has one or more HVRs derived from SEQ ID NO: 20.
• the HVR derived from SEQ ID NO: 19 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 68, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 253-259 in Table D).
• the HVR derived from SEQ ID NO: 20 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 69, an H2 of SEQ ID NO: 70, an H3 of SEQ ID NO: 71 , or any combination thereof (e.g. antibodies 260-266 in Table D).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 20 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 19.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 68, an L2 of SEQ ID NO: 24, an L3 of SEQ ID NO: 25, or any combination thereof (e.g. antibodies 267-315 in Table D).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 19 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 20.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 19, 20, 68, 24, 25, 69, 70, and 71 , which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• each of the exemplary antibodies described above may also contain a variant Fc region, including but not limited to a variant Fc region that is modified to alter the natural interaction with the microglia FcR.
• an isolated antibody of Group II recognizes an epitope listed in Tables 4-7, for example, as described for the exemplary antibody HJ155, HJ156, HJ159, HJ1513, or HJ1518.
• SEQ ID NO: 47 SEQ ID NO: 24 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 48
• SEQ ID NO: 47 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 50
• SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 50
• SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 25 SEQ ID NO: 49 SEQ ID NO: 50
• SEQ ID NO: 47 SEQ ID NO: 25 SEQ ID NO: 50 SEQ ID NO: 47 SEQ ID NO: 25 SEQ ID NO: 48 SEQ ID NO: 50
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 53 SEQ ID NO: 54
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 52 SEQ ID NO: 53
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 53 SEQ ID NO: 54
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 54
• SEQ ID NO: 51 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 52 SEQ ID NO: 54
• SEQ ID NO: 24 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54
• SEQ ID NO: 24 SEQ ID NO: 53 103 SEQ ID NO: 24 SEQ ID NO: 53 SEQ ID NO: 54
• SEQ ID NO: 62 SEQ ID NO: 59 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 61 SEQ ID NO: 62 223 SEQ ID NO: 59 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 60 SEQ ID NO: 62
• an anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 1 13.
• the heavy chain variable region further comprises SEQ ID NO: 109 and/or SEQ ID NO: 1 1 1 .
• the heavy chain variable region further comprises SEQ ID NO: 1 10 and/or SEQ ID NO: 1 12.
• the antibody has a light chain variable region comprising SEQ ID NO: 107.
• the light chain variable region can further comprise (a) SEQ ID NO: 105; and/or (b) SEQ ID NO: 106.
• an anti-ApoE antibody has a heavy chain variable region comprising SEQ ID NO: 1 14.
• the heavy chain vartiable region further comprises SEQ ID NO: 109 and/or SEQ ID NO: 1 1 1 .
• the heavy chain variable region further comprises SEQ ID NO: 1 10 and/or SEQ ID NO: 1 12.
• the antibody has a light chain variable region comprising SEQ ID NO: 108.
• the light chain variable region can further comprise (a) SEQ ID NO: 105; and/or (b) SEQ ID NO: 106.
• an anti-ApoE antibody is selected from Table E.
• SEQ ID NO: 106 SEQ ID NO: 107 SEQ ID NO: 109 SEQ ID NO: 111 SEQ ID NO: 113
• SEQ ID NO: 110 SEQ ID NO: 112 SEQ ID NO: 114
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 1 15 or a VH that has one or more HVRs derived from SEQ ID NO: 1 16.
• the HVR derived from SEQ ID NO: 1 15 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 of SEQ ID NO: 1 17, or any combination thereof (e.g. antibodies 1 -7 in Table F).
• the HVR derived from SEQ ID NO: 1 16 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 1 18, an H2 of SEQ ID NO: 1 19, an H3 of SEQ ID NO: 120, or any combination thereof (e.g. antibodies 8-14 in
• the antibody comprising one or more HVRs derived from SEQ ID NO: 1 16 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 1 15.
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 of SEQ ID NO: 1 17, or any combination thereof (e.g. antibodies 15-63 in Table F).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 1 15 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 1 16.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 85%, 90%, 95%, or 99% sequence identity with a known human framework sequence).
• the present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 1 15, 1 16,
• 105, 106, 1 17, 1 18, 1 19, and 120 which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.
• an anti-ApoE antibody of this group comprises a VL that has one or more HVRs derived from SEQ ID NO: 121 or a VH that has one or more HVRs derived from SEQ ID NO: 122.
• the HVR derived from SEQ ID NO: 121 may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO:
• the HVR derived from SEQ ID NO: 122 may be H1 , H2, H3, or any combination thereof.
• the VH may comprise an H1 of SEQ ID NO: 124, an H2 of SEQ ID NO: 125, an H3 of SEQ ID NO: 126, or any combination thereof (e.g. antibodies 71 -77 in Table F).
• the antibody comprising one or more HVRs derived from SEQ ID NO: 122 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 121.
• VL light chain variable region
• the HVR may be L1 , L2, L3, or any combination thereof.
• the VL may comprise an L1 of SEQ ID NO: 105, an L2 of SEQ ID NO: 106, an L3 of SEQ ID NO: 123, or any combination thereof (e.g. antibodies 78-126 in Table F).
• the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 121 and/or a VH with 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 122.
• the anti-ApoE antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 85%, 90%, 95%, or 99% sequence identity with a known human framework sequence).
• each of the exemplary antibodies described above may also contain a variant Fc region, including but not limited to a variant Fc region that is modified to alter the natural interaction with the microglia FcR.
• an isolated antibody of Group III recognizes an epitope listed in Tables 4-7.
• SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 120
• SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 117 SEQ ID NO: 119 SEQ ID NO: 120
• SEQ ID NO: 120 SEQ ID NO: 106 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 120
• SEQ ID NO: 105 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 120
• SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 126
• SEQ ID NO: 106 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 126
• SEQ ID NO: 106 SEQ ID NO: 123 SEQ ID NO: 125 1 10 SEQ ID NO: 106 SEQ ID NO: 123 SEQ ID NO: 125 SEQ ID NO: 126

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