WO2024107749A1 - Protéines de fusion se liant à l'amyloïde et au récepteur de la transferrine et leurs utilisations - Google Patents

Protéines de fusion se liant à l'amyloïde et au récepteur de la transferrine et leurs utilisations Download PDF

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WO2024107749A1
WO2024107749A1 PCT/US2023/079673 US2023079673W WO2024107749A1 WO 2024107749 A1 WO2024107749 A1 WO 2024107749A1 US 2023079673 W US2023079673 W US 2023079673W WO 2024107749 A1 WO2024107749 A1 WO 2024107749A1
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amino acid
fusion protein
seq
acid sequence
set forth
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PCT/US2023/079673
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Suganya SELVARAJAH
Andrew Vick
Jonathan S. Wall
Aziz Gauhar
Shu-Fen COKER
Pawel STOCKI
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Attralus, Inc.
University Of Tennessee Research Foundation
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Publication of WO2024107749A1 publication Critical patent/WO2024107749A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to variable domain of immunoglobulin new antigen receptor (VNAR)-Fc-peptide fusion proteins and methods of treating amyloid-related disorders by administering VNAR-Fc-peptide fusion proteins.
  • VNAR immunoglobulin new antigen receptor
  • amyloid deposits e.g., Alzheimer’s disease
  • Other diseases related to amyloid accumulation in the brain include Parkinson’s disease, frontotemporal lobar degeneration (FTLD), cerebral amyloid angiopathy (CAA) and various tauopathies including posttraumatic stress disorders (PTSD), dementia pugilistica, and chronic traumatic encephalopathy (CTE).
  • FTLD frontotemporal lobar degeneration
  • CAA cerebral amyloid angiopathy
  • PTSD posttraumatic stress disorders
  • dementia pugilistica dementia pugilistica
  • CTE chronic traumatic encephalopathy
  • Beta amyloid (AP) amyloid AP amyloid
  • tau amyloid tau amyloid
  • a-synuclein aggregates Several types of amyloid are present in these disease, including Beta amyloid (AP) amyloid, tau amyloid, and a-synuclein aggregates.
  • AP Beta amyloid
  • tau amyloid tau amyloid
  • a-synuclein aggregates e.g., a-synuclein aggregates.
  • VNAR-Fc-peptide fusion proteins that bind to amyloid comprising a polypeptide comprising an amyloid-reactive peptide, an antibody Fc polypeptide, and a VNAR domain that binds to a transferrin receptor (TfR-1). Fusion proteins provided herein allow for efficient brain penetration at low therapeutic doses compared to existing therapies. Fusion proteins provided herein also allow for high affinity binding of brain specific amyloids, for example, Ap amyloid, Tau amyloid, and a-synuclein aggregates.
  • VNAR- Fc-peptide fusion proteins that (1) grant access to therapeutic targets in the brain (i.e., enabling blood-brain barrier transport) and (2) produce a therapeutic effect (i.e., reducing the prevalence and or propagation of brain amyloids).
  • a fusion protein that binds to amyloid comprising a polypeptide comprising an amyloid-reactive peptide, an antibody Fc polypeptide, and a VNAR domain that binds to a transferrin receptor (TfR-1).
  • the fusion protein comprises in N-terminal to C-terminal direction the VNAR domain, the antibody Fc polypeptide, and the amyloid reactive peptide.
  • the fusion protein is a dimer comprising two polypeptides.
  • each polypeptide comprises a VNAR domain, an antibody Fc polypeptide, and an amyloid-reactive peptide.
  • the dimer is formed by a disulfide bond between two polypeptide chains in the antibody Fc domain.
  • the amyloid reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13.
  • the antibody Fc domain is a human Fc domain. In any of the proceeding embodiments, the antibody Fc domain is a human IgGl, IgG2, or IgG4 Fc domain. In any of the proceeding embodiments, the antibody Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 14.
  • amyloid-reactive peptide is linked to the antibody Fc domain by a spacer.
  • a fusion protein comprising in N-terminal to C-terminal direction the VNAR, the antibody Fc domain, the spacer, and the amyloid-reactive peptide.
  • the spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-24.
  • said VNAR domain is a TfR-binding VNAR domain capable of specifically binding to a human TfR-1 without substantially interfering with transferrin binding to and/or transport by said human TfR-1.
  • said VNAR domain is a TfR-binding VNAR domain capable of specifically binding to a human TfR-1 without substantially interfering with transferrin binding to and/or transport by said human TfR-1 and capable of cross reacting with mouse TfR-1.
  • said VNAR domain is a TfR-binding VNAR domain capable of binding human TfR-1 with an EC50 ranging from about 1 nM to about 800 nM.
  • said VNAR domain is a TfR-binding VNAR domain designated as Clone C or one of its variants (described in WO2018/031424 and WO2019/089395). In some embodiments, said VNAR domain is a TfR-binding VNAR domain designated as Clone H or one of its variants (described in WO2018/031424 and WO2019/089395). In some embodiments, said VNAR domain is a TfR-binding VNAR domain designated as Clone 8 or one of its variants (described in W02020/056327).
  • said VNAR domain is a TfR-binding VNAR domain designated as TXB4(described in WO2019/089395). In some embodiments, said VNAR domain is a TfR- binding VNAR domain designated as VNAR-txpl (described in WO2022/103769). In some embodiments, said VNAR domain is a TfR-binding VNAR domain described in W02016/077840, WO2018/031424, WO2019/089395, W02020/056327, WO2022/103769, and WO2023/023166.
  • the fusion protein comprises a polypeptide comprising a VNAR domain described in WO2016/077840, WO2018/031424, WO2019/089395, W02020/056327, WO2022/103769, and WO2023/023166.
  • said VNAR domain is represented by the formula, from N to C terminus, FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4.
  • the CDR1 region has an amino acid sequence of DSNCALSS (SEQ ID NO:29) and the CDR3 region has an amino acid sequence of VVGTWCMSWRDV (SEQ ID NO:33).
  • the HV2 region has an amino acid sequence of TNEENISKG (SEQ ID NO:30).
  • the HV4 region has an amino acid sequence of SGSKS (SEQ ID NO:31).
  • said VNAR domain comprises an amino acid sequence of any one of SEQ ID Nos: 26-28.
  • said VNAR domain comprises a deimmunized VNAR scaffold.
  • said VNAR scaffold (described in WO2023/023166) comprises an amino acid sequence, from N to C terminus, of ⁇ RVDQTPQTITKETGESLTINCVLR (SEQ ID NO:34)-CDR1 33 TYWYRKKSGSTNEENISKGGRYVETVNSGSKSFSLKINDLTVEDSGTYRCN (SEQ ID NO:56)-CDR3- 96 YGGGTAVTVNA (SEQ ID NO:41).
  • CDR1 and CDR3 are a Type II cognate pair for a TfR-binding VNAR domain, where at least one of regions 1, 2 or 3 has one, two or three amino acids changes to reduce or remove the immunogenicity of an MHC class II binding site.
  • region 1 is amino acid residues 10 to 18 of SEQ ID NO:26 (i.e., residues ITKETGESL (SEQ ID NO:57)).
  • region 2 is amino acid residues 54 to 63 of SEQ ID NO:26 (i.e., residues YVETVNSGSK (SEQ ID NO:58)).
  • region 3 is amino acid residues 96-105 of SEQ ID NO:26 (i.e., residues YGGGTAVTVN (SEQ ID NO:59)).
  • said amino acid change in region 1 is I10A, I10E, I10S, or llOT.
  • said amino acid change in region 2 is a one amino acid change of Y55H, Y55N, Y55S or Y55T.
  • said amino acid change in region 2 is a two amino acid change of N60D and S63T, R54K and N60E, or R54K and N60Q.
  • said amino acid change in region 3 is a one amino acid change of Y97S, G99D or A 102V. In some embodiments, said amino acid change in region 3 is a three amino acid change of Y97S, G99D and A 102V.
  • the amino acid sequence between CDR1 and CDR3 is TYWYRKKSGSTNEENISKGGRYVETVDSGTKSFSLKINDLTVEDSGTYRCN (SEQ ID NO:60) or TYWYRKKSGSTNEENISKGGRKYVETVESGSKSFSLKINDLTVEDSGTYRCN (SEQ ID NO:61).
  • At least one amino acid is changed in each of regions 1, 2 and 3.
  • said amino acid changes are selected from the group consisting of (i) I10A, N60D, S63T, Y97S, G99D and A102V; (ii) I10A, R54K, N60E, Y97S, G99D and A102V; (iii) I10A, R54K, N60Q, Y97S, G99D and A102V; (iv) I10A, Y55H, Y97S, G99D and A102V; (v) I10A, Y55N, Y97S, G99D and A102V; (vi) I10A, Y55S, Y97S, G99D and A102V; (vii) I10A, Y55T, Y97S, G99D and A102V; (viii) I10S, Y55H, Y97S, G99D and A102V; and (ix) HOT, Y55H, Y97S, G99D and
  • said VNAR domain comprises the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • said VNAR domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-28 and 44-53.
  • said VNAR domain is represented by the formula, from N to C terminus, FW1-CDR1-FW2-HV2- FW2’-HV4-FW3-CDR3-FW4, and wherein the CDR1 region comprises an amino acid sequence of DSNCALSS (SEQ ID NO:29) and the CDR3 region comprises an amino acid sequence of VVQYPVYPNYFWCDV (SEQ ID NO:55).
  • the HV2 region comprises an amino acid sequence of TNEENISKG (SEQ ID NO:30).
  • the HV4 region comprises an amino acid sequence of SGSKS (SEQ ID NO:31).
  • said VNAR domain comprises the amino acid sequence of SEQ ID NO:49.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54. [0029] In any of the proceeding embodiments, the fusion protein has pan amyloid reactivity.
  • the fusion protein binds two or more types of amyloid deposits, wherein the types of amyloid deposits are selected from the group consisting of AA, AL, AH, ATTR, AB2M, Wild type TTR, AApoAI, AApoAII, AGel, ALys, ALect2, Afib, ACys, ACal, AMedin, AIAPP, APro, Alns, APrP, Ap, a-synuclein (AaSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALK4, and A1X1.
  • the fusion protein binds Ap and tau (ATau) amyloid deposits.
  • the fusion protein binds Ap and a-synuclein (AaSyn) amyloid deposits. In some embodiments, the fusion protein binds tau (ATau) and a-synuclein (AaSyn) amyloid deposits. In some embodiments, the fusion protein binds Ap, tau (ATau), and a-synuclein (AaSyn) amyloid deposits.
  • the fusion protein binds to one or more of Ap, tau (ATau), and a-synuclein (AaSyn) amyloid deposits with an EC50 ranging from about 0.5 nM to about 10 nM.
  • nucleic acid encoding the fusion protein of any of the proceeding embodiments.
  • nucleic acid of the proceeding embodiment is provided herein.
  • a host cell comprising the vector of the proceeding embodiment.
  • the host cell is a mammalian cell.
  • the mammalian call is a Chinese hamster ovary (CHO) cell.
  • a method of making a fusion protein comprising culturing the host cell of some of the proceeding embodiments under conditions suitable for expression of the fusion protein. In some embodiments, the method further comprises recovering the fusion protein.
  • a method of treating an amyloid disease in an individual comprising administering the fusion protein of some of the proceeding embodiments to the individual.
  • the individual has amyloid deposits in the brain.
  • the fusion protein is capable of uptake across the blood brain barrier.
  • the individual has Tau, Ap, and/or a-synuclein amyloid fibrils.
  • the individual has two or more of Tau, Ap, and/or a-synuclein amyloid fibrils.
  • the individual has Tau, Ap, and a-synuclein amyloid fibrils.
  • the amyloid disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Parkinson's Disease dementia (PDD), Ap amyloidosis, AL amyloidosis, AH amyloidosis, Ap2M amyloidosis, ATTR amyloidosis, transthyretin amyloidosis, AA amyloidosis, AApoAI amyloidosis, AApoAII amyloidosis, AGel amyloidosis, ALys amyloidosis, ALEct2 amyloidosis, AFib amyloidosis, ACys amyloidosis, ACal amyloidosis, AMed amyloidosis, AIAPP amyloidosis, APro amyloidosis, Alns amyloidosis, or APrP amyloidosis, progressive supranuclear palsy (PSP), cor
  • PDP Parkinson's Disease
  • the treatment with the fusion protein results in the clearance or partial clearance of amyloid in the brain of the individual. In some embodiments, the treatment with the fusion protein results in the clearance or partial clearance of Ap, Tau, and/or a-synuclein amyloid in the brain of the individual. In some embodiments, the treatment with the fusion protein results in the clearance or partial clearance of two or more of Ap, Tau, and/or a-synuclein amyloid in the brain of the individual. In some embodiments, the treatment with the fusion protein results in the clearance or partial clearance of Ap, Tau, and a-synuclein amyloid in the brain of the individual. In some embodiments, the individual is human.
  • the treatment with the fusion protein results in a reduction in the propagation of Tau and a-synuclein related pathology in the brain of the individual. In some embodiments, the treatment with the fusion protein results in a reduction in the propagation of Ap, Tau, and a-synuclein related pathology in the brain of the individual. In some embodiments, reduction in the propagation occurs in a dopaminergic neuron in the brain of the individual. In some embodiments, the dopaminergic neuron is a tyrosine hydroxylase (TH) positive dopaminergic neuron. In some embodiments, the dopaminergic neuron is located in the substantia nigra in the brain of the individual. In some embodiments, the individual is human.
  • TH tyrosine hydroxylase
  • FIG. 1 shows the results of a binding experiment testing the affinity of Fc- peptide protein for recombinant microtubule-associated protein tau (MAPT) P301S protein aggregates.
  • FIGS. 2A-2C show immunohistochemical staining to detect Fc-peptide protein associated with amyloid fibrils in human tissue sections from an individual diagnosed with Alzheimer’s disease.
  • FIG. 2A shows Fc-peptide protein binding to Ap plaques and tanglelike pathology in a human brain tissue section.
  • FIG. 2B shows Fc-peptide protein binding to Ap diffuse plaques in a human brain tissue section.
  • FIG. 2C shows Fc-peptide protein binding to Ap core plaques in a human brain tissue section.
  • FIGS. 3A-3D show the results of a binding experiment testing the affinity of Fc-peptide protein and aducanumab homolog for synthetic amyloid fibrils.
  • FIG. 3A shows the amyloid binding results for AP(l-40) synthetic fibrils.
  • FIG. 3B shows the amyloid binding results for rVX6Wil synthetic fibrils.
  • FIG. 3C shows the amyloid binding results for synthetic a- synuclein aggregates.
  • FIG. 3D shows the amyloid binding results for synthetic tau fibrils.
  • FIG. 4A shows a workflow diagram for assessing in vivo binding of Fc- peptide protein to cerebral amyloid following intravenous (IV) injection of 5xFAD mice.
  • FIG. 4B shows immunohistochemical staining to detect Fc-peptide protein associated with Ap plaques in 5xFAD mouse brain tissue.
  • FIG. 5 shows a cartoon diagram of a VNAR-Fc-peptide fusion protein.
  • FIGS. 6A-6B show the results of binding experiments testing the affinity of VNAR-Fc-peptide fusion proteins and Fc-peptide protein for transferrin receptor (TfR-1).
  • FIG. 6A shows the TfR-1 binding results for fusion protein 1 and Fc-peptide protein.
  • FIG. 6B shows the TfR-1 binding results for fusion protein 2 and Fc-peptide protein.
  • FIG. 7 shows the results of a binding experiment testing the affinity of VNAR-Fc-peptide fusion proteins for AP(l-40) synthetic fibrils.
  • FIGS. 8A-8C show immunohistochemical staining to detect Fc-peptide protein associated with amyloid fibrils in human tissue sections from individuals with Alzheimer’s disease.
  • FIG. 8A shows Fc-peptide protein binding to Ap fibrils in a human brain tissue section.
  • FIG. 8B shows fusion protein 2 binding to Ap fibrils in a human brain tissue section.
  • FIG. 8C shows fusion protein 1 binding to Ap fibrils in a human brain tissue section.
  • FIG. 9A shows the quantified brain penetrance (i.e., in vivo brain exposure) in wild type mice after injection with VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) compared to Fc-peptide protein.
  • FIG. 9B shows the fold-increase brain penetrance (i.e., in vivo brain exposure) for VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) over Fc-peptide protein control.
  • FIGS. 10A-10C show the results of a binding experiment testing the affinity of Fc-peptide protein and aducanumab homolog for synthetic amyloid fibrils.
  • FIG. 10A shows the amyloid binding results for AP(l-40) synthetic fibrils.
  • FIG. 10B shows the amyloid binding results for synthetic tau fibrils.
  • FIG. 10C shows the amyloid binding results for synthetic a- synuclein fibrils.
  • FIG. 11A shows the results of an in vitro tau propagation assay testing the impact of Fc-peptide protein on tau propagation in tau RD P301S cells.
  • FIG. 11B shows the results of an in vitro tau propagation assay testing the impact of VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) on tau propagation in tau RD P301S cells.
  • VNAR-Fc-peptide fusion protein e.g., fusion protein 2
  • FIG. 12 shows the activation of the human complement complex by VNAR- Fc-peptide fusion protein (e.g., fusion protein 2) in the presence of synthetic A
  • VNAR- Fc-peptide fusion protein e.g., fusion protein 2
  • FIG. 13 shows the results of an in vitro alpha- sy nuclein amyloid aggregate assay in cultured primary dopaminergic neurons, where cells are treated with either Fc-peptide protein or VNAR-Fc-peptide fusion protein (e.g., fusion protein 2).
  • FIG. 14 shows the results of an ex vivo phagocytosis assay performed with VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) and human Fcl control for A
  • Phagocytosis is detected by labeling with the pH sensitive dye succinimidyl-pHrodo red fluorophore where increasing fluorescence emission is indicative of enhanced phagocytosis.
  • FIG. 15 shows the results of a rat primary microglial assay for assessing phagocytosis of synthetic AP(l-42) fibrils in the presence of Fc-peptide protein or VNAR-Fc- peptide fusion protein (e.g., fusion protein 2).
  • FIGS. 16A shows a workflow diagram for assessing in vivo binding of Fc- peptide protein and VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) to cerebral amyloid following intraperitoneal (IP) injection of 5xFAD mice at 10 mg/kg dose of Fc-peptide protein, 14 mg/kg does of fusion protein 2, or 70 mg/kg dose of fusion protein 2.
  • FIGS. 16B-16G show immunohistochemical staining to detect Fc-peptide protein or VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) in harvested brain tissue after treatment.
  • FIGS. 16B-16C show brain tissue samples 1 and 2 treated with 50 mg/kg Fc-peptide protein.
  • FIGS. 16F-16G show brain tissue samples 1 and 2 treated with 70 mg/kg VNAR-Fc-peptide fusion protein (e.g., fusion protein 2).
  • FIG. 17A shows the quantified plasma level in wild type mice after injection with VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) compared to Fc-peptide protein.
  • FIG. 17B shows the quantified brain penetrance (i.e., in vivo brain exposure) in wild type mice after injection with VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) compared to Fc- peptide protein.
  • FIG. 17C shows the fold-increase brain penetrance (i.e., in vivo brain exposure) for VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) over Fc-peptide protein control.
  • FIGS. 18A-18B show the results of an in vivo mouse study for assessing the number of dopaminergic neurons and the amount of alpha- synuclein in dopaminergic neurons.
  • Mice underwent surgery to inject alpha- synuclein amyloid directly into the brain at day 0 and were administered either Fc-peptide protein or VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) every other day from day -7 to day 28 (5 weeks).
  • FIG. 18A shows the number of dopaminergic neurons in mice treated with Fc-peptide protein or VNAR-Fc-peptide fusion protein (e.g., fusion protein 2).
  • FIG. 18B shows the percent alpha- synuclein in dopaminergic neurons in mice treated with Fc-peptide protein or VNAR-Fc-peptide fusion protein (e.g., fusion protein 2).
  • VNAR-Fc-peptide fusion proteins allow for efficient brain penetration at low therapeutic doses compared to existing therapies.
  • High affinity monoclonal antibodies to human transferrin receptor (TfR-1) have poor brain penetration, while the use of a low affinity, monovalent monoclonal antibody requires large therapeutic doses to achieve similar levels, both of which are major limitations for human translation.
  • the fusion proteins provided herein comprise a high affinity bivalent variable domain of new antigen receptor (VNAR) antibody polypeptide that binds to the TfR-1 and has the potential to achieve high brain penetration, helping to overcome the clinical limitations of other existing therapeutics.
  • VNAR new antigen receptor
  • VNAR-Fc-peptide fusion proteins provide for high affinity binding of brain specific amyloids, for example, Ap amyloid, Tau amyloid, and a-synuclein aggregates.
  • Amyloid-reactive peptides have been described previously to bind synthetic amyloids and amyloid extracts associated with systemic amyloidosis.
  • fusion proteins comprising an amyloidreactive peptide that enable pan amyloid reactivity for targeting brain amyloids and systemic amyloids for clearance.
  • Existing therapies for treating amyloid-related diseases of the brain are extremely limited.
  • Aducanumab was recently approved as the first Ap specific monoclonal antibody for the treatment of Alzheimer’s disease, yet, as discussed above, monoclonal antibodies require intravenous administration of large therapeutic doses to achieve their results. Further, aducanumab is limited to Ap amyloid reactivity, whereas amyloid-reactive peptides demonstrate robust pan amyloid reactivity. Despite improvements in technology and scientific understanding, identifying effective therapeutic for treating brain disease remains one of the most difficult clinical challenges in human medicine.
  • VNAR-Fc- peptide fusion proteins that address these longstanding challenges by (1) granting access to the therapeutic target (i.e., enabling blood-brain barrier transport) and (2) producing a therapeutic effect (i.e., reducing the prevalence and or propagation of brain amyloids).
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value of the range and/or to the other particular value of the range. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. In certain example embodiments, the term “about” is understood as within a range of normal tolerance in the art.
  • amyloids amyloid deposits, amyloid fibrils, and amyloid fibers refer to insoluble fibrous protein aggregates sharing specific structural traits.
  • the protein aggregates have a tertiary structure, for example, that is formed by aggregation of any of several different proteins and that consists of an ordered arrangement of P sheets stacked perpendicular to a fiber axis. See Sunde et al., J. Mol. Biol. (1997) 273:729-39. Abnormal accumulation of amyloids in organs may lead to amyloidosis.
  • amyloids Although they are diverse in their occurrence, all amyloids have common morphologic properties in that they stain with specific dyes such as Congo red and have a characteristic red-green birefringent appearance in polarized light after staining. Amyloids also share common ultrastructural features and common x-ray diffraction and infrared spectra.
  • Amyloidogenic refers to producing or tending to produce amyloid deposits.
  • certain soluble monomeric proteins can undergo extensive conformational changes leading to their aggregation into well-ordered, unbranching, 8- to 10-nm wide fibrils, which culminate in the formation of amyloid aggregates.
  • More than thirty proteins, for example, have been found to form amyloid deposits (or amyloids) in man.
  • Other proteins of the class can form amyloid deposits and are thus amyloidogenic.
  • light chain protein some may be deemed more “amyloidogenic” than others based upon the ease with which they form amyloid fibrils. Certain light chain proteins are deemed non-amyloidogenic or less amyloidogenic because of their inability to readily form amyloid fibrils in patients or in vitro.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject and subject includes both human and veterinary individuals.
  • an animal is an individual suffering from an amyloid disease.
  • Clearance refers to reducing or removing by a measurable degree.
  • the clearance of an amyloid deposit as described herein relates to reducing or removing the deposit to a measurable or discernable degree. Clearance may result in 100% removal, but is not required to. Rather, clearance may result in less than 100% removal, such as about 10%, 20%, 30%, 40%, 50%, 60% or more removal.
  • Effective amount or therapeutically effective amount The amount of agent that is sufficient to prevent, treat (including prophylaxis'), reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease, for example to prevent, inhibit, and/or amyloidosis.
  • an “effective amount” is sufficient to reduce or eliminate a symptom of a disease.
  • An effective amount can be administered one or more times.
  • Inhibit To reduce by a measurable degree. Inhibition does not, for example, require complete loss of function or complete cessation of the aspect being measured. For example, inhibiting plaque formation can mean stopping further growth of the plaque, slowing further growth of the plaque, or reducing the size of the plaque.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating,” with reference to a disease or pathological condition refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible individual, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the individual, or by other parameters well known in the art that are specific to the particular disease.
  • Label refers to any detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
  • Specific, nonlimiting examples of labels include fluorescent tags, chemiluminescent tags, haptens, enzymatic linkages, and radioactive isotopes.
  • a protein that is “detectably-labeled,” for example, means that the presence of the protein can be determined by a label associated with the protein.
  • Isolated An “isolated” biological component, such as a peptide (for example one or more of the peptides disclosed herein), cell, nucleic acid, or serum samples has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a cell as well as chemically synthesized peptide and nucleic acids.
  • an isolated peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even at least 99% of the peptide or protein concentration.
  • join refers to any method known in the art for functionally connecting proteins and/or protein domains.
  • one protein domain may be linked to another protein domain via a covalent bond, such as a polypeptide bond in a recombinant fusion protein, with or without intervening sequences or domains.
  • Joined also includes, for example, the integration of two sequences together, such as placing two nucleic acid sequences together in the same nucleic acid strand so that the sequences are expressed together.
  • Nucleic acid A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.
  • nucleotide polymers in which the nucleotides and the linkages between them include non- naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O- methyl ribonucleotides, peptide- nucleic acids (PNAs), and the like.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”
  • Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • a nucleotide is one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • nucleotide sequences the left-hand end of a single- stranded nucleotide sequence is the 5 ‘-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5 ’-direction.
  • the direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5 ’-end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the coding RNA transcript are referred to as “downstream sequences.”
  • cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • non-coding strand used as the template for transcription
  • a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds.
  • polypeptide or “protein” as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • Fusion protein refers to a polypeptide comprising an amyloid-reactive peptide, an antibody Fc polypeptide, and a VNAR domain that binds to a transferrin receptor (TfR-1).
  • TfR-1 transferrin receptor
  • the fusion protein may also be referred to as a VNAR-Fc-peptide fusion protein.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate).
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art.
  • Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.
  • the term vector includes plasmids, linear nucleic acid molecules, and as described throughout adenovirus vectors and adenoviruses.
  • a subject or an individual refers to a mammal, for example, a human.
  • the individual may be a human patient.
  • An individual may be a patient having a disease or condition and may be in need of treatment.
  • VNAR-Fc-peptide fusion proteins that are useful for treating amyloid disorders, which involve amyloid accumulation in the central nervous system.
  • the fusion proteins provided herein beneficially have pan-amyloid reactivity and are able to react with multiple different forms of amyloid, for example, Ap and tau.
  • the fusion proteins comprise a polypeptide comprising a peptide that is able to bind to various forms of amyloid, linked to an antibody Fc domain that promotes and/or enhances phagocytosis of amyloid deposits.
  • the fusion protein comprises a polypeptide comprising a variable domain of new antigen receptor (VNAR) domain that binds to the transferrin receptor (TfR-1), which allows uptake across the blood-brain barrier.
  • VNAR variable domain of new antigen receptor
  • VNAR-Fc-peptide fusion proteins comprising a polypeptide that comprises amyloid-reactive peptides that are able to bind to diverse amyloid forms, including amyloid present in amyloid deposits in the central nervous system (CNS).
  • the fusion protein is a dimer comprising two monomers, each of which comprising an amyloid-reactive peptide, an antibody Fc polypeptide, and a variable domain of new antigen receptor (VNAR) domain binds to a transferrin receptor (TfR-1), where the antibody Fc domain is a dimer formed from two heavy chain Fc polypeptides containing CH domains.
  • VNAR new antigen receptor
  • TfR-1 transferrin receptor
  • the first and/or the second amyloid-reactive peptide comprises an amino acid sequence as set forth in Table 1.
  • one or more of the peptides shown in Table 1 can be joined to an antibody Fc domain through one or more N-termini of the antibody Fc domain or one or more C-termini of the antibody Fc domain, thereby forming an amyloid-reactive peptide-Fc fusion protein.
  • the first and/or the second amyloid-reactive peptide comprises two or more of the peptides shown in Table 1, which can be joined to a single antibody Fc polypeptide.
  • two of the amyloid reactive peptides can be joined with a single antibody Fc polypeptide.
  • one or more of the amyloid-reactive peptides set forth in Table 1 can be joined to a VNAR that binds to the transferrin receptor (TfR-1) through its N-terminus or its C-terminus.
  • the first and/or the second amyloid-reactive peptide comprises two or more of the peptides set forth in Table 1, which can be joined to a single VNAR that binds to TfR-1.
  • two of the amyloid-reactive peptides can be joined with a single VNAR that binds to TfR-1.
  • amyloid-reactive domain of amyloid-reactive peptide when administered to an individual, targets the fusion protein to amyloid deposits.
  • the antibody Fc domain then triggers an immune response at the site of the amyloid, thereby resulting in removal of the amyloid, such as by opsonization.
  • an amyloid-reactive peptide linked to an antibody Fc domain is believed to have a longer half-life than the amyloid-reactive peptides alone.
  • contacting an amyloid deposit with a fusion protein of the present disclosure results in a half-life that is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to contacting an amyloid deposit with the amyloid-reactive peptide alone.
  • the fusion protein comprising the amyloid-reactive peptide and antibody Fc domain can exert its immuno stimulatory effects longer at the site of the amyloid deposit, thereby increasing the immune response at the site of the amyloid deposit.
  • the amyloid-reactive peptides of the fusion proteins provided herein may comprise or consist of from about 10 to about 55 amino acids.
  • the amyloid-reactive peptides of the present invention may, for example, comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids.
  • Such peptides are described, for example, in international patent application WO2016032949, which is hereby incorporated herein in its entirety.
  • the first and/or the second amyloid-reactive peptide comprises the amino acid sequences set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the first and/or the second amyloid-reactive peptide comprises the amino acid sequences set forth in any one of SEQ ID NOs: 1-13. In some embodiments, the first and the second amyloid-reactive peptide comprise an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the first and the second amyloid-reactive peptide comprises an amino acid sequence set forth in SEQ ID NO:2.
  • the first and the second amyloidreactive peptide comprises an amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the first and the second amyloid-reactive peptide comprises an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the first and the second amyloidreactive peptide are the same. In some embodiments, the first and the second amyloid-reactive peptide are different.
  • the amino acids forming all or a part of the amyloid-reactive peptides linked to the antibody Fc polypeptides and/or the VNARs that bind to the transferrin receptor (TfR-1) may be stereoisomers and modifications of naturally occurring amino acids, non-naturally occurring amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like.
  • the amino acids forming the peptides of the present invention may be one or more of the 20 common amino acids found in naturally occurring proteins, or one or more of the modified and unusual amino acids.
  • the first amyloid-reactive peptide is linked to the N- terminus of the first antibody Fc polypeptide, and the second amyloid-reactive peptide is linked to the N-terminus of the second antibody Fc polypeptide.
  • the first amyloid-reactive peptide is linked to the C-terminus of the first antibody Fc polypeptide, and the second amyloid-reactive peptide is linked to the C-terminus of the second antibody Fc polypeptide.
  • the first and second antibody Fc polypeptides are connected in the hinge region by a variable number of disulfide bonds depending on the subclass of IgG (IgGl, IgG2, IgG3, and IgG4).
  • the first antibody Fc polypeptide and second antibody Fc polypeptide are linked by one or more disulfide bonds between cysteines at amino acid positions 6 and 9 as set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptides of the fusion proteins provided herein bind to amyloid deposits or fibrils located in the CNS, such as Ap, tau, or a- synuclein. In some embodiments, the amyloid-reactive peptides bind to amyloid deposits or fibrils located in the brain. In some embodiments, the amyloid-reactive peptides bind to one or more amyloidogenic peptides in amyloids. In some embodiments, the amyloid-reactive peptides binds to heparin sulfate glycosoaminoglycans in the amyloid.
  • the amyloid-reactive peptides binds to heparin sulfate proteoglycans (HSPG) in the amyloid. In some embodiments, the amyloid-reactive peptides bind to human fibrils. In some embodiments, the amyloid-reactive peptides bind to synthetic fibrils. In some embodiments, the amyloidreactive peptides bind to rVZ.6Wil fibrils, Perl25 wtATTR extract, KEN hATTR extract, SHI ALA liver extract, and/or TAL ALK liver extract.
  • HSPG heparin sulfate proteoglycans
  • amyloids bound by the amyloid-reactive peptides comprise an amyloidogenic Z.6 variable domain protein (VX6Wil) or an amyloidogenic immunoglobulin light chain (AL), AP(l-40) amyloid-like fibril or an amyloidogenic Ap precursor protein, or serum amyloid protein A (AA).
  • VX6Wil amyloidogenic Z.6 variable domain protein
  • AL amyloidogenic immunoglobulin light chain
  • AP(l-40) amyloid-like fibril or an amyloidogenic Ap precursor protein or serum amyloid protein A (AA).
  • AA serum amyloid protein A
  • the amyloid-reactive peptides bind to rVA6Wil, Ap, AP(l-40), IAAP, ALK, ALA, or ATTR amyloid.
  • the amyloid-reactive peptides bind to ALK4, AL amyloid.
  • amyloids bound by the amyloid-reactive peptides comprise amyloidogenic forms of immunoglobulin heavy chain (AH), p2-microglobulin (Ap2M), transthyretin variants (ATTR), apolipoprotein Al (AApoAI), apolipoprotein All (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin ((ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (Alns), prior protein (APrP); a-synuclein (AaSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALK4, A1X1, and other amyloidogenic peptid
  • amyloidogenic peptides bound by the amyloid-reactive peptides can be a protein, a protein fragment, or a protein domain.
  • the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins.
  • the amyloids are part of the pathology of a disease.
  • the amyloid-reactive peptides of the fusion proteins have pan-amyloid reactivity and are able to bind to diverse amyloid types in various amyloid tissues.
  • the amyloid-reactive peptides are able to bind to amyloid in the central nervous system (CNS).
  • the amyloid-reactive peptides are able to bind to amyloid in the brain.
  • the amyloid-reactive peptides are able to bind to Ap fibrils, tau fibrils, and/or a-synuclein aggregates.
  • the amyloidreactive peptides are able to bind to two or more of Ap fibrils, tau fibrils, and a-synuclein aggregates. In some embodiments, the amyloid-reactive peptides are able to bind to Ap fibrils, tau fibrils, and a-synuclein aggregates.
  • the amyloid-reactive peptides of the fusion proteins bind to human amyloid fibrils, include those present in the central nervous system (CNS), with a half maximal effective concentration (EC50) that is less than at or about 1, 10, 100, or 1000 nM.
  • the amyloid-reactive peptides bind to human amyloid fibrils with an EC50 that is at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 100, 250, 500, 750, or 1000 nM, including any value or range between these values.
  • the amyloid-reactive peptides bind to human amyloid fibrils with an EC50 that is less than about 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 80 nM, or 100 nM. In some embodiments, the amyloid-reactive peptides bind to human amyloid fibrils with an EC50 that is between 0.01 and 500, 0.05 and 400, 0.1 and 300, 0.5 and 200, 1 and 100, 2 and 75, 3 and 50, 4 and 40, 5 and 30, 6 and 20, 7 and 15, or 8 and 12 nM.
  • the amyloidreactive peptides of the fusion proteins bind to human amyloid fibrils with an EC50 that is less than the EC50 of a control antibody Fc domain binding to human amyloid fibrils. In some embodiments, the amyloid-reactive peptides bind to amyloid deposits or fibrils to a greater extent than a human Fcl control. In some embodiments, the human amyloid fibrils that are bound by the amyloid-reactive peptide of the fusion proteins are Ap amyloid, tau amyloid, and/or a-synuclein aggregates.
  • the human amyloid fibrils that are bound by the amyloid-reactive peptide of the fusion proteins are two or more of Ap amyloid, tau amyloid, and a-synuclein aggregates. In some embodiments, the human amyloid fibrils that are bound by the amyloid-reactive peptide of the fusion proteins are Ap amyloid, tau amyloid, and a-synuclein aggregates.
  • Methods for calculating EC50s are known in the art, and include, for example, surface plasmon resonance. In some embodiments, the EC50 is determined by measuring binding to rVX6Wil fibrils.
  • the VNAR-Fc-peptide fusion proteins provided herein comprise a polypeptide comprising an antibody Fc polypeptide.
  • the most common immunoglobulin isotype in humans is IgG, which is composed of two identical heavy chain polypeptides and two identical light chain polypeptides. Disulfide bonds link both heavy chain polypeptides to each other. In addition, a disulfide bond also links each light chain polypeptide to a heavy chain polypeptide.
  • Heavy chain polypeptides contain four distinct domains including the variable heavy (VH), constant heavy 1 (CHI), constant heavy 2 (CH2), and constant heavy 3 (CH3) domains. Each light chain contains a variable light (VL) and a variable heavy (VH) domain.
  • variable domains of the heavy and light chains provide the antibody with antigen binding activity and are responsible for the diversity and specificity of immunoglobulins.
  • the heavy chain constant domains primarily CH2 and CH3, are involved in nonantigen binding functions of antibodies, and constitute the Fc region.
  • the Fc region is capable of binding complement, which may trigger phagocytosis or complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the Fc region can also bind to Fc receptors, which may trigger phagocytosis or antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • the Fc region is known to improve the maintenance of the antibody during circulation.
  • the antibody Fc domain of the fusion protein is a dimer formed from two heavy chain Fc polypeptides containing CH domains.
  • the two heavy chains are connected in the hinge region by a variable number of disulfide bonds depending on the subclass of IgG (IgGl, IgG2, IgG3, and IgG4).
  • the first antibody Fc polypeptide and second antibody Fc polypeptide are linked by one or more disulfide bonds between cysteines at amino acid positions 6 and 9 as set forth in SEQ ID NO: 14.
  • the first and/or second antibody Fc polypeptide are linked to an amyloid-reactive polypeptide.
  • the first and/or second antibody Fc polypeptides are linked to a variable domain of new antigen receptor (VNAR) domain that binds to transferrin receptor (TfR-1).
  • VNAR new antigen receptor
  • the first and/or second antibody Fc polypeptide of the fusion protein is a human IgGl, IgG2, or IgG4 Fc. In some embodiments, the first and/or second antibody Fc polypeptide is a human IgGl Fc. In some embodiments, the first and second antibody Fc polypeptides are human IgGl Fc polypeptides. In some embodiments, the first and/or second antibody Fc polypeptide comprises the amino acid sequence set forth in Table 2. In some embodiments, the first and/or second antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the first and second antibody Fc polypeptides comprise the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the first and second antibody Fc polypeptides consist of the amino acid sequence set forth in SEQ ID NO: 14.
  • the first and/or second antibody Fc polypeptide of the fusion protein is an Fc polypeptide variant with enhanced effector function(s).
  • the first and/or second antibody Fc polypeptide is an Fc polypeptide variant with an enhanced ability to promote phagocytosis.
  • the first and/or second antibody Fc polypeptide is an Fc polypeptide variant with an enhanced ability to bind to an FcyR.
  • the first and/or second antibody Fc polypeptide is an Fc polypeptide variant with enhanced ability to recruit complement.
  • the first and/or second antibody Fc polypeptide comprises one or more amino acid substitutions, insertions, and/or deletions that confer enhanced effector function and/or enhanced binding to an FcyR.
  • amino acid substitutions, insertions, and/or deletions have been described, for example, in International Publication Nos. W02004/099249, W02005/063815, W02006/019447, W02006/020114, W02007/041635, W02009/058492, W02009/086320, and U.S. Publication Nos. US20070224192 and US20080161541, each of which are hereby incorporated by reference.
  • the antibody Fc domain which is a dimer of two antibody Fc polypeptides, comprises one or more amino acid substitutions, insertions, and/or deletions and has an enhanced ability to promote phagocytosis.
  • the first and/or second antibody Fc polypeptide is glycoengineered.
  • the glycoengineered antibody Fc domain has an enhanced ability to promote phagocytosis.
  • the VNAR-Fc-peptide fusion protein comprises a polypeptide comprising an amino acid spacer between the antibody Fc polypeptide and the amyloid-reactive peptide. Spacer sequences functionally separate protein domains from one other, which may improve epitope availability, flexibility, and overall function of the fusion protein.
  • the first and/or second amyloid-reactive peptide is linked to the first and/or second antibody Fc polypeptide via a spacer.
  • the first and/or second variable domain of new antigen receptor (VNAR) domain that binds to the transferrin receptor (TfR-1) is linked to the first and/or second antibody Fc polypeptide via a spacer.
  • the first and/or second amyloid-reactive peptide is linked to the first and/or second VNAR that binds to the TfR-1 via a spacer.
  • the spacer is a peptide spacer.
  • the fusion protein comprises two or more spacer sequences. In some embodiments, the two or more spacer sequences are the same. In some embodiments, the two or more spacer sequences are each unique spacers.
  • the amino acid spacers of the fusion proteins provided herein possess different physical traits.
  • the spacer is a flexible spacer.
  • the spacer comprises glycine and serine residues.
  • the spacer comprises a GGGGS motif.
  • the spacer comprises a glycine serine linker.
  • the spacer is a glycine serine linker.
  • the spacer is uncharged.
  • the spacer is a rigid spacer.
  • the spacer comprises or consists of from about 3 to about 55 amino acids.
  • the spacers of the present invention may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids.
  • the spacer is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, or 100 amino acids in length, including any value or range between these values.
  • the spacer comprises 5 amino acids.
  • the spacer comprises an amino acid sequence as set forth in Table 3 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the spacer comprises the amino acid sequence set forth in any of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the spacer comprises an amino acid sequence as set forth in Table 3. In some embodiments, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24.
  • the fusion proteins comprise a polypeptide comprising a variable domain of new antigen receptor (VNAR) domain that binds to the transferrin receptor (TfR-1), an antibody Fc polypeptide, a spacer, and an amyloid-reactive peptide, where the spacer is between the antibody Fc polypeptide and the amyloid-reactive peptide and the spacer comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15- 24.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15.
  • the spacer of the fusion protein consists of the amino acid sequence set forth in SEQ ID NO: 15.
  • the fusion protein provided herein comprises two or more amino acid spacers.
  • the fusion proteins comprise a polypeptide comprising a variable domain of new antigen receptor (VNAR) domain that binds to the transferrin receptor (TfR-1), a first spacer, an antibody Fc polypeptide, a second spacer, and an amyloid-reactive peptide.
  • VNAR variable domain of new antigen receptor
  • the two or more spacers are flexible spacers.
  • the two or more spacers comprise glycine and serine residues.
  • the two or more spacers comprise a GGGGS motif.
  • the two or more spacers comprise a glycine serine linker.
  • the two or more spacers are glycine serine linkers. In some embodiments, the two or more spacers are uncharged. In some embodiments, the two or more spacers are rigid spacers. In some embodiments the two or more spacers comprise or consists of from about 3 to about 55 amino acids.
  • the two or more spacers of the present invention may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids.
  • the two or more spacers are about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, or 100 amino acids in length, including any value or range between these values.
  • the two or more spacers each comprise 5 amino acids.
  • the two or more spacers each comprise an amino acid sequence as set forth in Table 3 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the two or more spacers each comprise the amino acid sequence set forth in any of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the two or more spacers each comprise an amino acid sequence as set forth in Table 3.
  • the two or more spacers each comprise the amino acid sequence set forth in any one of SEQ ID NOs: 15-24.
  • an Fc-peptide protein comprising an amyloid-reactive peptide, a spacer, and an antibody Fc polypeptide
  • the Fc-peptide protein comprises the amino acid sequences set forth is Table 4, as described previously in PCT/US 2022/072112 (WO2022/236286), which is hereby incorporated by reference in its entirety.
  • the Fc-peptide protein comprises the amino acid sequences set forth is SEQ ID NO:25.
  • the Fc-peptide protein is a monomer that forms a homodimer of two monomers, each consisting of the sequence set forth is SEQ ID NO:25.
  • each Fc-peptide protein monomer comprises in N-terminal to C-terminal direction the antibody Fc polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 (Table 4, unformatted sequence), a spacer comprising the amino acid sequence set forth in SEQ ID NO: 15 (Table 4, underlined sequence), and the amyloid-reactive peptide comprising the amino acid sequence set forth in SEQ ID NO:2 (Table 4, bold sequence).
  • two Fc-peptide protein monomers are connected in the hinge region of the antibody Fc polypeptides by a variable number of disulfide bonds.
  • the Fc-peptide protein has pan amyloid reactivity and reacts with multiple different forms of amyloid, for example, Ap, tau, and a-synuclein aggregates.
  • the Fc-peptide protein comprise a peptide that binds to various forms of amyloid, linked to an antibody Fc polypeptide that promotes and/or enhances phagocytosis of amyloid deposits.
  • the Fc-peptide protein is useful for treating amyloid disorders, which involve amyloid accumulation.
  • BBB Blood Brain Barrier
  • BBB blood-brain barrier
  • RMT receptor-mediated transcytosis
  • Transferrin receptor (TfR-1) is a dimeric transmembrane glycoprotein receptor in the brain that helps to regulate brain iron levels.
  • TfR-1 has been the target of numerous in vitro and in vivo studies aiming to deliver drugs to the brain. Iron delivery to the brain is accomplished via binding and intracellular trafficking of the iron binding protein transferrin (Tf) by TfR-1. Challenges using anti-TfR-1 agents to deliver drugs to the brain via RMT include specificity to the brain tissue, potential lysosomal degradation and significant transport into the brain parenchyma.
  • Transferrin receptor (TfR-1) is of special interest because it is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB).
  • BCECs brain capillary endothelial cells
  • BBB blood-brain barrier
  • TfR-1 Targeting TfR-1 at one or more of the apical domain, helical domain, and/or protease-like domain can enable receptor-mediated transcytosis (RMT), a principal pathway for transport of macromolecules essential for brain function across the BBB.
  • RTT receptor-mediated transcytosis
  • VNAR-Fc-peptide fusion proteins comprising a polypeptide comprising a variable domain of new antigen receptor (VNAR) domain that binds to a transferrin receptor (TfR-1) and an amyloid reactive peptide.
  • VNAR new antigen receptor
  • TfR-1 transferrin receptor
  • the VNAR of the fusion protein binds TfR-1 on the membrane of a mammalian cell and TfR-1 -specific binding mediates transport of the polypeptide and at least one associated heterologous molecule across the cell membrane (e.g., a fusion protein comprising the polypeptide that binds to the transferrin receptor).
  • TfR-1 -positive cell or cell type i.e., one with the transferrin receptor localized at the cell membrane
  • TfR-1 may thus be used to target delivery of heterologous molecules across its membrane by association (e.g., a complex or conjugate) with a polypeptide that binds to the TfR-1.
  • the cell membrane is part of the blood brain barrier (BBB) and TfR-1 -mediated transport across the BBB of a heterologous molecule, such as a fusion protein comprising the polypeptide that binds to the transferrin receptor (TfR-1), is enabled.
  • BBB blood brain barrier
  • TfR-1 transferrin receptor
  • Antibodies are powerful tools in research and diagnostics due to high affinity and specificity towards target antigen.
  • a conventional antibody also known as an immunoglobulin (Ig)
  • Ig immunoglobulin
  • the heavy chain of a conventional IgG consists of three constant domains (CHI, CH2 and CH3) and a variable domain (VH), whereas the light chain consists of a constant domain (CL) and a variable domain (VL).
  • camelid heavy-chain antibodies HCAbs
  • immunoglobulin new antigen receptors IgNARs
  • HCAbs camelid heavy-chain antibodies
  • IgNARs immunoglobulin new antigen receptors
  • VNARs variable domains of these shark antibodies
  • VNARs contain autonomous function as single-domain antibodies, which are promising therapeutic candidates due to their small size, high specificities for a cognate antigen, and high physiochemical stability.
  • VNARs have structural diversity in their antigen binding sites and are capable of accessing more cryptic epitopes and catalytic clefts of enzymes.
  • variable domain of an IgNAR i.e., the VNAR
  • VNAR contains only two complementarity-determining regions (CDRs), CDR1 and CDR3. Diversity of VNAR is achieved by long variable protruding CDR3, while the CDR1 is connected through two hypervariable regions (HV), HV2 and HV4.
  • HV hypervariable regions
  • HV2 hypervariable regions
  • the structure of CDR3 combined with supporting intermolecular disulphide bridging has conferred VNARs to ability to access confined epitopes.
  • VNARs are the smallest naturally occurring immunoglobulin-based protein scaffolds (roughly 12 kDa), providing a simple, highly stable scaffold useful in targeting new and difficult antigens.
  • VNARs are categorized into four isotypes based on the position and the number of non-canonical cysteine residues. Provided herein are Type II VNARs, having a cysteine residue in both CDR1 and CDR3 that form a disulfide bond and orients the two VNAR loops.
  • Non-limiting examples of antibody-like backbones that may be used according to the invention include monospecific and bispecific fragments such as multimerizing scFv fragments (diabodies, triabodies, tetrabodies), disulfide stabilized antibody variable (Fv) fragments, disulfide stabilized antigen-binding (Fab) fragments consisting of the VL, VH, CL and CHI domains, bivalent F(ab’)2 fragments, Fd fragments consisting of the heavy chain and CHI domains, dimeric CH2 domain fragments (CH2D), FC antigen binding domains (Fcabs), single chain FV-CH3 minibodies, bispecific minibodies, isolated complementary determining region 3 (CDR3) fragments, constrained FR3-CDR3-FR4 polypeptides, SMTP domains, and any genetically manipulated counterparts of the foregoing that retain TfR-1 binding function (see e.g., Weiner L, Cell 148: 1081-4 (2012); Ahmad
  • VNARs immunoglobulin new antigen receptor
  • TfR-1 transferrin receptor 1
  • VNARs immunoglobulin new antigen receptors
  • the fusion protein provided herein comprises a polypeptide comprising a VNAR domain that binds to the TfR-1.
  • VNAR domain has the general structure, from N to C terminus, given by the formula FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4, where the FWs are framework regions, CDRs are complementarity determining regions and HVs are hypervariable regions that collectively form the variable domain of a shark IgNAR (“VNAR”).
  • the VNAR of the present invention may be directed to Type II VNAR domains where the FW1, FW2, FW2’, FW3 and FW4 regions have naturally occurring VNAR sequences or altered VNAR sequences with amino acid substitutions, insertions or deletions (typically, but not limited to, no more than 1-10 amino acids changes, insertions or deletions) provided that such alterations maintain the overall tertiary structure of the Type II VNAR, and provided that such VNAR domains have combination of CDR1, HV2, HV4, and CDR3 regions shown in Table 6.
  • the amino acid sequences of the CDR1, HV2, HV4, and CDR3 regions of those VNAR domains are also provided in Table 6.
  • VNAR scaffolds i.e., the FW and HV regions of the VNAR domain
  • VNAR domains of the fusion protein provided herein may comprise amino acid substitutions, insertions or deletions provided that such alterations maintain the overall primary and tertiary structure of the Type II VNAR and do not create a human T-cell epitope.
  • the VNAR domains of the fusion protein provided herein can optionally have a His-Tag (or other convenient tag for purification purposes). In some cases, such tags are removable.
  • fusion proteins that comprise a polypeptide comprising a VNAR domain that binds to the transferrin receptor (TfR-1) joined to an antibody Fc domain and an amyloid-reactive peptide, which may efficiently cross the blood brain barrier (BBB).
  • TfR-1 transferrin receptor
  • BBB blood brain barrier
  • the VNAR that binds to the TfR-1 exhibits broader primate specificity.
  • the VNAR that binds to the TfR-1 exhibits cross-species reactivity.
  • the VNAR that binds to the TfR-1 exhibits cross-species reactivity with mouse, rat, monkey and/or human TfR-1.
  • the VNAR that binds to the TfR-1 comprises VNAR antibody domain.
  • single chain variable domain antibodies include, but are not limited to, a shark or other cartilaginous fish antibodies, camelid antibodies and nanobodies.
  • the VNAR is a shark single domain VNAR.
  • VNAR is a Type II VNAR polypeptide obtained by in vitro selection against recombinant human TfR-1 using semisynthetic VNAR phage display libraries, as described in WO2022/103769.
  • the polypeptide that binds to the TfR-1 is capable of reaching higher concentrations in the brain than existing therapeutic and exploratory compositions (e.g., monoclonal antibodies).
  • the polypeptide that binds to the TfR-1 is a deimmunized variant for use in a mammalian subject.
  • the mammalian subject is a human.
  • the fusion proteins comprising the polypeptide comprising the VNAR that binds to the TfR-1 is capable of crossing the BBB, where the VNAR that binds to the TfR-1 enables effective and efficient BBB trafficking.
  • the fusion protein comprising the polypeptide comprising the VNAR that binds to the TfR-1 is useful in treating amyloid disease in brain.
  • the fusion protein comprising the polypeptide comprising the VNAR that binds to the TfR-1 is useful in treating a subject having amyloid deposits in the brain.
  • fusion proteins comprising the polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), an amyloid reactive peptide, and an antibody Fc polypeptide, capable of specifically binding to human TfR-1 without substantially interfering with transferrin binding to and/or transport by human TfR-1.
  • the VNAR of the fusion protein is represented by the formula, from N to C terminus, FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4, where FW1, FW2, FW2’, FW3, and FW4 are framework (FW) region sequences, CDR1 and CDR3 are complimentary- determining regions (CDR) sequences, and HV2 and HV4 are hypervariable regions (HV) sequences.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in Table 5.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 26-28 and 44-53. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequence set forth in Table 5. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:44.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:45. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:46. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:47.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 50.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:52. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:53.
  • the underlined sequences of Table 5 represent VNAR CDR and HV sequences (SEQ ID NOs: 29-33), where the bold and underlined sequences are HV4 sequences (SEQ ID NOs: 31 and 32).
  • fusion proteins comprising the polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), an amyloid reactive peptide, and an antibody Fc polypeptide, where the VNAR specifically binds to TfR- 1.
  • the VNAR of the fusion protein does not bind, or does not bind with high affinity, to TfR-2.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in Table 5.
  • the VNAR of the fusion protein comprises an amino acid sequences having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 26-28 and 44-53. In some embodiments, the VNAR of the fusion protein comprises an amino acid sequence set forth in Table 5.
  • the VNAR-Fc-peptide fusion proteins comprising a polypeptide that comprises a VNAR domain that binds to the transferrin receptor (TfR-1) comprising one or more complimentary-determining regions (CDR) sequences.
  • CDR sequences of conventional antibodies are highly variable regions of the heavy and light chains in immunoglobulins antibodies that determine antigen specificity and represent the location where these molecules bind to their specific antigen.
  • IgNAR i.e., a shark antibody
  • only one set of CDRs are present, located at the N-terminal portion of the structure.
  • fusion proteins comprising a polypeptide that comprises a VNAR comprising one or more CDR sequence that possess an amino acid length between 4 and 30, 6 and 28, 8 and 26, 10 and 24, 12 and 22, 14 and 20, or 16 and 18 residues.
  • the CDR sequences are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.
  • the CDR sequences are between 4 and 20 amino acids in length.
  • the CDR sequences are between 5 and 15 amino acids in length.
  • the CDR sequences comprise an amino acid sequence set forth in Table 6A comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the CDR sequences comprise an amino acid sequence set forth in Table 6A.
  • the CDR sequences of a control VNAR comprise an amino acid sequence set forth in Table 6B.
  • the VNAR that binds to the transferrin receptor (TfR- 1) of the VNAR-Fc-peptide fusion proteins provided herein comprises one or more of a CDR1 sequence and CDR3 sequence.
  • the VNAR that binds to TfR-1 comprises a CDR1 sequence and a CDR3 sequence.
  • the VNAR that binds to TfR-1 comprises a CDR1 sequence and a CDR3 sequence, each having an amino acid sequence set forth in Table 6A, where the CDR1 and/or CDR3 sequences comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises a CDR1 sequence having the amino acid sequence set forth in SEQ ID NO:29 and a CDR3 sequence having the amino acid sequence set forth in SEQ ID NO:33, each comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29 and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the VNAR that binds to the transferrin receptor (TfR- 1) of the VNAR-Fc-peptide fusion proteins provided herein comprises one or more hypervariable (HV) region sequences. Similar to the CDR sequences, the HV sequences are highly variable, are located at the terminal portion of the polypeptide that binds to TfR-1, and play an essential role in determining antigen specificity. HV sequences are typically located between the CDR sequences. In some embodiments, the VNAR that binds to TfR-1 comprises a VNAR domain, where the VNAR domain comprises one or more CDR sequence and one or more HV sequence.
  • VNARs that bind to TfR-1 comprising one or more HV sequence possessing an amino acid length between 4 and 30, 6 and 28, 8 and 26, 10 and 24, 12 and 22, 14 and 20, or 16 and 18 residues.
  • the HV sequences are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.
  • the HV sequences are between 4 and 20 amino acids in length.
  • the HV sequences are between 5 and 15 amino acids in length.
  • the HV sequences comprise an amino acid sequence set forth in Table 6A comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the HV sequences comprise an amino acid sequence set forth in Table 6A.
  • the HV sequences of a control VNAR comprise an amino acid sequence set forth in Table 6B.
  • the VNAR that binds to the transferrin receptor (TfR- 1) of the VNAR-Fc-peptide fusion proteins provided herein comprises one or more of a HV2 sequence and HV4 sequence.
  • the VNAR that binds to TfR-1 of the fusion protein comprises a HV2 sequence and a HV4 sequence.
  • the VNAR that binds to TfR-1 comprises HV2 and HV4 sequences, each comprising an amino acid sequence set forth in Table 6 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises an HV2 sequence having the amino acid sequence set forth in SEQ ID NO:30 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30. In some embodiments, the VNAR that binds to TfR-1 comprises an HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises an HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:32 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, the VNAR that binds to TfR-1 comprises an HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:32.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33, and where one or more of the CDR1, HV2, HV4, and CDR3 sequences comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the VNAR that binds to the TfR-1 comprises a CDR1 sequence consisting of the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence consisting of the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence consisting of the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence consisting of the amino acid sequence set forth in SEQ ID NO:33.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the VNAR that binds to the TfR-1 comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:29, a HV2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, a HV4 consisting of the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:33.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID N0:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55.
  • the VNAR that binds to the TfR-1 comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:29, a HV2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, a HV4 consisting of the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:55.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:26.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:27.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:28.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR that binds to the transferrin receptor (TfR- 1) of the fusion proteins provided herein comprises one or more framework (FW) region sequence.
  • Framework regions (FW) are highly conserved regions of the variable portion of the antibody, which support proper folding of the antibody allowing the CDR regions to be stabilized and specific for their target.
  • the framework regions of the VNAR that binds to TfR-1 can be any natural or artificial framework sequence provided that it preserves the structural integrity of VNAR (e.g., a Type II VNAR domain), as well as any deimmunized VNAR scaffold disclosed herein.
  • the VNAR that binds to TfR-1 comprises one or more of an FW1, FW2, FW2’, FW3, and FW4 sequence. In some embodiments, the VNAR comprises FW1, FW2, FW2’, FW3, and FW4 sequences.
  • the FW sequences described herein can be murine sequences, or derivatives thereof that are deimmunized for use in non-human primates and human subjects. In some embodiments, the FW sequences are murine sequences and/or derived from a mouse. In some embodiments, the FW sequences have been mutated at one or more amino acid positions to reduce immune related responses and/or deimmunize VNAR use in non-human primates and human subjects.
  • the FW sequences comprise an amino acid sequence set forth in Table 7.
  • the VNAR that binds to TfR-1 comprises FW1, FW2, FW2’, FW3, and FW4 sequences set forth in Table 7, where one or more FW sequence comprises 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to transferrin receptor (TfR-1) of the fusion protein provided herein comprises an FW 1 comprising the amino acid sequence set forth in SEQ ID NO:34 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises an FW1 comprising the amino acid sequence set forth in SEQ ID NO: 34.
  • the VNAR that binds to TfR-1 comprises an FW1 comprising the amino acid sequence set forth in SEQ ID NO:35 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises an FW1 comprising the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the VNAR that binds to TfR-1 comprises an FW2 comprising the amino acid sequence set forth in SEQ ID NO:36 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an FW2 comprising the amino acid sequence set forth in SEQ ID NO:36.
  • the VNAR that binds to TfR-1 comprises an FW2’ comprising the amino acid sequence set forth in SEQ ID NO:37 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an FW2’ comprising the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the VNAR that binds to TfR-1 comprises an FW2’ comprising the amino acid sequence set forth in SEQ ID NO:38 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises an FW2’ comprising the amino acid sequence set forth in SEQ ID NO:38. In some embodiments, the VNAR that binds to TfR-1 comprises an FW2’ comprising the amino acid sequence set forth in SEQ ID NO:39 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an FW2’ comprising the amino acid sequence set forth in SEQ ID NO:39.
  • the VNAR that binds to TfR-1 comprises an FW3 comprising the amino acid sequence set forth in SEQ ID NO:40 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an FW3 comprising the amino acid sequence set forth in SEQ ID NO:40. In some embodiments, the VNAR that binds to TfR-1 comprises an FW4 comprising the amino acid sequence set forth in SEQ ID NO:41 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises an FW4 comprising the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the VNAR that binds to TfR-1 comprises an FW4 comprising the amino acid sequence set forth in SEQ ID NO:42 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the VNAR that binds to TfR-1 comprises an FW4 comprising the amino acid sequence set forth in SEQ ID NO:42.
  • the VNAR-Fc-peptide fusion protein comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises, from N to C terminus, a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where FW1, FW2, FW2’, FW3, and FW4 are framework (FW) regions, CDR1 and CDR3 are complimentary-determining regions (CDR), and HV2 and HV4 are hypervariable regions (HV).
  • TfR-1 transferrin receptor
  • the VNAR that binds to TfR-1 comprises, from N to C terminus, a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:31, and a CDR3 set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 comprises, from N to C terminus, a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:31, and a CDR3 set forth in SEQ ID NO:33.
  • the VNAR that binds to TfR-1 comprises a CDR1 and a CDR3 set, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to TfR-1 comprises a CDR1 and a CDR3, and an amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to TfR-1 of the fusion proteins provided herein comprises, from N to C terminus, a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3- CDR3-FW4 sequence, where the polypeptide comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:31, and a CDR3 set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the polypeptide that binds to TfR-1 comprises, from N to C terminus, a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3- CDR3-FW4 sequence, where the polypeptide comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:31, and a CDR3 set forth in SEQ ID NO:33.
  • the polypeptide that binds to TfR-1 comprises a CDR1 and a CDR3, where the polypeptide has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the polypeptide that binds to TfR-1 comprises a CDR1 and a CDR3, and an amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to TfR-1 of the fusion proteins provided herein comprises, from N to C terminus, a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3- CDR3-FW4 sequence, where the VNAR comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:32, and a CDR3 set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 of the fusion protein provided herein comprises, from N to C terminus, a FW1-CDR1-FW2- HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:32, and a CDR3 set forth in SEQ ID NO:33.
  • the VNAR that binds to TfR-1 comprises a CDR1 and a CDR3, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the VNAR that binds to TfR-1 comprises a CDR1 and a CDR3, and an amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 of the fusion proteins provided herein comprises a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 consists of the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 of the fusion proteins provided herein comprises a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 consists of the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 of the fusion proteins provided herein comprises a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to the TfR-1 consists of the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to the TfR-1 of the fusion proteins provided herein comprises a FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4 sequence, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR that binds to the TfR-1 consists of the amino acid sequence set forth in SEQ ID NO:49.
  • VNAR-Fc-peptide fusion protein comprising a polypeptide that comprises two or more VNAR domains that binds to the transferrin receptor (TfR-1).
  • TfR-1 transferrin receptor
  • the two or more VNARs that binds to the TfR-1 are the same VNAR.
  • the two or more VNARs that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the two or more VNARs that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the two or more VNARs that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • the two or more VNARs that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • the two or more VNARs of the fusion proteins provided herein each comprise the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the two or more VNARs of the fusion proteins provided herein, each comprising the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the two or more VNARs of the fusion proteins provided herein, each comprising the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the two or more VNARs of the fusion proteins provided herein, each comprising the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the two or more VNARs of the fusion proteins provided herein are unique polypeptides, each comprising an amino acid sequence set forth in any one of SEQ ID NOs: 26- 28.
  • the VNAR-Fc-peptide fusion protein comprises a polypeptide comprising a VNAR that binds to TfR-1 with a high binding affinity.
  • the VNAR binding affinity for TfR-1 is measured as an equilibrium dissociation constant (KD).
  • the VNAR binds to TfR-1 with a binding affinity of less than 500, 400, 300, 200, 100, or 1 nM.
  • the VNAR binds to TfR-1 with a binding affinity between 0.01 and 500, 0.05 and 400, 0.1 and 300, 0.5 and 200, 1 and 100, 2 and 75, 3 and 50, 4 and 40, 5 and 30, 6 and 20, 7 and 15, or 8 and 12 nM. In some embodiments, the VNAR binds to TfR-1 with a binding affinity between 0.01 and 20, 0.05 and 10, 0.1 and 8, 0.5 and 6, 1 and 4, or 2 and 3 nM. In some embodiments, the VNAR binds to TfR-1 with a binding affinity no more than 10, 5, 3, or 1 nM.
  • the VNAR that binds to TfR-1 with a binding affinity described herein comprises a CDR1, a HV2, a HV4, and a CDR3 set forth in Table 5 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 with a binding affinity described herein comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to TfR-1 with a binding affinity described herein comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to TfR-1 with a binding affinity described herein comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to TfR-1 with a binding affinity described herein comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR-Fc-peptide fusion protein comprising a polypeptide comprising a VNAR capable of binding to non-human primate transferrin receptor (TfR-1).
  • the non-human primate is a macaque (e.g., a cynomolgus monkey or cynomolgus macaque).
  • the VNAR of the fusion proteins provided herein is a small protein domain with a mass between 8 and 16, 9 and 15, 10 and 14, or 11 and 13 kDa. In some embodiments, the VNAR has a mass of about 8, 9, 10, 11, 12, 13, 14, 15, or 16 kDa. In some embodiments, the VNAR has a mass of 8, 9, 10, 11, 12, 13, 14, 15, or 16 kDa. In some embodiments, the VNAR has a mass between 10 and 14 kDa. In some embodiments, VNAR of the fusion protein provided herein is highly stable. In some embodiments, the small size and mass of the VNAR makes it a useful domain for targeting new and/or difficult antigens.
  • the fusion proteins comprise a polypeptide comprising a VNAR that binds to TfR-1 is capable of uptake across the BBB, or the cell membrane of a TfR-1 -expressing cell.
  • the fusion protein capable of crossing the BBB is used to treat amyloid deposits in the brain and associated central nervous system diseases.
  • the fusion protein capable of crossing the BBB comprises a VNAR comprising a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:31, and a CDR3 set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to TfR-1 of the fusion protein comprises a CDR1 and a CDR3, and an amino acid sequence set forth in SEQ ID NO:26.
  • the fusion protein capable of crossing the BBB comprises a polypeptide comprising a VNAR comprising a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:31, and a CDR3 set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the polypeptide has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • the polypeptide that binds to TfR-1 comprises a CDR1 and a CDR3 and an amino acid sequence set forth in SEQ ID NO:28.
  • the fusion proteins provided herein are used to treat amyloid deposits in the brain and associated central nervous system diseases.
  • the fusion protein capable of crossing the BBB comprises a polypeptide comprising a VNAR comprising a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:32, and a CDR3 set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the VNAR has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to TfR-1 comprises a CDR1 and a CDR3, and an amino acid sequence set forth in SEQ ID NO:27.
  • the fusion protein provided herein are used to treat amyloid deposits in the brain and associated central nervous system diseases.
  • the fusion protein capable of crossing the BBB comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 set forth in SEQ ID NO:29, a HV2 set forth in SEQ ID NO:30, a HV4 set forth in SEQ ID NO:32, and a CDR3 set forth in SEQ ID NO:33.
  • the VNAR that binds to TfR-1 of the fusion protein comprises a CDR1 and a CDR3, and where the VNAR that binds to the TfR-1 consists of an amino acid sequence set forth in SEQ ID NO:27.
  • the fusion protein provided herein are used to treat amyloid deposits in the brain and associated central nervous system diseases.
  • the fusion protein capable of crossing the BBB comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3 of SEQ ID NO:27.
  • the fusion proteins provided herein are used to treat amyloid deposits in the brain and associated central nervous system diseases.
  • the fusion proteins provided herein are used in the manufacture of a medicament for treating amyloid deposits in the brain and associated central nervous system diseases in a patient.
  • the fusion protein capable of crossing the BBB comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3 of SEQ ID NO:49.
  • the fusion proteins provided herein are used to treat amyloid deposits in the brain and associated central nervous system diseases.
  • the fusion proteins provided herein are used in the manufacture of a medicament for treating amyloid deposits in the brain and associated central nervous system diseases in a patient.
  • Polypeptides that bind to the transferrin receptor (TfR-1) listed in Table 5 may be fused together with one or more amino acid domains.
  • VNAR-Fc- peptide fusion proteins comprising a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1) are described.
  • Fusion protein comprising a polypeptide comprising a VNAR that binds to the TfR-1, an antibody Fc polypeptide, and an amyloid reactive peptide are also described.
  • VNAR-Fc-peptide fusion protein comprising a polypeptide comprising one or more of the amino acid domains described herein, including but not limited to an amyloid-reactive peptide, an antibody Fc polypeptide, and a VNAR that binds to the transferrin receptor (TfR-1).
  • the fusion protein has pan amyloid reactivity and are able to react with multiple different forms of amyloid, for example, Ap, tau, and a-synuclein aggregates.
  • the fusion protein comprises an amyloid-reactive peptide that is able to bind to various forms of amyloid, linked to an antibody Fc domain that promotes and/or enhances phagocytosis of amyloid deposits.
  • the fusion protein is useful for treating amyloid disorders, which involve amyloid accumulation.
  • the fusion protein comprises a VNAR that binds to the TfR-1 and is capable of crossing the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • the fusion protein is useful for treating amyloid deposits in the brain and central nervous system (CNS) diseases involving amyloid accumulation.
  • the fusion protein is a dimer comprising two monomers, where each fusion protein monomer comprises in the N-terminal to C-terminal direction a VNAR that binds to the transferrin receptor (TfR-1), an antibody Fc polypeptide, and an amyloid-reactive peptide.
  • Fusion protein monomers can interact to form dimers covalently or non-covalently. Examples of protein monomer interactions include, by are not limited to, disulfide bonds, hydrostatic salt bridge, hydrophobic interactions, chemical crosslinking, or inclusion of an amino acid spacer from the first monomer to the second monomer.
  • the dimer of fusion protein monomers is formed by a disulfide bond between a first antibody Fc polypeptide and a second antibody Fc polypeptide.
  • the first antibody Fc polypeptide and second antibody Fc polypeptide form a dimer by covalent linkage in the Fc hinge region.
  • the first antibody Fc polypeptide and second antibody Fc polypeptide are linked by a disulfide bond.
  • an antibody Fc domain is the first antibody Fc polypeptide and the second antibody Fc polypeptide covalently or non-covalently linked together.
  • the first antibody Fc polypeptide and second antibody Fc polypeptide are linked by one or more disulfide bonds between cysteines at amino acid positions 6 and 9 as set forth in SEQ ID NO: 14.
  • the fusion protein dimer may be a homodimer. In some embodiments, the fusion protein dimer may be a heterodimer.
  • the fusion protein comprises a polypeptide comprising a VNAR that binds the transferrin receptor (TfR-1) fused to an Fc polypeptide of a conventional antibody. Such fusions can be made at the N terminus or the C terminus of the antibody Fc polypeptide.
  • a first antibody Fc polypeptide and a second antibody Fc polypeptide form a dimer (i.e., an antibody Fc domain), where the polypeptide that binds TfR-l can be fused at the N terminus or the C terminus of either or both of the antibody Fc polypeptides.
  • the Fc domain is a mammalian Fc domain, including primate Fc domains. In some embodiments, the Fc domain is a human Fc domain, a cynomolgus macaque Fc domain or a murine Fc domain. In some embodiments, the Fc domain is an human Fc domain. In some embodiments, the Fc domain is from an human IgG domain, for example, a human IgGl, IgG2, or IgG4 domain. In some embodiments, the antibody Fc domain comprises a human Fc domain. In some embodiments, the antibody Fc domain comprises a human IgGl, IgG2, or IgG4 Fc domain.
  • the fusion protein monomer comprises an antibody Fc polypeptide comprising an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14.
  • two antibody Fc polypeptides are covalently linked by disulfide bonds to form the antibody Fc domain of the fusion protein dimer.
  • the VNAR-Fc-peptide fusion protein dimer comprises two amyloid-reactive peptides, where each fusion protein monomer comprises an amyloidreactive peptide.
  • the fusion protein dimer comprises two amyloid-reactive peptides that are the same peptide, where the amyloid-reactive peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the fusion protein dimer comprises two amyloid-reactive peptides that are the same peptide, where the amyloid-reactive peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13.
  • the fusion protein dimer comprises two amyloid-reactive peptides that are the same peptide, where the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the fusion protein dimer comprises two amyloid-reactive peptides that are the same peptide, where the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein dimer comprises two amyloidreactive peptides that are unique, where each amyloid-reactive peptide comprises a unique amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the unique amyloid-reactive peptides each comprise a unique amino acid sequence set forth in any one of SEQ ID NOs: 1-13.
  • the fusion protein monomer comprises an amyloidreactive peptide and an antibody Fc polypeptide.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13.
  • the amyloid-reactive peptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1-13.
  • the antibody Fc polypeptide of the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody Fc polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 14 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the fusion protein dimer comprises two monomers joined by a noncovalent or covalent linkage in the Fc hinge region.
  • the VNAR-Fc-peptide fusion protein monomer comprises an amyloid-reactive peptide and an antibody Fc polypeptide, where the amyloidreactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2 and the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14.
  • the fusion protein monomer comprises an amyloid-reactive peptide and an antibody Fc polypeptide, where the amyloid-reactive peptide consists of the amino acid sequence set forth in SEQ ID NO:2 and the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14.
  • the fusion protein monomer comprises an amyloidreactive peptide and an antibody Fc domain, where the amyloid-reactive peptide is linked to the Fc domain by a spacer.
  • amino acid spacers include but are not limited to those set forth in SEQ ID NOs: 15-24.
  • Spacers of the present invention may comprise at least 5, 10, 15, 20, 25 or 30 amino acids in length. Spacers of the present invention may comprise between 3 and 50, 5 and 45, 7 and 40, 10 and 35, 12 and 30, or 15 and 25 amino acids.
  • the spacer is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids in length.
  • the spacer of the fusion protein monomer described herein may be a flexible spacer or a rigid spacer.
  • the spacer of the fusion protein monomer described herein may be a short spacer or a long spacer.
  • the amino acid spacer comprises an amino acid sequence as set forth in Table 3 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amino acid spacer comprises an amino acid sequence as set forth in Table 3. Spacers described herein can be used to link two or more amino acid domains together.
  • the fusion protein monomer comprises in N-terminal to C-terminal direction the antibody Fc polypeptide, a spacer, and an amyloid-reactive peptide.
  • the antibody Fc polypeptide comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions, and the amyloid-reactive peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the fusion protein monomer comprises in N-terminal to C-terminal direction the antibody Fc polypeptide, a spacer, and an amyloid-reactive peptide.
  • the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24
  • the amyloid-reactive peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13.
  • the fusion protein monomer comprises in N-terminal to C-terminal direction the an amyloid-reactive peptide, a spacer, and the antibody Fc polypeptide.
  • the fusion protein monomer comprises in N-terminal to C-terminal direction the antibody Fc polypeptide, a spacer, and an amyloid-reactive peptide
  • the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide comprises the amino acid sequence set forth in ID NO:2.
  • the fusion protein monomer comprises in N-terminal to C-terminal direction the antibody Fc polypeptide, a spacer, and an amyloid-reactive peptide, where the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14, the spacer consists of the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide consists of the amino acid sequence set forth in ID NO:2.
  • the fusion protein is a dimer comprising two monomers, where each fusion protein monomer comprises in the N-terminal to C-terminal direction a VNAR that binds to the transferrin receptor (TfR-1), an antibody Fc polypeptide, and an amyloid-reactive peptide.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the TfR-1 fused to the antibody Fc polypeptide. Such fusions can be made at the N terminus or the C terminus of the VNAR that binds to the TfR-1.
  • the fusion protein monomer further comprises an amyloid-reactive peptide linked to the antibody Fc polypeptide.
  • the amyloid-reactive peptide of the fusion protein is linked to the antibody Fc polypeptide by an amino acid spacer.
  • the fusion protein monomer comprises in the N-terminal to C-terminal direction a VNAR that binds to the TfR-1, an antibody Fc polypeptide, a spacer, and an amyloidreactive peptide.
  • the fusion protein dimer is formed from two monomers comprising two VNARs that bind to TfR-1, each fused to an antibody Fc polypeptide linked by an amino acid spacer to an amyloid-reactive peptide.
  • the fusion protein dimer comprises two fusion protein monomers that are linked together covalently or non- covalently.
  • the two fusion protein monomers are linked together covalently at the antibody Fc hinge region.
  • the two fusion protein monomers are linked together by one or more disulfide bond between the antibody Fc polypeptides of each protein fusion monomer.
  • the one or more disulfide bonds between cysteines at amino acid positions 6 and 9 as set forth in SEQ ID NO: 14 link together the two protein fusion monomers. In some embodiments, the two disulfide bonds between cysteines at amino acid positions 6 and 9 of the first antibody Fc polypeptide and the second antibody Fc polypeptide link together the two protein fusion monomers.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33, and where one or more of the CDR1, HV2, HV4, and CDR3 sequences comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the polypeptide that binds to the TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:28.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the polypeptide that binds to the TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:27.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the polypeptide that binds to the TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises a CDR1 and a CDR3, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:49.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein monomer comprises a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), where the VNAR comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:49.
  • the protein fusion monomer further comprises an antibody Fc polypeptide, a spacer, and an amyloid reactive peptide, where two monomers are linked together to form the protein fusion dimer.
  • the fusion protein comprising a polypeptide comprising a VNAR that binds to the transferrin receptor (TfR-1), an antibody Fc polypeptide, and an amyloid-reactive peptide can be assembled in different linear and non-linear arrangements.
  • the fusion protein comprises in the N- terminal to C-terminal direction the VNAR that binds to the TfR-1, the antibody Fc polypeptide, and the amyloid-reactive peptide.
  • the fusion protein comprises in the N- terminal to C-terminal direction the amyloid-reactive peptide, the antibody Fc domain, and the VNAR that binds to the TfR-1.
  • the fusion protein further comprises an amino acid spacer, where the fusion protein comprises in the N-terminal to C-terminal direction the VNAR that binds to the TfR-1, the antibody Fc domain, a spacer, and the amyloid-reactive peptide. In another embodiment, the fusion protein comprises in the N-terminal to C-terminal direction the amyloid-reactive peptide, the antibody Fc domain, a spacer, and the VNAR that binds to the TfR-1.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide
  • the VNAR that binds the TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33, and where one or more of the CDR1, HV2, HV4, and CDR3 sequences comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to the TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloidreactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloidreactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:49.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein may further comprise one or more amino acid spacer that link together two or more amino acid domains, for example, the antibody Fc polypeptide and the amyloid-reactive peptide.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, a spacer, and the amyloid-reactive peptide, where the spacer comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the spacer consists of the amino acid sequence set forth in SEQ ID NO: 15.
  • the fusion protein may comprise a dimer of monomers, where each monomer comprise a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloidreactive peptide.
  • the fusion protein that is a dimer comprises two or more amino acid spacers that each link together two or more protein domains, for example, the antibody Fc domain and the amyloid-reactive peptide.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, a spacer, and the amyloidreactive peptide, where the spacer comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the spacer comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24.
  • the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15.
  • the spacer consists of the amino acid sequence set forth in SEQ ID NO: 15.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33, and where one or more of the CDR1, HV2, HV4, and CDR3 sequences comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 sequence comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 sequence comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the polypeptide that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloidreactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the polypeptide that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, where one or more of the CDR1, HV2, HV4, and CDR3 comprise 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloidreactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:49.
  • TfR-1 transferrin receptor
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:49.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the VNAR-Fc-peptide fusion protein may assume one or more linear or non-linear orientations.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide.
  • the fusion protein comprises in the N-terminal to C-terminal direction the amyloid-reactive peptide, the spacer, the antibody Fc polypeptide, and the VNAR that binds to the transferrin receptor (TfR-1).
  • the antibody Fc polypeptide may be linked two or more amyloid-reactive peptides. In some embodiments, the two or more spacers link together the antibody Fc polypeptide and the two or more amyloid-reactive peptides.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the VNAR-Fc-peptide fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, and the amyloid-reactive peptide, where the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein comprises the amino acid sequence set forth in Table 8.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the polypeptide that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein comprises the amino acid sequence set forth in Table 8.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the VNAR-Fc-peptide fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, and the amyloid-reactive peptide, where the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:54.
  • the fusion protein comprises the amino acid sequence set forth in Table 8.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:54.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the polypeptide that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:54.
  • the fusion protein comprises the amino acid sequence set forth in Table 8.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:54.
  • amino acid sequence of the fusion protein in provided in Table 8 where underlined sequence represents the VNAR that binds to the transferrin receptor (TfR-1), underlined and bolded sequence represents CDR and HV sequences within the VNAR, italic sequence represent the antibody Fc polypeptide sequence, unformatted sequence represent the spacer, and bolded sequence represents the amyloid-reactive peptide.
  • VNAR sequence is underlined; CDR1, HV2, HV4, and CD3 sequences are underlined and bolded; antibody Fc domain is italicized; and the amyloid reactive peptide is bolded.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein comprises the amino acid sequence set forth in Table 8. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 comprises the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises an amino acid sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein comprises the amino acid sequence set forth in Table 8. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43. [0207] In some embodiments, the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:29, a HV2 consisting of the amino acid sequence set forth in SEQ ID NO:30, a HV4 consisting of the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:33, the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14, the
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to TfR-1 consists of the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14, the spacer consists of the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide consists of the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:43.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer is a homodimer, where each fusion protein monomer comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein dimer is a homodimer, where each fusion protein monomer consists of the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the fusion protein dimer is a heterodimer.
  • the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide
  • the VNAR that binds to TfR-1 comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:29, a HV2 consisting of the amino acid sequence set forth in SEQ ID NO:30, a HV4 consisting of the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:55
  • the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer consists of the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide consists of the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to TfR-1 consists of the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 14, the spacer consists of the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide consists of the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer is a homodimer, where each fusion protein monomer comprises the amino acid sequence set forth in SEQ ID NO:54. In some embodiments, the fusion protein dimer is a homodimer, where each fusion protein monomer consists of the amino acid sequence set forth in SEQ ID NO:54. In some embodiments, the fusion protein dimer is a heterodimer.
  • VNAR-Fc-peptide fusion protein possess important advantages over existing compositions and therapeutics intended to treat amyloid-related disease, especially in instances when the amyloid is located in the CNS.
  • the provided fusion proteins enable efficient brain penetration at low therapeutic doses compared to existing therapies.
  • the fusion proteins provided herein comprise a high affinity bivalent variable domain of new antigen receptor (VNAR) antibody polypeptide that binds to the TfR-1 and has the potential to achieve high brain penetration, helping to overcome the clinical limitations of other existing therapeutics.
  • VNAR new antigen receptor
  • the fusion proteins provided herein enable high affinity binding of brain specific amyloids, for example, Ap amyloid, Tau amyloid, and a-synuclein aggregates.
  • fusion proteins comprising an amyloid-reactive peptide that enable pan amyloid reactivity for targeting brain amyloids and systemic amyloids for clearance.
  • fusion proteins that address these longstanding challenges by (1) granting access to the therapeutic target (i.e., enabling blood-brain barrier transport) and (2) producing a therapeutic effect (i.e., reducing the prevalence and or propagation of brain amyloids).
  • the VNAR-Fc-peptide fusion protein monomer comprises in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:43.
  • the VNAR-Fc-peptide fusion protein monomer comprises in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the VNAR-Fc-peptide fusion protein is capable of uptake across the blood-brain barrier (BBB).
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 enables receptor-mediated transcytosis (RMT) to transport cargo through the brain endothelial cells toward brain parenchyma.
  • TfR-1 transferrin receptor
  • RMT receptor-mediated transcytosis
  • the fusion protein further comprises an amino acid spacer that links together the antibody Fc polypeptide and the amyloid-reactive peptide.
  • interactions between the fusion protein and TfR-1 induces RMT and enables efficient uptake of the fusion protein across the BBB.
  • interactions between the fusion protein and TfR- 1 induces RMT and enables efficient brain penetration of the fusion protein at low therapeutic doses.
  • the fusion protein capable of uptake across the bloodbrain barrier (BBB), comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the VNAR that binds to the TfR-1 comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR consists of the amino acid sequence set forth in SEQ ID NO:27. In some embodiment, the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein monomer capable of uptake across the blood-brain barrier (BBB), comprises a polypeptide comprising in the N-terminal to C- terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer is capable of uptake across the blood-brain barrier (BBB) and efficient brain penetration.
  • BBB blood-brain barrier
  • the fusion protein comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc polypeptide, and the amyloid-reactive peptide, wherein the fusion protein has pan amyloid reactivity.
  • the fusion protein further comprises an amino acid spacer that links together the antibody Fc polypeptide and the amyloid-reactive peptide.
  • the fusion protein monomer having pan amyloid reactivity, comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • TfR-1 transferrin receptor
  • the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer comprises the amino acid sequence set forth in SEQ ID NO:
  • the amyloid-reactive peptide comprises the amino acid
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer having pan amyloid reactivity, comprises a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer has pan amyloid reactivity.
  • the VNAR-Fc-peptide fusion proteins bind to amyloid deposits or amyloid fibrils. In some embodiments, the fusion proteins bind to one or more amyloidogenic peptides in amyloids, heparin sulfate glycosoaminoglycans in the amyloid, human amyloid fibrils, and/or synthetic amyloid fibrils. In some embodiments, the fusion proteins bind to rVA6Wil fibrils, Perl25 wtATTR extract, KEN hATTR extract, SHI AL liver extract, and/or TAL ALK liver extract.
  • amyloids bound by the fusion proteins comprise an amyloidogenic A6 variable domain protein (VA6Wil) or an amyloidogenic immunoglobulin light chain (AL), AP(l-40) amyloid-like fibril or an amyloidogenic Ap precursor protein, or serum amyloid protein A (AA).
  • VA6Wil amyloidogenic A6 variable domain protein
  • AL amyloidogenic immunoglobulin light chain
  • serum amyloid protein A AA
  • the amyloid-reactive peptides of the fusion proteins described herein bind to rVA6Wil, Ap, AP(l-40), IAAP, ALK, ALA, or ATTR amyloid.
  • the fusion proteins provided herein bind to ALK4, ALA amyloid.
  • the amyloids bound by the fusion proteins comprise amyloidogenic forms of immunoglobulin heavy chain (AH), p2-microglobulin (Ap2M), transthyretin variants (ATTR), apolipoprotein Al (AApoAI), apolipoprotein All (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin ((ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (Alns), prior protein (APrP); a-synuclein (AaSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALK4, A1X1, and other amyloidogenic peptides.
  • AH immunoglobulin
  • the fusion proteins have pan amyloid reactivity.
  • the amyloidogenic peptides bound by the fusion proteins can be a protein, a protein fragment, or a protein domain.
  • the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins.
  • the amyloids are part of the pathology of a disease described herein.
  • the fusion protein that binds to amyloid deposits or amyloid fibrils comprises a polypeptide comprising in the N-terminal to C- terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, where the VNAR that binds to the TfR-1 has at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.
  • the VNAR that binds to the TfR-1 of the fusion protein comprises CDR1, HV2, HV4, and CDR3 sequences, where the VNAR comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the antibody Fc polypeptide of the fusion protein comprises a sequence having at least 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14.
  • the spacer of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-24 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-13 comprising 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions.
  • the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the VNAR-Fc-peptide fusion proteins bind to amyloid deposits or amyloid fibrils. In some embodiments, the fusion proteins bind to one or more amyloidogenic peptides in amyloids, heparin sulfate glycosoaminoglycans in the amyloid, human amyloid fibrils, and/or synthetic amyloid fibrils. In some embodiments, the fusion proteins bind to rVA6Wil fibrils, Perl25 wtATTR extract, KEN hATTR extract, SHI AL liver extract, and/or TAL ALK liver extract.
  • amyloids bound by the fusion proteins comprise an amyloidogenic A6 variable domain protein (VA6Wil) or an amyloidogenic immunoglobulin light chain (AL), AP(l-40) amyloid-like fibril or an amyloidogenic Ap precursor protein, or serum amyloid protein A (AA).
  • VA6Wil amyloidogenic A6 variable domain protein
  • AL amyloidogenic immunoglobulin light chain
  • serum amyloid protein A AA
  • the fusion proteins provided herein bind to rVA6Wil, Ap, AP(l-40), IAAP, ALK, ALA, or ATTR amyloid.
  • the fusion proteins provided herein bind to ALK4, ALA amyloid.
  • the amyloids bound by the fusion proteins comprise amyloidogenic forms of immunoglobulin heavy chain (AH), p2-microglobulin (Ap2M), transthyretin variants (ATTR), apolipoprotein Al (AApoAI), apolipoprotein All (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin ((ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (Alns), prior protein (APrP); a-synuclein (AaSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALK4, A1A1, and other amyloidogenic peptides.
  • AH immunoglobulin
  • the fusion proteins have pan amyloid reactivity.
  • the amyloidogenic peptides bound by the fusion protein can be a protein, a protein fragment, or a protein domain.
  • the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins.
  • the amyloids are part of the pathology of a disease described herein.
  • the fusion protein monomer that binds to amyloid deposits or amyloid fibrils comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO: 27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer that binds to amyloid deposits or amyloid fibrils comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer binds to amyloid deposits or amyloid fibrils.
  • the fusion proteins provided herein bind to amyloid deposits or fibrils, specifically Ap, Tau, and a-synuclein amyloid fibrils.
  • amyloids bound by the fusion proteins comprise an AP(l-40) amyloid-like fibril or an amyloidogenic Ap precursor protein.
  • the amyloids bound by the fusion proteins comprise amyloidogenic forms of a-synuclein (AaSyn), tau (ATau), and other amyloidogenic peptides.
  • the fusion proteins provided herein have pan amyloid reactivity.
  • amyloidogenic peptides bound by the fusion protein can be a protein, a protein fragment, or a protein domain.
  • the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins.
  • the amyloids are part of the pathology of a disease described herein.
  • the fusion protein monomer that binds to amyloid deposits or amyloid fibrils comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the amyloids are part of the pathology of a disease described herein.
  • the fusion protein monomer that binds to amyloid deposits or amyloid fibrils comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer binds to amyloid deposits or amyloid fibrils.
  • the VNAR-Fc-peptide fusion proteins target any of the amyloids described herein for clearance, where the fusion proteins comprise a polypeptide comprising the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, and the amyloid-reactive peptide.
  • the fusion proteins further comprise an amino acid spacer that links together the antibody Fc polypeptide and the amyloid-reactive peptide.
  • the amyloid is an amyloid substrate, amyloid fibril, and/or amyloid deposit located in the brain.
  • the fusion proteins can participate in receptor-mediated transcytosis (RMT) to cross the blood-brain barrier (BBB).
  • RMT receptor-mediated transcytosis
  • BBB blood-brain barrier
  • the fusion proteins target amyloid substrates in the brain, including but not limited to Ap, Tau, and a-synuclein amyloid fibrils, for clearance.
  • the fusion proteins target amyloid substrates for clearance that are associated with systemic amyloidosis, in one or more organs other than the brain.
  • the fusion proteins provided herein target amyloids for clearance that are part of the pathology of a disease described herein.
  • the fusion proteins target amyloids for clearance that are part of the pathology of one or more neurological and/or degenerative disease, for example, Alzheimer’s disease, cerebral amyloid angiopathy (CAA), inflammatory cerebral amyloid angiopathy, cerebral amyloidoma.
  • Alzheimer’s disease cerebral amyloid angiopathy (CAA)
  • CAA cerebral amyloid angiopathy
  • inflammatory cerebral amyloid angiopathy cerebral amyloidoma.
  • the fusion protein monomer that targets amyloids for clearance comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer that targets amyloids for clearance comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer targets amyloids in the brain and other organs for clearance.
  • the VNAR-Fc-peptide fusion proteins provided herein induce phagocytosis of the amyloids, where the fusion protein comprises a polypeptide comprising the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, and the amyloid-reactive peptide.
  • the fusion proteins further comprise an amino acid spacer that links together the antibody Fc polypeptide and the amyloid-reactive peptide.
  • the phagocytosis is by macrophages.
  • the amyloids are amyloid substrates, amyloid fibrils, and/or amyloid deposits located in the brain.
  • proteins can participate in receptor-mediated transcytosis (RMT) to cross the blood-brain barrier (BBB).
  • RMT receptor-mediated transcytosis
  • BBB blood-brain barrier
  • the fusion proteins induce phagocytosis of one or more amyloid substrates in the brain, including but not limited to Ap, Tau, and a- synuclein amyloid fibrils.
  • the fusion proteins induce phagocytosis of amyloid substrates that are associated with systemic amyloidosis, in one or more organs other than the brain.
  • the fusion proteins provided herein induce phagocytosis of amyloids that are part of the pathology of a disease.
  • the fusion proteins provided herein induce phagocytosis of amyloids that are part of the pathology of one or more neurological and/or degenerative disease, for example, Alzheimer’ s disease, cerebral amyloid angiopathy (CAA), inflammatory cerebral amyloid angiopathy, cerebral amyloidoma.
  • Alzheimer’ s disease cerebral amyloid angiopathy (CAA)
  • CAA cerebral amyloid angiopathy
  • inflammatory cerebral amyloid angiopathy cerebral amyloidoma.
  • the fusion protein monomer that induces phagocytosis of amyloids comprises a polypeptide in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer that induces phagocytosis of amyloids comprises a polypeptide in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer induce phagocytosis of amyloids in the brain and other organs.
  • the VNAR-Fc-peptide fusion protein provided herein promotes amyloid clearance by opsonization, where opsonization involves the coating of molecule target with a signaling and/or immune-related biomolecule (e.g., an antibody or a fragment thereof) as a mechanism for increasing their susceptibility to ingestion by phagocytes (i.e., phagocytosis). Biomoleucles that induce or enhance phagocytosis are referred to as opsonins, for example, IgG.
  • the fusion protein provided herein is an opsonin for amyloid substrates, including amyloid substrates located in the central nervous system and brain.
  • the VNAR-Fc-peptide fusion protein promotes amyloid clearance by opsonization, where the fusion protein comprises a polypeptide comprising the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, and the amyloid-reactive peptide.
  • the fusion proteins further comprise an amino acid spacer that links together the antibody Fc polypeptide and the amyloid-reactive peptide.
  • the amyloid is an amyloid substrate, amyloid fibril, and/or amyloid deposit located in the brain.
  • the fusion proteins can participate in receptor-mediated transcytosis (RMT) to cross the blood-brain barrier (BBB).
  • RMT receptor-mediated transcytosis
  • BBB blood-brain barrier
  • the fusion proteins promote amyloid clearance by opsonization of one or more amyloid substrates in the brain, including but not limited to Ap, Tau, and a-synuclein amyloid fibrils.
  • the fusion proteins promote amyloid clearance by opsonization of amyloid substrates that are associated with systemic amyloidosis, in one or more organs other than the brain.
  • the fusion proteins provided herein promote amyloid clearance by opsonization of amyloids that are part of the pathology of a disease described herein.
  • the fusion proteins describe herein promotes amyloid clearance by opsonization of amyloids that are part of the pathology of one or more neurological and/or degenerative disease, for example, Alzheimer’s disease, cerebral amyloid angiopathy (CAA), inflammatory cerebral amyloid angiopathy, cerebral amyloidoma.
  • Alzheimer’s disease cerebral amyloid angiopathy (CAA)
  • CAA cerebral amyloid angiopathy
  • inflammatory cerebral amyloid angiopathy cerebral amyloidoma.
  • the fusion protein monomer that promotes amyloid clearance by opsonization comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer that promotes amyloid clearance by opsonization comprises a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the fusion protein dimer promotes amyloid clearance by opsonization of amyloids in the brain and other organs.
  • compositions comprising any of the fusion proteins provided herein.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • nucleic acids encoding any of the fusion proteins of the present disclosure.
  • the nucleic acid provided herein are in one or more vectors.
  • the vector comprises the nucleic acid(s) encoding a fusion protein of the present disclosure.
  • the nucleic acid encoding the VNAR-Fc-peptide fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the nucleic acid encoding the VNAR-Fc-peptide fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two encoded fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the encoded fusion protein monomers forms a homodimer.
  • the fusion protein expression vector may be introduced into one or more appropriate production cell lines known in the art. Introduction of the expression vector may be accomplished by co-transfection via electroporation or any other suitable transformation technology available in the art. Fusion protein producing cell lines can then be selected and expanded and antibodies purified. The purified fusion protein can then be analyzed by standard techniques such as SDS-PAGE or size exclusion chromatography (SEC).
  • a host cell comprising a nucleic acid encoding any of the fusion proteins of the present disclosure.
  • Suitable host cells for cloning or expression of the fusion protein-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • the fusion protein may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • polypeptides in bacteria see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254)
  • the fusion protein may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • Suitable host cells for the expression of glycosylated fusion protein are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • the host cell comprises a vector comprising a nucleic acid encoding a VNAR-Fc-peptide fusion protein monomer, where the fusion protein monomer comprises a polypeptide comprsing in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • TfR-1 transferrin receptor
  • the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer
  • the fusion protein monomer encoded by the nucleic acid comprises the amino acid sequence set forth in SEQ ID NO:43.
  • two encoded fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the host cell comprises a vector comprising a nucleic acid encoding a VNAR-Fc-peptide fusion protein monomer, where the fusion protein monomer comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • TfR-1 transferrin receptor
  • the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14
  • the spacer comprises the
  • the fusion protein monomer encoded by the nucleic acid comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two encoded fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the method comprises culturing a host cell of the present disclosure under conditions suitable for expression of the vector encoding the fusion protein. In some embodiments, the method further comprises recovering the fusion protein.
  • protein recovery involves disrupting the host cell, for example by osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris is removed by centrifugation or filtration. The fusion proteins can then be further purified.
  • the fusion protein of the disclosure is purified by various methods of protein purification, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the fusion protein is conjugated to marker sequences, such as a peptide to facilitate purification.
  • a marker amino acid sequence is a hexa-histidine peptide, which can bind to a nickel-functionalized agarose affinity column with micromolar affinity.
  • a hemagglutinin “HA” tag which corresponds to an epitope derived from the influenza hemagglutinin protein can be used.
  • VNAR-Fc-peptide fusion protein may be made by any technique known to those of skill in the art, including chemical synthesis or recombinant means using standard molecular biological techniques.
  • the methods provided herein comprises treating amyloid deposits located in the brain and/or CNS.
  • Aggregation of amyloidogenic proteins is a pathological hallmark of various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotropic lateral sclerosis, frontotemporal lobar degeneration, and tauopathies.
  • Amyloidogenic proteins implicated in the aggregation of protein deposits in neurodegenerative disease include, amyloid P (A
  • Amyloid oligomers may describe a dimer of two amyloidogenic proteins as well as an association of hundreds of amyloidogenic monomers. Consequently, in some neurodegenerative diseases a single disease-causing amyloid can result in a defined pathology, whereas other neurodegenerative diseases may involve the assembly of multiple overlapping disease-causing amyloids. Several amyloid pathologies can coexist in the same brain with or without clinical presentation and may even occur in individuals without disease. In some neurodegenerative diseases, amyloid oligomers may form structurally stable intermediates in a wide range of molecular sizes and can function as neuronal toxins rather than structurally stable fibrils.
  • amyloid oligomers The formation of amyloid oligomers is responsible for the molecular pathogenesis of the associated diseases and are thus potent targets for therapeutics. Oligomer formation causes impaired synaptic function and neuronal death through various mechanisms including, oxidative stress, inflammation, apoptosis, and dysfunction of proteostasis.
  • fusion protein comprises a polypeptide comprising a VNAR that binds transferrin receptor (TfR- 1), an antibody Fc polypeptide, and an amyloid reactive peptide.
  • provided herein is a method of treating an amyloid disease comprising administering a therapeutically effective amount of any of the VNAR-Fc- peptide fusion proteins described herein to an individual in need thereof.
  • the amyloid deposits contribute to the pathology of a disease.
  • the disease is a neurological disease or degenerative disease, such as Alzheimer’s disease.
  • the amyloid deposits are indicative of amyloidosis or an amyloid-related disease in an individual.
  • the fusion protein binds to amyloids in an individual with an amyloidosis.
  • the amyloidosis is localized to a specific tissue or organ system, such as the liver, the heart, brain, or the central nervous system.
  • the amyloidosis is a systemic amyloidosis. In some embodiments, the amyloidosis is a familial amyloidosis. In other embodiments, the amyloidosis is a sporadic amyloidosis.
  • the amyloidosis or amyloid-related disease is AA amyloidosis, AL amyloidosis, AH amyloidosis, Ap amyloidosis, ATTR amyloidosis, hATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis of type II diabetes, Alzheimer’s disease, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, cerebral beta-amyloid angiopathy, spongiform encelohalopathy, thyroid tumors, Parkinson’s disease, dementia with Lewis bodies, a tauopathy, Huntington’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), dementia with Lewy bodies (DLB), and Lewy body variant AD (LBVAD), Parkinson's Disease dementia (PD
  • the amyloid related disease is selected from the group consisting of AL, AH, A
  • treatment with the fusion protein results in the clearance of amyloid.
  • treatment with the fusion protein promotes and/or induces phagocytosis. In some embodiments, treatment with the fusion protein promotes amyloid clearance by opsonization. In some embodiments, the fusion protein binds to amyloids associated with normal aging. In other embodiments, the fusion protein is used in the treatment of an amyloidosis or amyloid-related disease in an individual.
  • provided herein is a method of treating an amyloid- related disorder in an individual comprising administering a fusion protein provided herein, wherein the individual has amyloid in the brain.
  • the individual being treated has amyloid in the brain, kidney, liver, and/or heart.
  • the individual has Alzheimer’s disease.
  • the individual has one or more of Ap amyloid, tau fibrils, and a-synuclein aggregates.
  • the individual has Ap amyloid.
  • the individual has tau fibrils or a-synuclein aggregates.
  • the individual has Parkinson’s disease.
  • the individual has ALk deposits in the kidney.
  • the individual has ALK deposits in the kidney. In some embodiments, the individual has ALk deposits in the liver. In some embodiments, the individual has ALK deposits in the liver. In some embodiments, the individual has ATTR deposits in the heart. In some embodiments, the individual has ALK deposits in the heart. In some embodiments, the individual has fibrils in the spleen.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-re
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the method of treating an amyloid-related disorder in an individual comprises administering a fusion protein dimer.
  • amyloid-related disorder is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Parkinson's Disease dementia (PDD), frontotemporal lobar degeneration (FTLD) progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), dementia with Lewy bodies (DLB), Lewy body variant AD (LBVAD), and other tauopathies.
  • PDD Parkinson's Disease dementia
  • FTLD frontotemporal lobar degeneration
  • PSP frontotemporal lobar degeneration
  • CBD corticobasal degeneration
  • FTDP-17T frontotemporal dementia and parkinsonism linked to chromosome 17
  • DLB Lewy body variant AD
  • tauopathies is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Parkinson's Disease dementia (PDD), frontotemporal lobar degeneration (FTLD) progressive supranuclear palsy (PSP
  • the fusion protein monomer used in the method of treating an amyloid related disorder comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide of the fusion protein comprises the amino acid sequence set
  • the fusion protein monomer used in the method of treating an amyloid related disorder comprises a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the fusion protein monomer used in the method of treating an amyloid related disorder comprises a polypeptide comprising in the N-terminal to C- terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:
  • the fusion protein monomer used in the method of treating an amyloid related disorder comprises a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the method of treating an amyloid-related disorder in an individual selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Parkinson's Disease dementia (PDD), frontotemporal lobar degeneration (FTLD) progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), dementia with Lewy bodies (DLB), Lewy body variant AD (LBVAD) and other tauopathies comprises administering a fusion protein dimer.
  • PDD Parkinson's Disease dementia
  • FTLD frontotemporal lobar degeneration
  • PSP frontotemporal lobar degeneration
  • CBD corticobasal degeneration
  • FTDP-17T frontotemporal dementia and parkinsonism linked to chromosome 17
  • DLB dementia with Lewy bodies
  • LVAD Lewy body variant AD
  • other tauopathies comprises administering a fusion protein dimer.
  • treatment with the fusion protein according to the methods provided herein results in the clearance or partial clearance of an amyloid in the brain of the individual.
  • the amyloid comprises Ap, Tau, and/or a-synuclein amyloid.
  • the amyloid comprises Ap, Tau, and a-synuclein amyloid.
  • treatment with the fusion protein according to the methods provided herein results in the clearance or partial clearance of Ap, Tau, and/or a-synuclein amyloid in the brain of the individual.
  • the amyloid comprises Ap, Tau, and a-synuclein amyloid.
  • treatment with the fusion protein according to the method provided herein results in the clearance or partial clearance of Ap, Tau, and a-synuclein amyloid in the brain of the individual.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloidreactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, the antibody Fc poly
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO: 27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of
  • the method of treating an amyloid- related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • Parkinson’s disease for example, is characterized by defects in motor symptom caused by the loss of dopaminergic neurons in the substantia nigra (SN).
  • PD is characterized by the accumulation of misfolded intraneuronal fibrillar a-synuclein that forms Lewy-bodies structures and the degeneration of dopaminergic neurons, where alpha-synuclein (a-synuclein, or a-syn) amyloid aggregates mediate toxicity that occurs via several intracellular mechanisms. Accumulation of alpha-synuclein in the SN activates microglial cells and potentiates the loss of dopaminergic neurons via neuro-inflammation.
  • treatment with the fusion protein according to the methods provided herein results in a reduction in the propagation of an amyloid related pathology in the brain of the individual.
  • the amyloid related pathology comprises an Ap, Tau, and/or a-synuclein related pathology.
  • the amyloid related pathology comprises an Ap, Tau, and a- synuclein related pathology.
  • treatment with the fusion protein according to the methods provided herein results in a reduction in the propagation of Ap, Tau, and/or a- synuclein related pathology in the brain of the individual.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc- peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C- terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc- peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C- terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • treatment with the fusion protein according to the methods provided herein results in a reduction in the propagation of an amyloid related pathology in the brain of the individual, where reduction in the propagation occurs in a one more regions or cells in the brain of the individual.
  • the one or more regions or cells comprise a dopaminergic neuron.
  • the dopaminergic neuron is a tyrosine hydroxylase (TH) positive dopaminergic neuron.
  • the dopaminergic neuron is located in the substantia nigra in the brain of the individual.
  • treatment with the fusion protein according to the methods provided herein results in a reduction in the propagation of an Ap, Tau, and/or a-synuclein related pathology in the brain of the individual, where reduction in the propagation occurs in a dopaminergic neuron located in the substantia nigra in the brain of the individual.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:32, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-
  • the method of treating an amyloid- related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc domain, the spacer, and the amyloid-reactive peptide, where the VNAR that binds to TfR-1 comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a HV2 comprising the amino acid sequence set forth in SEQ ID NO:30, a HV4 comprising the amino acid sequence set forth in SEQ ID NO:31, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, the antibody Fc polypeptide of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer of the
  • the method of treating an amyloid-related disorder in an individual comprises administering a VNAR-Fc-peptide fusion protein monomer comprising a polypeptide comprising in the N-terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR- 1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • the individual has amyloid deposits in the brain, spleen, kidney, liver, and/or heart.
  • the fusion protein provided herein targets amyloids.
  • the fusion protein described herein promotes and/or induces phagocytosis of a targeted amyloid.
  • the fusion protein described herein targets amyloids promotes amyloid clearance by opsonization.
  • the fusion protein provided herein is administered via an intradermal, subcutaneous, intramuscular, intracardiac, intravascular, intravenous, intraocular, intra-arterial, epidural, intraspinal, extracorporeal, intrathecal, intraperitoneal, intrapleural, intraluminal, intravitreal, intracavemous, intraventricular, intra-bone, intra- articular, intracellular, or pulmonary route.
  • the fusion protein provided herein is administered in sufficient amounts to target amyloid substrates in distinct locations throughout the body, for example, the brain and/or the kidneys. In some embodiments, the fusion protein provided herein is administered in sufficient amounts to induce phagocytosis of targeted amyloid by cells of the immune system (e.g., macrophages). In some embodiments, the fusion protein provided herein is administered in sufficient amounts to promotes amyloid clearance by opsonization by cells of the immune system (e.g., macrophages).
  • the individual is a mammal such as primate, bovine, rodent, or pig. In some embodiments, the individual is a human.
  • the amyloid deposit is in an individual.
  • targeting the amyloid deposit for clearance results in clearance of the amyloid deposit.
  • clearance results from opsonization of the amyloid deposit.
  • the method of targeting an amyloid deposit for clearance results in phagocytosis of the amyloid.
  • the individual is a human.
  • the individual has amyloid accumulation and/or and amyloid-related disease (e.g., Alzheimer’s disease).
  • the VNAR-Fc-peptide fusion protein monomer used in the method of targeting an amyloid deposit comprises a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:27, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:43.
  • the VNAR-Fc-peptide fusion protein monomer used in the method of targeting an amyloid deposit comprises a polypeptide comprising in the N- terminal to C-terminal direction the VNAR that binds to the transferrin receptor (TfR-1), the antibody Fc polypeptide, the spacer, and the amyloid-reactive peptide, where the VNAR that binds the TfR-1 comprises a CDR1, HV2, HV4, and CDR3, and the amino acid sequence set forth in SEQ ID NO:49, the antibody Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, the spacer comprises the amino acid sequence set forth in SEQ ID NO: 15, and the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:54.
  • two fusion protein monomers can come together to form a fusion protein dimer, where the two fusion protein monomers are linked by one or more disulfide bond in the antibody Fc hinge region.
  • the method of targeting an amyloid deposit contacting the amyloid deposit with a fusion protein dimer.
  • contacting the amyloid deposit with the fusion protein dimer involves administering the fusion protein to an individual.
  • the fusion protein is used to treat individuals with amyloid deposits resulting in amyloid accumulation and/or amyloidosis in the CNS including, but not limited to, secondary amyloidosis, age-related amyloidosis, Alzheimer's disease (AD), mild cognitive impairment (MCI), Lewy body dementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type), the Guam Parkinson-Dementia complex, cerebral amyloid angiopathy (CAA), Huntington's disease, progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, transmissible spongiform encephalopathy, HIV-related dementia, amyotrophic lateral sclerosis (ALS), inclusion-body myositis (IBM), and ocular diseases relating to beta- amyloid deposition (i.e., macular degeneration, drusen-related optic neuropathy, and cataract).
  • the fusion protein is used to treat individuals with amyloid deposits
  • the VNAR-Fc-peptide fusion protein is used to treat an individual with one or more clinical indications of a neuronal amyloid disease including vascular dementia, dysautonomia, ataxia, motor impersistence, sleep disturbances, mood disorders, cognitive disturbances, pain and sensory disturbances, hallucinations and illusions, metabolic abnormalities, nutritional deficiencies, infection, side effects from some medications, normal pressure hydrocephalus, psychiatric disease, a dysexecutive neuropsychological profile, parkinsonism, autonomic features, an antecedent rapid eye movement sleep behavioral disorder, sensitivity to pharmacologic dopamine blockade; behavioral features or changes such as, apathy, loss of empathy, compulsions, altered eating habits, personality, emotion, executive control, compulsive/ritualistic behaviors, atrophy within brain regions including, the lateral parietal and occipital cortices, the dorsolateral prefrontal cortex, the hippocampus, the amygdala
  • vascular dementia including lateral
  • This example describes the binding assessment of an Fc-peptide protein to recombinant microtubule-associated protein tau (MAPT) P301S protein, a protein aggregate associated with neurodegenerative disease and cognitive decline.
  • MTT microtubule-associated protein tau
  • This example demonstrates that Fc-peptide proteins can bind cerebral amyloid with high potency.
  • MAPT P301S protein aggregates are observed in individuals that have frontotemporal lobar degeneration (FTLD) and other tauopathies, which contributes to reduced microtubule assembly in the brain and progressive cognitive decline.
  • FTLD frontotemporal lobar degeneration
  • an Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 was expressed by stably transfected CHO cells grown under perfusion culture conditions and purified at day 7. The Fc-peptide protein was purified by Protein A.
  • MAPT P301S protein aggregates were purchased from a commercial source (Abeam).
  • Synthetic MAPT P301S protein aggregates were used as the substrate for binding of the Fc-peptide protein.
  • the Fc-peptide protein was added to the wells in 2-fold serial dilution starting at 400 nM. Detection of bound Fc-peptide protein was assessed by measuring time-resolved fluorescence, following addition of a biotinylated goat anti-human Fc-reactive secondary antibody and streptavidin-europium conjugate (FIG. 1). The mean and standard deviation (SD) of three replicates were calculated and the potency (EC50) was determined following fitting with a sigmoidal four parameter logistic (4PE) equation with logarithmic x-axis (Prism).
  • This example describes the assessment of biotinylated Fc-peptide proteins binding to brain tissues containing cerebral Ap amyloid and tau tangle pathology. This example demonstrates that Fc-peptide proteins binds to cerebral Ap amyloid diffuse and core plaques with high specificity, and evidence of uptake in tangles.
  • Formalin-fixed paraffin embedded sections were prepared from brain tissues containing Ap amyloid. Tissue samples were derived from a patient confirmed to have Alzheimer’s disease with evidence of scattered neurons showing degenerative changes, amyloid plaques, and tangles within hippocampal neurons. The tissues were stained with biotinylated Fc- peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 (2 pg/mE in PBS), using standard immunohistochemical methods and visualized following addition of diaminobenzidine.
  • An Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 bound specifically to the Ap plaques and tangle-like pathology (FIG. 2A), Ap diffuse plaques (FIG. 2B), and Ap core plaques (FIG. 2C) amyloid in brain tissue samples from patients with Alzheimer’s disease.
  • This example describes the binding assessment of Fc-peptide protein and aducanumab binding to synthetic brain amyloid fibrils. This example demonstrates that Fc- peptide proteins binds to more diverse cerebral amyloids with higher potency than aducanumab.
  • Ap amyloid, tau amyloid, and a-synuclein aggregates are observed in individuals suffering from neurodegenerative disease (e.g., Alzheimer’s disease), which contributes to progressive cognitive decline.
  • neurodegenerative disease e.g., Alzheimer’s disease
  • Aducanumab is an AP-specific antibody for the treatment of Alzheimer’s disease.
  • An Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 and an aducanumab homolog were assessed for amyloid binding of different cerebral amyloids associated with neurodegenerative disease using the in vitro assay described in Example 1.
  • the Fc-peptide protein was expressed by stably transfected CHO cells grown under perfusion culture conditions and purified at day 7.
  • the Fc-peptide protein was purified by Protein A. Ap fibrils were prepared according to methods described previously (Stine et al., “Preparing synthetic Ap in different aggregation states,” Methods Mol. Biol., 2011; 670: 13-32). Synthetic tau, and a- synuclein aggregates protofibrils were purchased from a commercial source (rPeptide).
  • AP(l-40) synthetic fibrils, rVX6Wil, synthetic tau, and a-synuclein aggregates were used as the substrate for the Fc-peptide protein or aducanumab binding analysis.
  • a Fc- peptide protein or aducanumab was added to the wells in 2-fold serial dilution starting at 400 nM. Detection of bound Fc-peptide protein or aducanumab was assessed by measuring time- resolved fluorescence, following addition of a biotinylated goat anti-human Fc-reactive secondary antibody and streptavidin-europium conjugate (FIGS. 3A-3D). The mean and standard deviation (SD) of three replicates were calculated and the potency (EC50) was determined following fitting with a sigmoidal four parameter logistic (4PL) equation with logarithmic x-axis (Prism) (Table 9).
  • Binding potency of Fc-peptide protein to AP(l-40) synthetic fibrils was substantially similar to an aducanumab homolog (FIG. 3A). However, in contrast to the aducanumab reagent, Fc-peptide protein also bound rVX6Wil (FIG. 3B), a-synuclein aggregates (FIG. 3C), and synthetic tau (FIG. 3D) with high potency (EC50 of 0.21-0.84 nM).
  • This example describes the binding of Fc-peptide protein to cerebral amyloid in vivo following intravenous (IV) injection of 5xFAD mice and brain tissue assessment. This example demonstrates that Fc-peptide protein engage cerebral amyloid targets in vivo in a mouse model.
  • 5xFAD transgenic mice which represent a well-established in vivo model for studying Alzheimer’s disease, were injected with labelled ( 125 I) Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 to determine Fc-peptide protein brain penetrance (FIG. 4A).
  • FIG. 5 shows the domain arrangement of the fusion proteins that were produced, where fusion protein 1 comprises the amino acid sequence set forth in SEQ ID NO:43 and where fusion protein 2 comprises the amino acid sequence set forth in SEQ ID NO:54 (Table 8).
  • Fusion protein 1 and fusion protein 2 were cloned and purified using the Exp293F expression system (Thermo Fisher), following the manufacturer's manual. On day 1 post transfection, and 7 hours after adding the enhancers, cell cultures were supplemented with 2% FCS ultra-low IgG (Gibco). After 3 days of growth, cells were centrifuged at 6000 x g for 30 minutes, and the collected supernatants were centrifuged again at 4300 x g for 30 minutes at 4°C. Supernatants were filtered using 0.22 pm membrane filters and loaded onto HiTrap MabSelect SuRe (GE Healthcare) column pre-equilibrated against PBS, pH 7.4.
  • Protein A affinity bound proteins were eluted with 0.1 M Glycine, pH 2.7. Next, the samples were concentrated using Protein Concentrator PES, 30K MWCO (Pierce) and loaded onto HiLoad 26/600 Superdex 200 column (GE Healthcare). PBS pH 7.4 was used as mobile phase, and elution flow rate was set to 1 ml/min. The eluted protein was subsequently buffer exchanged to 50 mM sodium acetate pH 5.5 using HiPrep 26/10 column (GE Healthcare) before loading onto HiTrap SP HP cation exchange column (GE Healthcare).
  • the proteins were eluted with buffer B (1 M NaCl in 50 mM sodium acetate pH 5.5) in steps: 5 column volumes (CV) of 10% B, 20 CV of a gradient 10% B to 30% B, 5 CV of a gradient 30% B to 50% B, and 5 CV of 100% B. Collected fractions were buffer exchanged to PBS pH 7.4 using HiPrep 26/10 column (GE Healthcare). Then, the samples were concentrated to approximately 1 mg/ml using protein concentrator PES, 3 OK MWCO (Pierce) and stored in -80°C.
  • buffer B (1 M NaCl in 50 mM sodium acetate pH 5.5
  • This example describes the binding assessment of human and mouse VNAR- Fc-peptide fusion proteins and a Fc-peptide protein binding to transferrin receptor (TfR-1). This example demonstrates that VNAR-Fc-peptide fusion proteins bind to TfR-1 with high potency, and Fc-peptide protein does not bind to TfR-1.
  • TfR-1 is a transmembrane receptor in the brain that can be targeted to deliver drugs to the brain via receptor-mediated transcytosis (RMT).
  • RMT receptor-mediated transcytosis
  • Plates were washed three times with 300 pl of PBST (PBS with 0.1% Tween20) and blocked with 2% BSA in PBST for 1 hour at room temperature. Next, the plates were washed and incubated with serially diluted human VNAR-Fc-peptide protein or mouse VNAR-Fc-peptide protein. Each plate was also washed and incubated with serially diluted Fc-peptide protein as a control. After 1 hour incubation the plates were washed and incubated for 1 hour with 1:5000 diluted detection antibody, anti-hFc-HRP (Sigma) or anti-mFc-HRP (Sigma), before the final wash.
  • Antibody signals were developed using HRP substrate in colorimetric reaction with 1-StepTM Ultra TMB-ELISA Substrate Solution (Thermo) and stopped with 1% HC1 solution. Plates were analyzed using microplate reader (Thermo) with reading at 450 nm. 4-parametric non-linear regression analysis was used to define EC50 values (Prism).
  • Binding potency of fusion protein 1 to TfR-1 was substantially higher than Fc-peptide protein control (FIG. 6A; 1.7 nM).
  • the binding potency of fusion protein 2 to TfR-1 was substantially higher than Fc-peptide protein control (FIG. 6B; 1.4 nM).
  • This example describes the binding assessment of human and mouse VNAR- Fc-peptide proteins binding to AP(l-40) synthetic fibrils. This example demonstrates that VNAR-Fc-peptide proteins bind to cerebral amyloids associated with neurodegenerative disease with high potency, similar to Fc-peptide proteins.
  • fusion protein 1 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:43
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54
  • Fusion protein 1 or fusion protein 2 was added to the wells in 2-fold serial dilution starting at 400 nM.
  • Detection of bound fusion proteins was assessed by measuring time-resolved fluorescence, following addition of a biotinylated goat anti-human Fc-reactive secondary antibody and streptavidin-europium conjugate. The mean and standard deviation (SD) of three replicates were calculated and the potency (EC50) was determined following fitting with a sigmoidal four parameter logistic (4PL) equation with logarithmic x-axis (Prism).
  • VNAR-Fc-peptide fusion proteins demonstrated significant binding potency to AP(l-40) synthetic fibrils (FIG. 7). Fusion protein 1 bound to AP(l-40) synthetic fibrils with an EC50 of 0.16 nM, and fusion protein 2 bound to AP(l-40) synthetic fibrils with an EC50 of 0.9 nM.
  • This example describes the assessment of biotinylated VNAR-Fc-peptide fusion proteins binding to brain tissue samples from patients with Alzheimer’s disease. This example demonstrates that VNAR-Fc-peptide fusion proteins bind to cerebral amyloids associated with neurodegenerative disease with high potency, similar to Fc-peptide proteins.
  • Formalin-fixed paraffin embedded sections were prepared from brain tissues containing Ap amyloid. Tissue samples were derived from patients confirmed to have Alzheimer’s disease. The tissues were stained with biotinylated fusion protein 1 (i.e., a VNAR- Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:43) (2 pg/mL in PBS), biotinylated fusion protein 2 (i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54) (2 pg/mL in PBS), or biotinylated Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 (2 pg/mL in PBS) using standard immunohistochemical methods and visualized following addition of diaminobenzidine.
  • biotinylated fusion protein 1 i.e., a VNAR- Fc-peptide fusion protein comprising the amino acid sequence set
  • Fc-peptide protein bound specifically to the diffuse and core plaques composed of Ap in brain tissue samples from patients with Alzheimer’s disease as well as the Ap amyloid in the vascular walls (FIG. 8A).
  • fusion protein 1 and fusion protein 2 bound specifically to the Ap plaques in brain tissue samples and in the vascular walls (FIG. 8B and FIG. 8C).
  • VNAR-Fc-peptide fusion proteins demonstrate specific reactivity of the VNAR-Fc-peptide fusion proteins with endogenous brain tissue amyloid deposits. These data support the use of the VNAR-Fc-peptide fusion proteins in targeting brain amyloids and treating brain amyloid related diseases (e.g., Alzheimer’s disease).
  • This example describes the assessment of in vivo brain exposure in mice when injected with VNAR-Fc-peptide fusion protein (e.g., fusion protein 2) compared to Fc-peptide protein.
  • VNAR-Fc-peptide fusion protein e.g., fusion protein 2
  • fusion protein 2 has nearly a 10-fold increase in brain penetrance compared to Fc-peptide protein following intravenous (IV) route of administration.
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54; 1.03 mg/kg
  • Fc-peptide protein i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25; 0.74 mg/kg.
  • Eysates were spun down at 17,000 x g for 20 minutes, and the supernatant was collected and blocked in 2.5% milk in PBST overnight at 4°C.
  • Blocked brain lysates 100 pF were added to the blocked plates and incubated for 1 hour at RT. Plates were washed with PBST and incubated with a goat anti-human Fc-peroxidase antibody diluted 1:5000 (Sigma) in blocking buffer for 30 minutes. Plates were washed and developed with SureBlue TMB substrate, the reaction stopped with 1% HC1, and absorbance measured at 450 nm.
  • the concentrations of fusion protein 2 and Fc-peptide protein were determined from standard curves prepared individually for each protein.
  • the Fc-peptide protein achieved a concentration of 0.52 nM (1.4% plasma level) in the mouse brain after IV injection (FIG. 9A).
  • the concentration of fusion protein 2 in mouse brain after IV injection was significantly higher, reaching a concentration of 4.83 nM (FIG. 9A).
  • fusion protein 2 demonstrated a 9.27-fold increase in brain penetrance compared to Fc-peptide protein (FIG. 9B).
  • VNAR-Fc-peptide fusion protein e.g., fusion protein 2
  • fusion protein 2 significantly increases the brain penetrance and local concentration in vivo.
  • These data support the use of VNAR-Fc-peptide fusion proteins in targeting brain amyloids and treating brain amyloid related diseases (e.g., Alzheimer’s disease).
  • Example 10 In Vitro Binding of Cerebral Amyloids by Fc-Peptide Protein and Fusion Proteins
  • This example describes a binding assessment of Fc-peptide protein and an aducanumab homolog binding to synthetic brain amyloid fibrils. It also describes a binding assessment of Fc-peptide protein and a VNAR-Fc-peptide fusion protein binding of synthetic cerebral amyloid fibrils. This example demonstrates that the Fc-peptide protein and the VNAR- Fc-peptide fusion protein (e.g., fusion protein 2) both bind to more diverse cerebral amyloids with higher potency than an aducanumab homolog.
  • VNAR- Fc-peptide fusion protein e.g., fusion protein 2
  • an Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25 and an aducanumab homolog were assessed for amyloid binding of different cerebral amyloids associated with neurodegenerative disease using the in vitro assay described in Example 1.
  • the in vitro assay was also used to assess the binding of different cerebral amyloid fibrils by the Fc-peptide protein and fusion protein 2 (i.e., a VNAR- Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54).
  • Fc- peptide protein and an aducanumab homolog were expressed and purified, and then mixed with AP(l-40) synthetic fibrils, synthetic tau, and a-synuclein aggregates substrate for the binding assessment, as described in Example 3.
  • Fusion protein 2 was also expressed and purified according to the methods described in Example 3, and then similarly mixed with AP(l-40) synthetic fibrils, synthetic tau fibrils, and a-synuclein fibrils substrate for the binding assessment.
  • This example describes the assessment of Fc-peptide protein and a VNAR-Fc- peptide fusion proteins’ impact on the propagation of tau aggregates.
  • This example demonstrates that in vitro formation and propagation of tau fibril aggregates is significantly reduced in the presence of Fc-peptide protein and VNAR-Fc-peptide fusion proteins (i.e., fusion protein 2), but not in control conditions (e.g., human Fc control, which lacks the amyloid reactive peptide).
  • Interneuronal tau propagation and aggregation of intracellular tau protein is a hallmark of aggregation disorders of the central nervous system (e.g., Alzheimer’s disease).
  • the propagation of tau aggregates can be assessed using a recombinant cell-based system. Once intracellular seeding of the fluorescent tau monomer occurs, the formation of large fibrillar tau aggregates can be assessed using microscopy or flow cytometry methods.
  • tau RD P301S cells (ATCC CRL-3275) were plated at 50,000 cells/well in 48-well plates in 0.5 mL of DMEM/F12 medium, complete with antibiotics and 1% FBS (v/v).
  • Fc-peptide protein and fusion protein 2 were expressed and purified according to the methods described in Example 3.
  • Human Fc control was expressed and purified according to standard protein purification methods. Three separate conditions were tested by adding the Fc-peptide protein, fusion protein 2, or a human Fc domain control to the wells at the indicated concentrations (FIGS. 11A-11B; 3 nM to 300 nM). After addition of the reagent, the plate was incubated for 20 minutes at 37°C.
  • VNAR-Fc-peptide fusion proteins impact on the human complement complex in the presence of AP(l-40) fibrils.
  • VNAR-Fc-peptide fusion proteins i.e., fusion protein 2
  • fusion protein 2 can activate the human complement complex in the presence of A
  • Activation of the complement complex is an important aspect in establishing an inflammatory gradient capable of recruiting phagocytic cells to the site of amyloid deposition.
  • fusion proteins 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54
  • 3(1-4O) fibrils were coated at 1 pg/ml on the wells of a 96-well microplate.
  • Either fusion protein 2 or human Fc control reagent (each at 30 nM), or PBS control alone were added to the wells. Fusion proteins 2 was expressed and purified according to the methods described in Example 3.
  • Citrate human plasma was added at a final concentration of 5%, 10%, or 20% as a source of complement.
  • biotinylated human C5b9 monoclonal antibody (1:4000 dilution; AbCam, Cambridge, UK) was added to the wells.
  • wells were incubated with 100 pL of europiumstreptavidin (Perkin Elmer, Waltham, MA, USA) and washed, followed by addition of 100 pL enhancement solution (Perkin Elmer) before measurement of time-resolved fluorescence emission using a Wallac VICTOR3 plate reader (Perkin Elmer).
  • VNAR-Fc-peptide fusion proteins e.g., fusion protein 2
  • a [3( 1 -40) fibrils such that the human complement complex was activated.
  • Example 13 Inhibition of Aggregated alpha-Synuclein Entry by Fc-Peptide Protein and Fusion Proteins in Primary Dopaminergic Neurons
  • This example describes the use of an in vitro model of Parkinson’s disease to assess the entry of aggregated alpha- synuclein (a-syn) into dopaminergic neurons in the presence of Fc-peptide protein and VNAR-Fc-peptide fusion proteins.
  • This example demonstrates that the entry of aggregated alpha- sy nuclein is inhibited by the presence of Fc-peptide proteins and VNAR-Fc-peptide fusion proteins (i.e., fusion protein 2).
  • the in vitro model of Parkinson’s disease used in this Example is effective for investigating the neurotoxic effects of alpha- synuclein, wherein the model recapitulates the chronic neuronal toxicity of alpha-synuclein oligomers and fibrils in a primary culture of mesencephalic neurons cultured in the absence of proliferating microglial cells.
  • This in vitro model used in this Example also reproduces essential neuropathological features associated with Parkinson’s disease (e.g., a loss of dopaminergic neurons), without the acute neuroinflammation response usually observed in the presence of microglial cells.
  • the embryonic midbrains were removed and placed in ice-cold medium of Leibovitz (L15) containing 2% of Penicillin-Streptomycin (PS) and 1% of bovine serum albumin (BSA).
  • PS Penicillin-Streptomycin
  • BSA bovine serum albumin
  • the midbrains were dissociated by trypsinization for 20 minutes at 37°C. The reaction was stopped by the addition of Dulbecco’s modified Eagle’s medium (DMEM) containing DNAse I grade II (0.5 mg/mL) and 10% of fetal calf serum (FCS).
  • DMEM Dulbecco’s modified Eagle’s medium
  • Cells were then mechanically dissociated by 3 passages with a 10 ml pipette. Cells were then centrifuged at 180 x g for 10 minutes at 4°C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded, and the cell pellets were re-suspended in a defined culture medium consisting of Neurobasal supplemented with B27 (2%), L-glutamine (2 mM) and 2% of PS solution and 10 ng/mL of Brain-derived neurotrophic factor (BDNF) and 1 ng/mL of Glial-Derived Neurotrophic Factor (GDNF). Viable cells were counted in a Neubauer cytometer using the trypan blue exclusion test.
  • BDNF Brain-derived neurotrophic factor
  • GDNF Glial-Derived Neurotrophic Factor
  • the cells were seeded at a density of 40,000 cells/well in 96 well-plates (pre-coated with poly-L- lysine) and maintained in a humidified incubator at 37 °C in 5% CO2/95% air atmosphere. Half of the medium was changed every 2 days with fresh medium.
  • Fc-peptide protein i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54
  • the Fc-peptide protein and fusion protein 2 were dissolved in PBS and then in the culture medium (at a final concentration of 1 nM or 5 nM) and pre-incubated with primary neurons for 1 hour before a-syn PFF exposure.
  • Fc-peptide protein and fusion protein 2 were expressed and purified according to methods described in Example 3.
  • Assay controls included untreated wells and wells that received the addition of alpha-synuclein fibrils alone (250 nM, 86 h).
  • a-syn PFF was added to a final concentration of 250 nM, diluted in culture medium still in presence of Fc-peptide protein or fusion protein 2 for 86 hours (see e.g., Henriques et al., 2022 for details).
  • Non-specific sites were blocked with a solution of PBS containing 0.1% of saponin and 1% FCS for 15 minutes at room temperature.
  • the cultures were incubated with a) monoclonal anti-Tyrosine Hydroxylase (TH) antibody produced in mouse at dilution of 1/10000 in PBS containing 1% FCS, 0.1 % saponin, for 2 hours at room temperature and b) polyclonal anti-a-syn antibody produced in rabbit at dilution of 1/200 in PBS containing 1% FCS, 0.1 % saponin, for 2 hours at room temperature.
  • TH monoclonal anti-Tyrosine Hydroxylase
  • Antibody staining was detected with Alexa Fluor 488 goat anti-mouse IgG at the dilution 1/800 and with Alexa Fluor 568 goat anti-rabbit IgG at the dilution 1/400 in PBS containing 1% FCS, 0.1 % saponin, respectively, for 1 hour at room temperature.
  • Cell nuclei were counterstained with the fluorescent dye Hoechst (1/1000, Sigma).
  • 20 pictures were taken using ImageXpress® (Molecular Devices) at 10X magnification using the same acquisition parameters. Analysis was performed by MetaXpress® (Molecular Devices) for all the images.
  • Fc-peptide protein and fusion protein 2 significantly reduced the amount of intracellular alpha-synuclein (a-syn) in primary cultured dopaminergic (TH) neurons (FIG. 13) compared to alpha-synuclein treated control. In each condition, a reduction at least 40% intracellular alpha-synuclein was observed compared to alpha-synuclein treated control.
  • Fc-peptide proteins and VNAR-Fc peptide fusion proteins can inhibit the entry of amyloid aggregates into dopaminergic neurons. These proteins may counteract the toxicity associated with amyloid aggregates found in neurodegenerative diseases. Substances that reduce a-synuclein or cellular entry of amyloids may also reduce the neurotoxic effects associated with amyloid aggregates.
  • Fc-peptide proteins or VNAR-Fc peptide fusion proteins as therapeutic agents for the treatment or prevention of amyloid aggregates in neurodegenerative diseases.
  • VNAR-Fc-peptide fusion proteins impact on phagocytosis of AP(l-40) amyloid fibrils.
  • VNAR-Fc-peptide fusion proteins i.e., fusion protein 2
  • fusion protein 2 enhance phagocytosis of Ap amyloid fibrils.
  • Phorbol myristate acetate (PMA; Sigma, P8139) was added to a final concentration of 50 ng/mL and the cells were allowed to differentiate for 24 hours at 37°C in a 5% CO2 incubator. After 24 hours, the medium containing PMA was carefully removed by manual aspiration and the wells were replenished with 1 mL of complete DMEM-F12 medium and incubated for about 48-72 hours.
  • the rate of phagocytosis of Ap fibrils was assayed in the presence of either human Fcl control or fusion protein 2 (i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54), where the fusion protein 2 was expressed and purified according to the methods described in Example 3.
  • the media was aspirated from the wells of the tissue culture plate, which were then rinsed once with 1 mL of DPBS (Hyclone, SH 30028.02).
  • a volume of 500 pL serum-free RPML1640 without phenol red was added into each well and the plate was incubated at 37°C until the start of the assay.
  • Test samples were prepared by the addition of 500 pL of RPML1640 into each tube followed by the addition of either human Fcl control or fusion protein 2 at the required concentration (0.78 nM to 100 nM). The samples were mixed well before the addition of pHrodo red-labeled (pHrodo TM Red SE, ThermoFisher, P36600) and the addition of AP(l-40) fibrils (20 pg) followed by a 5 minutes incubation at room temperature. The samples were then added into the wells of the 24-well plate yielding a final reaction volume of 1 mL. The plate was gently mixed and incubated at 37°C in 5% CO2 for 1 hour to facilitate phagocytosis.
  • pHrodo red-labeled pHrodo red-labeled
  • AP(l-40) fibrils 20 pg
  • VNAR-Fc-peptide fusion protein e.g., fusion protein 2
  • fusion protein 2 activates and enhances phagocytosis of amyloid substrates, including Ap amyloid fibrils.
  • VNAR-Fc-peptide fusion protein activates and enhances phagocytosis of amyloid substrates, including Ap amyloid fibrils.
  • This example describes the use of surface plasmon resonance (SPR) to assess the interaction between Fc-peptide protein or VNAR-Fc-peptide fusion proteins and activation and inhibitor receptors related to antibody-dependent cellular phagocytosis (ADCP).
  • SPR surface plasmon resonance
  • Fc-peptide protein and VNAR-Fc-peptide fusion proteins i.e., fusion protein 2
  • fusion protein 2 can both bind Fc-activation and inhibitory receptors similar to an IgG control antibody (Rituximab).
  • a surface plasmon resonance (SPR) assay was performed to optically analyze the molecular interactions between the phagocytosis inhibitor (FcyRIIb) and activating (FcyRIIa (H131)) receptors and the either Fc-peptide protein (i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25) or fusion protein 2 (i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54).
  • Fc-peptide protein i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54.
  • These receptors are expressed on macrophages or microglial cells and are involved in phagocytosis mechanisms and clearance of fibrils.
  • the receptors were captured by
  • the activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection.
  • the CM5 sensor chip was activated for 420 seconds with the mixture at a flow rate of 10 pL/min.
  • Human FcyRIIa (H131) or FcyRIIb in 10 mM NaAc (pH 5.5) was then injected to Fc2 of channels to obtain the target level about 1000 RU at a flow rate of 10 pL/min.
  • the chip was deactivated by 1 M ethanolamine-HCl at a flow rate of 10 pL/min for 420 seconds.
  • Buffer of the Fc-peptide protein, fusion protein 2, and Rituximab were exchanged to running buffer 2xHBS-EP+ (0.02 M HEPES, 0.3 M NaCl, 6 mM EDTA, 0.1% surfactant P20, pH 7.4) using desalting column.
  • concentration was determined by Nanodrop.
  • analyte concentration (10, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5120 nM for Fc- peptide protein ;40, 80, 160, 320, 640, 1280, 2560 and 5120 nM for fusion protein 2; and 20, 40, 80, 160, 320, 640, 1280, 2560, 5120 and 10240 nM for Rituximab) and running buffer 2xHBS- EP+ were injected to the chip using multi-cycle kinetics mode at a flow rate of 30 pL/min for an association phase of 60 s, followed by 90 s dissociation. No regeneration was performed.
  • Example 16 Phagocytosis of Ap Fibrils by Fusion Proteins in Rat Primary Culture of Hippocampal Neurons, Microglial Cells, and Astrocytes
  • This example describes the assessment of the ability of Fc-peptide protein and VNAR-Fc-peptide fusion proteins to mediate antibody -dependent cellular phagocytosis (ADCP) of amyloid fibrils in primary effector cells.
  • ADCP antibody -dependent cellular phagocytosis
  • a rat primary microglial assay was developed to evaluate the clearance of Ap amyloid fibrils by phagocytosis, which included the co-culturing of rat hippocampal neurons, microglial cells, and astrocytes.
  • microglial cells When microglial cells are exposed to Ap amyloid fibrils, they express activation markers such as SIRP alpha/CD172a on the cell membrane, and cells then exhibit the ability to phagocytose Ap amyloid fibrils.
  • staining for activation markers SIRP alpha/CD172a and for Ap amyloid fibrils was performed, providing areas of overlap to be visualized and Ap amyloid fibrils that are undergoing phagocytosis to be quantified.
  • Fc-peptide protein i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54
  • Fc-peptide protein and fusion protein 2 were expressed and purified as described in Example 3.
  • a control condition with only Ap amyloid fibrils was also included.
  • cells were fixed and analyzed for changes by using the microglial activation marker SIRP alpha/CD172a (OX 41) antibody.
  • AP(l-42) amyloid fibrils engulfed by phagocytosis within permeabilized microglial cells were stained using 6E10 antibody.
  • Microglial cells that phagocytosed AP(l-42) amyloid fibrils were measured by quantifying the area of SIRP alpha/CD172a (OX 41) that overlapped with Ap fibrils (6E10) staining.
  • Fc-peptide protein or fusion protein 2 significantly increased the percent of phagocytosed AP(l-42) amyloid fibrils at both concentrations tested compared to Ap treated control (FIG. 15 and Table 12).
  • a one-way ANOVA followed by Fisher’s LSD test showed that conditions with either Fc-peptide protein or fusion protein 2 have a significant amount of phagocytosed AP(l-42) amyloid fibrils.
  • Fc-peptide protein and VNAR-Fc-peptide fusion proteins e.g., fusion protein 2
  • Fc-peptide protein and VNAR-Fc-peptide fusion proteins enhance phagocytosis of the substrates by activating microglial cells relative to cells treated only with AP(l-42) amyloid fibrils.
  • This example describes the assessment of the target engagement of Fc-peptide protein and VNAR-Fc-peptide fusion proteins in a murine model of cerebral A amyloid deposition.
  • This example demonstrates the ability of Fc-peptide protein and VNAR-Fc-peptide fusion proteins (i.e., fusion protein 2) to cross the blood brain barrier and bind AP amyloid deposits in the brain.
  • the 5xFAD mouse model used in this example recapitulates major features of Alzheimer’s disease amyloid pathology and is useful for studying the intraneuronal AP-induced neurodegeneration and amyloid plaque formation.
  • the Alzheimer’s disease mouse model was used to investigate target engagement of Fc-peptide protein (i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25) and fusion protein 2 (i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54) with A(3 amyloid deposits in the brain, similar to methods described in Example 4.
  • Fc- peptide protein and fusion protein 2 were expressed and purified according to the methods described in Example 3.
  • the experimental design included three groups of 5xFAD female mice greater than 6 months of age, as shown in the flowchart in FIG. 16A.
  • 5xFAD transgenic mice were injected with Fc-peptide protein and fusion protein 2 to determine brain penetrance of Fc-peptide protein and fusion protein 2.
  • IP intraperitoneally
  • Fc-peptide protein at a 50 mg/kg dose
  • fusion protein 2 at a 14 mg/kg dose
  • fusion protein 2 at a 70 mg/kg dose (molar equivalent of Fc-peptide protein). Injections were repeated for all mice on days 4 and 8.
  • mice were euthanized and brain tissue was harvested for further analysis.
  • FIGS. 16B-16C Images of brain tissues (subiculum/hippocamus and white matter) harvested from the treated 5xFAD mice were analyzed to determine the presence of Fc-peptide protein and fusion protein 2 in the brain, as well as the distribution of amyloid deposits.
  • Immunohistochemical visualization showed that 50 mg/kg Fc-peptide protein (FIGS. 16B-16C; arrows), 14 mg/kg fusion protein 2 (FIGS. 16D-16E; arrows) and 70 mg/kg fusion protein 2 (FIGS. 16F-16G; arrows) crossed the blood brain barrier in mice and were specifically bound to A
  • fusion protein 2 in the amyloid deposits was qualitatively greater than that of Fc-peptide protein, including at the lower concentration of fusion protein 2 used (14 mg/kg). This difference in accumulation is likely due to the presence of the blood brain barrier shuttle in fusion protein 2, which enhances its ability to cross the blood brain barrier.
  • VNAR-Fc-peptide fusion proteins e.g., fusion protein 2
  • Fc-peptide proteins can cross the blood brain barrier and target brain amyloid deposits following IP route of administration.
  • These data demonstrate the potential utility of Fc- peptide protein and VNAR-Fc-peptide fusion proteins as a potential therapy for brain amyloids and related disorders.
  • This example describes the assessment of in vivo brain penetrance by Fc- peptide protein and VNAR-Fc-peptide fusion proteins. This example demonstrates significant brain penetrance by the VNAR-Fc-peptide fusion protein (i.e., fusion protein 2) compared to Fc- peptide protein.
  • Fc-peptide protein i.e., Fc-peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54
  • Lysates were spun down at 17,000 xg for 20 minutes; the supernatant was collected and blocked in 2.5% milk in PBST overnight at 4°C.
  • Blocked brain lysates (100 pL) were added to the blocked plates and incubated for 1 hour at room temperature. Plates were washed with PBST and incubated with a goat anti-human Fc-peroxidase antibody diluted 1:5000 (Sigma) in blocking buffer for 30 minutes. Plates were washed and developed with SureBlue TMB substrate, the reaction was stopped with 1% HC1, and the absorbance was measured at 450 nm. The concentrations were determined from standard curves prepared individually for each protein.
  • the plasma concentration of fusion protein 2 was higher than the plasma concentration of Fc-peptide protein (FIG. 17A), but this was not significant when considering experimental error.
  • the brain exposure for fusion protein 2 was significantly higher (10 nM) than the brain exposure observed for Fc-peptide protein (0.5 nM) (FIG. 17B).
  • fusion protein 2 showed an about 19X fold increase (FIG. 17C).
  • the plasma to brain concentration ratio for fusion protein2 was about 10X fold-increased compared to Fc-peptide protein.
  • Statistical analysis was performed using a two-tailed T-test with p ⁇ 0.05 being considered statistically significant.
  • VNAR-Fc-peptide fusion protein e.g., fusion protein 2
  • fusion protein 2 significantly increases the brain penetrance and local concentration in vivo.
  • These data support the use of VNAR-Fc-peptide fusion proteins in targeting brain amyloids and treating brain amyloid related diseases (e.g., Alzheimer’s disease).
  • This example describes the assessment of in vivo impact of by Fc-peptide proteins and VNAR-Fc-peptide fusion proteins on dopaminergic neurons (tyrosine hydroxylase neurons, TH neurons).
  • This example demonstrates dopaminergic neurons are protected in vivo from exposure to amyloid aggregates by VNAR-Fc-peptide fusion proteins (i.e., fusion protein 2) and not Fc-peptide protein.
  • mice received a stereotaxic injection of 2.5 pL of alpha-synuclein solution.
  • the mice were anesthetized by isoflurane (4%, for induction), in an induction chamber coupled with a vaporizer and an oxygen concentrator. Mice were placed on the stereotaxic frame. Anesthesia was maintained by isoflurane (2%) with a face mask coupled to the isoflurane vaporizer and an oxygen concentrator. The skull was exposed, and holes were drilled.
  • Human a- synuclein peptide (stock at 69 pM in water at -20°C) was reconstituted at 50 pM in NaCl, 0.9% (final concentration).
  • the a-synuclein preparations was bilaterally injected at the following coordinates: A-P, -0.3 cm; M-L, ⁇ 0.12 cm; D-V, -0.45 cm. Depth of anesthesia and rectal temperature was verified every 5 minutes. After surgery, mice were allowed to recover before being placed back in the cage.
  • Fc-peptide protein i.e., Fc- peptide protein comprising the amino acid sequence set forth in SEQ ID NO:25
  • fusion protein 2 i.e., a VNAR-Fc-peptide fusion protein comprising the amino acid sequence set forth in SEQ ID NO:54
  • IP intraperitoneal
  • TBS Tris-phosphate saline
  • the number of dopaminergic neurons is unchanged in mice treated with Fc- peptide protein compared to alpha- sy nuclein treated control (FIG. 18A), even at 50 mg/kg dose.
  • the number of dopaminergic (TH) neurons is significantly increased in mice treated with fusion protein 2 compared to alpha-synuclein treated control (FIG. 18A).
  • FIG. 18B the histological evaluation of alpha-synuclein in dopaminergic (TH) neurons is significantly lower in mice treated with fusion protein 2 at 10 mg/kg or Fc-peptide protein at 50 mg/kg dose compared to alpha-synuclein treated control.
  • VNAR-Fc-peptide fusion protein e.g., fusion protein 2
  • Fc-peptide fusion protein 2 provides protection for mice injected with alpha-synuclein in the brain, by increasing the number of dopaminergic neurons and reducing the alpha-synuclein in dopaminergic neurons post amyloid exposure.
  • Fc-peptide provides some protection, specifically in reducing the alpha-synuclein in dopaminergic neurons.
  • These data demonstrate the protective characteristics of VNAR-Fc-peptide fusion proteins against neurotoxicity induced by amyloids in an in vivo model of Parkinson’s disease.

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Abstract

La présente divulgation concerne, selon certains aspects, des compositions et des procédés d'utilisation de protéines de fusion de peptide Fc à domaine variable du nouveau récepteur d'antigène de l'immunoglobuline (VNAR) qui sont capables d'une absorption à travers la barrière hémato-encéphalique (BBB) et qui peuvent cibler des amyloïdes, notamment ceux associés à des maladies cérébrales, pour élimination.
PCT/US2023/079673 2022-11-16 2023-11-14 Protéines de fusion se liant à l'amyloïde et au récepteur de la transferrine et leurs utilisations WO2024107749A1 (fr)

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