Proteins targeting amyloid beta and methods of use

Abstract

This paper discloses a protein that targets amyloid-beta, the protein comprising an amyloid-beta binding domain, a TfR binding domain that specifically binds to the transferrin receptor (TfR) with an affinity of approximately 900 nM to approximately 10,000 nM, and an FcγR binding domain. Furthermore, it discloses a method for preparing the protein that targets amyloid-beta, as well as a method for using the protein that targets amyloid-beta to treat neurodegenerative diseases.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 492,124, filed on 24 March 2023. The entire contents of the aforementioned application are incorporated herein by reference.

[0002] Sequence List The sequence listing saved in the file 590729_SeqListing_ST26.xml is 34 kilobytes in size, was created on February 28, 2023, and is incorporated herein by reference. [Background technology]

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment, behavioral disorders, psychiatric symptoms, and impairment of activities of daily living. These clinical symptoms constitute dementia caused by AD.

[0004] One of the prominent features of this disease is the presence of amyloid-beta plaques. It is hypothesized that the accumulation of amyloid-beta plaques, resulting from an imbalance between amyloid-beta production and elimination in the brain, contributes to the onset of the disease.

[0005] Developing drugs to treat diseases like Alzheimer's requires delivery to the brain, making the process challenging due to the need to transport the drugs across the blood-brain barrier. Several potential disease-improving drug candidates are currently under investigation. However, there are currently no treatments that reliably improve the course of Alzheimer's disease. Antibodies against amyloid-beta may offer some therapeutic benefits, but they are associated with side effects such as edema (VE) and microhemorrhages (mH). Therefore, there is a need in this field for improved therapeutic agents and methods for treating neurodegenerative diseases such as Alzheimer's disease. [Overview of the project] [Means for solving the problem]

[0006] We disclose a protein that targets amyloid-beta, the protein comprising (a) an amyloid-beta binding domain, (b) a TfR binding domain that specifically binds to the transferrin receptor (TfR) with an affinity of approximately 900 nM to approximately 10,000 nM, and (c) an FcγR binding domain. The amyloid-beta targeting protein can cross the blood-brain barrier and bind to amyloid-beta. The TfR binding domain facilitates the transport of the amyloid-beta targeting protein across the blood-brain barrier, and thus facilitates its delivery to the brain. Contrary to expectations, the TfR binding domain, which specifically binds to TfR with an affinity of approximately 900 nM to approximately 10,000 nM, facilitates the delivery of the amyloid-beta binding polypeptide to the brain at a higher initial rate compared to the initial delivery rate of the amyloid-beta binding polypeptide linked to a TfR binding polypeptide having a higher affinity for TfR. In some embodiments, the TfR binding region specifically binds to TfR with affinities of approximately 900 nM to 8500 nM, 900 nM to 6500 nM, 900 nM to 5000 nM, 900 nM to 3500 nM, 900 nM to 2500 nM, 900 nM to 1600 nM, or 900 nM to 1300 nM. In some embodiments, the TfR binding region specifically binds to TfR with an affinity of approximately 1000 nM to 1200 nM. In some embodiments, the TfR binding region specifically binds to TfR with an affinity of approximately 1100 nM.

[0007] In some embodiments, the TfR binding domain specifically binds to the apical domain of TfR. In some embodiments, the amyloid-beta targeting protein is configured such that the TfR binding domain binds to TfR without inhibiting the binding of transferrin to TfR. In some embodiments, TfR is human TfR1.

[0008] Substances that can result in TfR binding include, for example, a TfR-binding polypeptide, a TfR-binding polypeptide linked to an Fc polypeptide, a first Fc polypeptide containing a CH3 domain modified to bind to TfR, an anti-TfR antibody or its antigen-binding fragment, or an anti-TfR antibody or its antigen-binding fragment linked to an Fc polypeptide. The Fc polypeptide may be derived from IgG, for example, human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the TfR bond is derived from an Fc polypeptide, which comprises a CH3 domain having amino acid substitutions (e.g., N384Y, Q386T, P387E, E388W, N389V, D413T, S415E, R416E, and N421F) at at least six, seven, eight, nine, or ten positions selected from 380, 384, 386, 387, 388, 398, 390, 413, 415, 416, and 421 according to the EU numbering system. In some embodiments, the TfR bond-generating entity is an Fc polypeptide, which comprises a CH3 domain having amino acid substitutions at eight, nine, or ten positions selected from 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421. In some embodiments, the TfR bond-generating entity is an Fc polypeptide, which comprises a CH3 domain having tyrosine or phenylalanine at position 384, threonine or asparagine at position 386, aspartic acid at position 387, tryptophan at position 388, serine, threonine, or valine at position 389, serine or asparagine at position 390, threonine or serine at position 413, serine or glutamine at position 415, glutamic acid at position 416, and phenylalanine or tyrosine at position 421, according to the EU numbering system. Exemplary CH3 domains modified to specifically bind to TfR with affinity of approximately 900 nM to approximately 10,000 nM (e.g., approximately 900 nM to approximately 2,500 nM or approximately 900 nM to approximately 1,300 nM) are given by amino acids 348-453 or 348-454 of any of SEQ ID NOs. 8, 10, and 17-31.In some embodiments, the TfR binding region is a CH3 domain having one of the substitution combinations shown in Table 1, and includes a CH3 domain having an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 348-453 or 348-454 of any of SEQ ID NOs. 8, 10, and 17-31. In some embodiments, the Fc polypeptide having a TfR binding domain comprises a modified CH3 domain which has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, and which, according to the EU numbering system, has tyrosine (Y) at position 384, threonine (T) at position 386, glutamic acid (E) at position 387, tryptophan (W) at position 388, valine (V) at position 389, threonine (T) at position 413, glutamic acid (E) at position 415, glutamic acid (E) at position 416, and phenylalanine (F) at position 421. In some embodiments, the Fc polypeptide having a TfR binding region comprises a modified CH3 domain, the modified CH3 domain having an amino acid sequence of amino acids 348-453 or 348-454 of any one of SEQ ID NOs. 8, 10, and 17-31. If the TfR binding is resulting from an antigen-binding fragment of an anti-TfR antibody, the fragment may be, but is not limited to, an F(ab)2 fragment, a Fab fragment, or a single-chain variable fragment (scFv).

[0009] FcγR binding can be mediated by Fc polypeptides. These Fc polypeptides may be derived from IgG, for example, human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc polypeptides that mediated FcγR binding do not include modifications that reduce FcγR binding.

[0010] In some embodiments, the amyloid-beta targeting protein comprises an Fc dimer, where the dimer comprises a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the Fc dimer is monovalent with respect to TfR binding (i.e., either the first or second Fc polypeptide, but not both, contains the TfR binding region).

[0011] In some embodiments, amyloid-beta targeting proteins are configured to have reduced binding to FcγR when they bind to TfR. Reducing FcγR binding when an amyloid-beta targeting protein binds to TfR can be achieved by using an Fc dimer, where the first Fc polypeptide of the Fc dimer comprises a modified CH3 domain to bind to TfR and one or more amino acid substitutions that reduce FcγR binding, and the second Fc polypeptide of the Fc dimer does not bind to TfR and does not contain any modifications that reduce FcγR binding. Examples of mutations that reduce FcγR binding include, but are not limited to, substitutions of L234A, L235A, and P329G (e.g., substitutions of L234A and L235A, or substitutions of L234A, L235A, and P329G) according to the EU numbering system.

[0012] Proteins targeting amyloid-beta containing Fc dimers may have mutations in the Fc dimer polypeptide that promote heterodimerization. Such mutations include, but are not limited to, knob mutations and hole mutations. Knob mutations may be substitutions of T366W according to the EU numbering system. Hole mutations may be substitutions of T366S, T368A, and Y407V according to the EU numbering system, but are not limited to these. One Fc polypeptide of the Fc dimer may be modified to have a knob mutation, and the other Fc polypeptide of the Fc dimer may be modified to have a hole mutation.

[0013] Exemplary Fc polypeptides having a TfR binding domain and having one or more substitutions that reduce binding to FcγR, suitable for use in protein formation targeting amyloid-beta, are provided by amino acids 228-453 or 228-454 of any one of SEQ ID NOs. 8, 10, and 17-31. In some embodiments, Fc polypeptides having a TfR binding domain include polypeptides having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-453 or 228-454 of any one of SEQ ID NOs. 8, 10, and 17-31, and having amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1. In some embodiments, the Fc polypeptide having a TfR binding domain includes polypeptides having at least 85%, at least 90%, or at least 95% identity to amino acids 228-454 of SEQ ID NO: 8 or 10, and having, according to the EU numbering system, alanine at position 234, alanine at position 235, optionally glycine at position 329, optionally tryptophan at position 366, tyrosine at position 384, threonine at position 386, glutamic acid at position 387, tryptophan at position 388, valine at position 389, threonine at position 413, glutamic acid at position 415, glutamic acid at position 416, and phenylalanine at position 421. In some embodiments, the Fc polypeptide having a TfR binding domain includes polypeptides having amino acid sequences of amino acids 228-453 or amino acids 228-454 of any of SEQ ID NOs: 8, 10, and 17-31.

[0014] Exemplary Fc polypeptides having an FcγR binding domain suitable for use in protein formation targeting amyloid-beta are provided by amino acids 228-454 of SEQ ID NO: 9. In some embodiments, the Fc polypeptide having an FcγR binding domain comprises polypeptides having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-454 of SEQ ID NO: 9, where the polypeptide optionally has serine at position 366, alanine at position 368, and valine at position 407, according to the EU numbering system. In some embodiments, the Fc polypeptide having an FcγR binding domain comprises polypeptides having the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 9.

[0015] The amyloid-beta binding region of a protein targeting amyloid-beta may be, but is not limited to, an anti-amyloid-beta antibody or its antigen-binding region. In some embodiments, the anti-amyloid-beta Fab binds to amyloid-beta plaques and amyloid-beta fibrils. The anti-amyloid-beta antibody or its antigen-binding region may be derived from an anti-amyloid-beta antibody known in the art. In some embodiments, the amyloid-beta binding region of a protein targeting amyloid-beta includes a CDR sequence of an anti-amyloid-beta antibody known in the art. The antigen-binding region may be, but is not limited to, Fab (or F(ab)2) or scFv. In some embodiments, the amyloid-beta binding region includes an anti-amyloid-beta Fab. In some embodiments, the anti-amyloid-beta Fab is linked to an Fc polypeptide having a TfR binding region or an FcγR binding region. In some embodiments, the amyloid-beta targeting protein comprises a first anti-amyloid-beta Fab linked to a first Fc polypeptide having a TfR binding domain, and a second anti-amyloid-beta Fab linked to a second Fc polypeptide having an FcγR binding domain, wherein the first and second Fc polypeptides form an Fc dimer.

[0016] In some embodiments, the amyloid-beta targeting protein comprises two antibody light chains: a first antibody heavy chain containing an Fc polypeptide having a CH3 domain that has been modified to have a TfR binding region and optionally one or more mutations that reduce FcγR binding; and a second antibody heavy chain containing an Fc polypeptide having an FcγR binding region.

[0017] In some embodiments, amyloid-beta-targeting proteins bind to amyloid-beta and mediate effector function via their FcγR receptor-binding domain. In some embodiments, amyloid-beta-targeting proteins bind to amyloid plaques, vascular amyloid-beta, or diffuse amyloid-beta deposits and mediate effector function via their FcγR receptor-binding domain. In some embodiments, amyloid-beta-targeting proteins are configured such that FcγR receptor binding is reduced when they bind to TfR. In some embodiments, amyloid-beta-targeting proteins do not substantially deplete reticulocytes when administered to a subject in vivo.

[0018] Any of the amyloid-beta targeting proteins disclosed can be provided in a pharmaceutically acceptable composition. A pharmaceutically acceptable composition may contain one or more pharmaceutically acceptable excipients. The pharmaceutical composition can be formulated for use in combination with additional agents useful for treating neurodegenerative diseases, such as Alzheimer's disease.

[0019] The present invention provides nucleic acids encoding proteins that target amyloid-beta. In some embodiments, the nucleic acids encoding the antibody heavy chain of the amyloid-beta-targeting protein encode a polypeptide having at least 85% identity, at least 90% identity, or at least 95% identity to any of SEQ ID NOs. 8, 10, and 17-31, wherein the encoded polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to the EU numbering system) as shown in Table 1. In some embodiments, the nucleic acid encoding the antibody heavy chain of a protein targeting amyloid beta encodes a polypeptide having at least 85% identity, at least 90% identity, or at least 95% identity to SEQ ID NO: 8 or 10, wherein the polypeptide encoded herein contains (a) alanine at position 234, alanine at position 235, glycine at position 329, tryptophan at position 366, tyrosine at position 384, tyrosine at position 384, threonine at position 386, glutamic acid at position 387, 3 (b) containing tryptophan at position 88, valine at position 389, threonine at position 413, glutamic acid at position 415, glutamic acid at position 416, and phenylalanine at position 421, or (b) containing alanine at position 234, alanine at position 235, tryptophan at position 366, tyrosine at position 384, threonine at position 386, glutamic acid at position 387, tryptophan at position 388, valine at position 389, threonine at position 413, glutamic acid at position 415, glutamic acid at position 416, and phenylalanine at position 421. In some embodiments, the nucleic acid encoding the antibody heavy chain of a protein targeting amyloid beta encodes a polypeptide having any of the amino acid sequences of SEQ ID NOs: 8, 10, and 17-31.

[0020] Also disclosed are cells comprising one or more nucleic acids encoding all or part of a protein that targets amyloid beta. In some embodiments, the cells comprise (a) a first nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to any of SEQ ID NOs: 8, 10, and 17-31; (b) a second nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to SEQ ID NO: 9; and (c) a third nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to SEQ ID NO: 7.

[0021] Disclosed is a method for producing an amyloid-beta targeting protein, the method comprising culturing a host cell containing one or more nucleic acids encoding an amyloid-beta targeting protein under conditions suitable for nucleic acid expression, and isolating the amyloid-beta targeting protein from the culture.

[0022] Disclosed is a method for reducing amyloid plaques in the brain of a subject, the method comprising administering an amyloid-beta-targeting protein to the subject. The amyloid-beta-targeting protein can be administered to a subject having cognitive impairment, memory loss, dementia, or loss of neural connectivity in the brain, or to a subject having or being at risk of developing a disease associated with the accumulation of amyloid-beta or amyloid plaques. In some embodiments, the subject has Alzheimer's disease, has been diagnosed with Alzheimer's disease, or is at high risk of developing Alzheimer's disease.

[0023] Disclosed are methods for treating neurodegenerative diseases in subjects, the methods comprising administering a protein that targets amyloid-beta to the subjects. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the methods further comprise administering at least one additional agent useful for treating Alzheimer's disease to the subjects.

[0024] Disclosed is a method for increasing amyloid-beta phagocytosis and / or increasing the recruitment of microglia to amyloid-beta-positive plaques in a subject, the method comprising administering an amyloid-beta-targeting protein to the subject.

[0025] Disclosed is a method for reducing amyloid-related imaging abnormalities (ARIA) in a subject, the method comprising administering a protein that targets amyloid beta to the subject. [Brief explanation of the drawing]

[0026] [Figure 1] This graph shows the binding of ATV to oligomeric amyloid beta 1-42, fibrous amyloid beta 1-42, and monomeric amyloid beta 1-40.

[0027] [Figure 2-1] This graph shows (A) plasma clearance and (B) brain pharmacokinetics (PK) of the injectable doses of the amyloid-beta-targeting proteins ATV35.23.3cisLALA:amyloid-beta and ATV35.23.4cisLALA:amyloid-beta, as well as (C) reticulocytes in TfRmu / hu mice after administration of the amyloid-beta-targeting proteins ATV35.23.3cisLALA:amyloid-beta and ATV35.23.4:amyloid-beta.

[0028] [Figure 2-2]This graph shows (D) plasma human IgG concentration, (E) brain human IgG concentration, (F) serum reticulocyte level, or (G) bone marrow reticulocyte level in TfRmu / hu mice after injection with anti-amyloid beta IgG antibody, ATV35.23.3:amyloid beta, ATV35.23.3cisLALA:amyloid beta, or ATV35.23.3LALA:amyloid beta.

[0029] [Figure 3] (A) This figure shows an embodiment of a protein that targets amyloid beta. (B) This graph shows the brain IgG concentration for the amyloid beta-targeting protein shown in A. (C) This graph shows the effect on reticulocytes in TfRmu / hu mice after administration of the amyloid beta-targeting protein shown in A.

[0030] [Figure 4] This graph shows (A) plasma human IgG concentration, (B) brain human IgG concentration, (C) human IgG intensity in plaques, (D) percentage of plaques colocalized with CD68, and (E) plaque area percentage in AppSAAKI TfRmu / hu mice after administration of anti-amyloid beta IgG, ATV:35.23.4cisLALA:amyloid beta, and ATV35.23.3cisLALA:amyloid beta.

[0031] [Figure 5] The graph shows plasma PK in wild-type mice after administration of the displayed ATV (amyloid-beta) molecule.

[0032] [Figure 6A] This graph shows the dynamic light scattering analysis of fluorescein (FAM)-labeled amyloid-beta 1-42 fibers.

[0033] [Figure 6B] This figure shows the FACS analysis of FAM-labeled amyloid-beta fibers in TfRmu / hu mice after administration of ATV (Amyloid-Beta) molecules.

[0034] [Figure 6C] The graphs shown are (C) amyloid-beta-positive microglia and (D) amyloid-beta signal intensity in microglia in TfRmu / hu mice after injection of the displayed ATV: amyloid-beta molecule. [Figure 6D] The graphs shown are (C) amyloid-beta-positive microglia and (D) amyloid-beta signal intensity in microglia in TfRmu / hu mice after injection of the displayed ATV: amyloid-beta molecule.

[0035] [Figure 7] The graph shows (A) immunohistochemical analysis, (B) recruitment of microglia to plaques, and (C) total plaque area in APPSAAKI;TfRmu / huKI mice after injection of the displayed ATV: amyloid-beta molecule.

[0036] [Figure 8A] This graph shows the concentrations of (A) reticulocytes and (B) hemoglobin in non-human primates after injection of the displayed ATV: amyloid-beta molecule (see legend in Figure 8C). [Figure 8B] This graph shows the concentrations of (A) reticulocytes and (B) hemoglobin in non-human primates after injection of the displayed ATV: amyloid-beta molecule (see legend in Figure 8C).

[0037] [Figure 8C] The displayed graph shows the concentration of red blood cells in non-human primates after administration of ATV (amyloid-beta) molecules.

[0038] [Figure 9] The graph shows plasma PK in non-human primates after administration of ATV (amyloid beta) molecules.

[0039] [Figure 10A]This graph shows the plasma PK profiles in 5XFAD;TfRmu / huKI mice treated with ATV35.23.4cisLALA, ATV35.23.4cisL ALAPG, or control IgG.

[0040] [Figure 10B] The graphs show (B) brain concentrations of ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG after administration of ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG, (C) immunohistochemical staining of plaques with ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG, and (D) mobilization of CD68 to 35-125 μm² plaques. [Figure 10C] The graphs show (B) brain concentrations of ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG after administration of ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG, (C) immunohistochemical staining of plaques with ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG, and (D) mobilization of CD68 to 35-125 μm² plaques. [Figure 10D] The graphs show (B) brain concentrations of ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG after administration of ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG, (C) immunohistochemical staining of plaques with ATV35.23.4cisLALA, ATV35.23.4cisLALAPG, or control IgG, and (D) mobilization of CD68 to 35-125 μm² plaques.

[0041] [Figure 11]This graph shows the number of 5xFAD:TfRmu / huKI mice exhibiting ARIA events after administration of anti-amyloid beta hIgG (10 mg / kg), anti-amyloid beta kLALA hIgG (10 mg / kg), ATV35.23.3:amyloid beta (3 mg / kg), and ATV35.23.3cisLALA:amyloid beta (3 mg / kg).

[0042] [Figure 12] This figure shows the difference in the pathways of entry into the brain between anti-amyloid beta and ATV:amyloid beta, due to the preferential expression of TfR in capillaries and venules. As a result, one day after a single peripheral injection, ATV:amyloid beta shows a broader distribution in the brain parenchyma (cyan), while anti-amyloid beta remains mainly distributed within the cerebrovascular system. [Modes for carrying out the invention]

[0043] I. Definition As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to "a polypeptide" may include two or more such molecules.

[0044] As used herein, the terms “about” and “approximately,” when used as modifiers for a quantity specified by a number or range, indicate the number and a reasonable deviation from that number, such deviation being known to those skilled in the art. In some embodiments, the term “about” means within a typical acceptable range in the art. In some embodiments, the term “about” means within one standard deviation or two standard deviations from the mean. In some embodiments, the term “about” means ±10%. In some embodiments, the term “about” refers to ±5%. As is obvious when the term “about” precedes a series of numbers or a range, “about” can modify each of the numbers in that series or range.

[0045] When used in connection with the present invention, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The polypeptide sequence of human transferrin receptor 1 is shown in SEQ ID NO: 15. Sequences of transferrin receptor protein 1 from other species are also known (e.g., chimpanzee, accession number XP 003310238.1; rhesus macaque, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP 035768.1; rat, P_073203.1; and chicken, P_990587.1). The term “transferrin receptor” also encompasses allelic variants of exemplary reference sequences (e.g., human sequences) encoded by the gene at the chromosomal locus of transferrin receptor protein 1. The full-length transferrin receptor protein includes a short N-terminal intracellular domain, a transmembrane domain, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is shown in Sequence ID No. 4.

[0046] An "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as its structural domain. An Fc polypeptide contains a constant region sequence including at least a CH2 domain and / or a CH3 domain. It may also contain at least a portion of a hinge region. Generally, Fc polypeptides do not contain a variable region.

[0047] A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, such as a substitution, deletion, or insertion, compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the original Fc polypeptide.

[0048] An "Fc polypeptide dimer" refers to a dimer of two Fc polypeptides. In some embodiments, an Fc polypeptide dimer can bind to an Fc receptor (e.g., FcγR). In an Fc polypeptide dimer, the two Fc polypeptides dimerize through an interaction between two CH3 antibody constant domains. In some embodiments, the two Fc polypeptides may also dimerize via one or more disulfide bonds formed between the hinge domains of the two dimerizing Fc domain monomers. An Fc polypeptide dimer can be a heterodimer or a homodimer. An Fc polypeptide dimer may contain two wild-type Fc polypeptides, a wild-type Fc polypeptide and a modified Fc polypeptide, or two modified Fc polypeptides. In the case of an Fc polypeptide dimer containing two modified Fc polypeptides, the two modified Fc polypeptides may be the same or different.

[0049] As used herein, the terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides. With respect to IgG antibodies, the CH3 domain polypeptide refers to the amino acid segment from approximately position 341 to approximately position 447, as numbered according to the EU numbering system, and the CH2 domain polypeptide refers to the amino acid segment from approximately position 231 to approximately position 340, as numbered according to the EU numbering system. The CH2 and CH3 domain polypeptides may also be numbered according to the EVIGT (ImMunoGeneTics) numbering system, in which case, according to the IMGT Scientific Chart numbering (IMGT website), the numbering of the CH2 domain is 1 to 110 and the numbering of the CH3 domain is 1 to 107. The CH2 and CH3 domains are part of the Fc region of immunoglobulins. With respect to IgG antibodies, the Fc region refers to the amino acid segment from approximately position 231 to approximately position 447, as numbered according to the EU numbering system. As used herein, the term “Fc region” may also include at least a portion of the hinge region of the antibody.

[0050] In this specification, the terms “wild-type,” “original,” and “naturally occurring” with respect to CH3 domains or CH2 domains are used to refer to domains having naturally occurring sequences.

[0051] The term "Fcγ receptor" or "FcγR" refers to a type of Fc receptor. Here, Fc receptors are classified based on the type of antibody they recognize. Examples of FcγRs include FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), and FcγRIIIB(CD16b). These differ in their molecular structure and therefore in their antibody affinities. FcγRs bind to the Fc portion of IgG class antibodies. FcγRs are important for inducing phagocytosis of opsonized microorganisms. FcγRs are present on the cell surface of immune system cells. FcγRs are involved in inducing effector functions of the immune system. FcγRs are activated when the Fc portion of an antibody binds to the receptor. FcγR mediates immune function, for example, by binding to antibodies attached to infected cells or invading pathogens, and stimulating phagocytic or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis or ADCC (antibody-dependent cell-mediated cytotoxicity).

[0052] The term "variable region" refers to a domain in the heavy or light chain of an antibody that originates from germline variable (V) genes, diversity (D) (heavy chain only) genes, and linking (J) genes (and does not originate from stationary (Cμ and Cδ) gene segments) and confers its specificity to binding to an antigen. Antibody variable regions typically consist of four conserved "framework" regions with three hypervariable "complementarity-determining regions (CDRs)" incorporated between them.

[0053] A variant of a given sequence is a change in the nucleic acid or amino acid sequence compared to a reference sequence (e.g., wild-type or most common). Variants can be naturally occurring sequences (e.g., allelic variants) or sequences that do not naturally occur. A non-naturally occurring variant domain refers to a variant domain or mutant domain that does not exist in natural cells and is produced by genetic modification of the original domain (e.g., using genetic engineering or mutagenesis). Changes in the nucleic acid or amino acid sequence (e.g., mutations) can include one or more substitutions, one or more insertions, one or more deletions, or combinations thereof.

[0054] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to mutations in which one amino acid is substituted with another amino acid that can be classified as having similar characteristics. Examples of classifications of such conservative amino acid groups include the following: The "charged / polar group" includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); the "aromatic group" includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Tip (tryptophan or W), and (histidine or H); and the "aliphatic group" includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), He (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Subgroups can also be identified within each group. For example, the group of charged amino acids or polar amino acids can be subdivided into subgroups such as the "positively charged subgroup" containing Lys, Arg, and His, the "negatively charged subgroup" containing Glu and Asp, and the "polar subgroup" containing Asn and Gln. In another example, the aromatic group or cyclic group can be subdivided into subgroups such as the "nitrogen ring subgroup" containing Pro, His, and Trp, and the "phenyl subgroup" containing Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups such as the "aliphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala, and the "aliphatic weakly polar subgroup" containing Met, Ser, Thr, and Cys.Examples of the classification of conservative mutations include amino acid substitutions of amino acids within the above subgroups, for example, without limitation, substitutions of Arg by Lys to maintain a positive charge or vice versa; substitutions of Asp by Glu to maintain a negative charge or vice versa; substitutions of Thr by Ser to maintain free -OH or vice versa; and substitutions of Asn by Gln to maintain free -NH2 or vice versa. In some embodiments, hydrophobic amino acids are substituted with naturally occurring hydrophobic amino acids, for example, at the active site, thereby preserving hydrophobicity.

[0055] "Binding affinity" refers to the strength of the non-covalent interaction between two molecules (for example, between a single binding region or site on a protein and its target). Binding affinity can be quantified by measuring the equilibrium dissociation constant (KD). The equilibrium dissociation constant (KD) is the same as the dissociation rate constant (k d time -1 ) and the association rate constant (k a time -1 M -1 This refers to the value obtained by dividing by ). KD can be determined by measuring the dynamics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) (e.g., the Biacore® system), dynamical exclusion assays (e.g., KinExA®), and biolayer interferometry (e.g., using the ForteBio® Octet® platform). "Binding affinity" includes not only normal binding affinity (e.g., reflecting a 1:1 interaction between a polypeptide and its target) but also apparent affinity for which a KD can be calculated that reflects strong binding (avidity).

[0056] The terms "specifically bind" or "selectively bind" to a target (e.g., amyloid beta, TfR, or FcγR) refer to a binding reaction in which a binding region (e.g., an amyloid beta binding region, a TfR binding region, or an FcγR binding region) binds to the target with a higher affinity, a higher avidity, and / or a longer duration than it binds to structurally different targets. When assayed under the same affinity assay conditions, the binding region can have an affinity for a particular target that is at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater than that for an unrelated target. The terms "specific binding", "specifically binds", or "specific" with respect to a particular target mean, for example, that a molecule has a dissociation constant KD of 10 -4 M or less (e.g., 10 -5 M, 10 -6 M (where 1000 nM), -7 10M (where 100 nM), -8 10M (where 10 nM), -9 10M (where 1 nM), -10 10M, -11 10M, or -12 10M). In some embodiments, the binding region specifically binds to a target (e.g., a protein) that is conserved across species (e.g., structurally conserved across species).

[0057] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.<00,00334>

[0058] Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). "Amino acid analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and carbon bonded to the R group) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimes" refer to compounds that have a different structure from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0059] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoimers of naturally occurring α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. In this specification, amino acids may be represented by either the commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0060] In this specification, the term “mutant” in relation to a mutant polypeptide or mutant polynucleotide is interchangeable with “variant.” A variant with respect to a given wild-type CH3 domain reference sequence or wild-type CH2 domain reference sequence may include naturally occurring allelic variants. A “not naturally occurring” CH3 domain or CH2 domain refers to a mutant domain or mutant domain that is not naturally present in cells and is produced by genetic modification (e.g., using genetic engineering or mutagenesis) of the original CH3 domain or CH2 domain polynucleotide or polypeptide. A “variant” includes any domain that contains at least one amino acid mutation relative to the wild type. Mutations can include substitutions, insertions, and deletions. A single amino acid substitution can be indicated by a single amino acid symbol indicating the amino acid before substitution, followed by a number indicating the amino acid position, and then a single amino acid symbol indicating the subsequent amino acid substitution (for example, T366W indicates that the threonine at position 366 has been replaced with tryptophan). Furthermore, single amino acid substitutions can also be indicated by a number indicating the position of the substituted amino acid and a single-letter amino acid symbol indicating the subsequent amino acid substitution (for example, 366W indicates tryptophan at that position).

[0061] A polypeptide is a polymer of two or more amino acid residues in a single-chain form. This term applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. Amino acid polymers may be entirely L-amino acids, entirely D-amino acids, or mixtures of L-amino acids and D-amino acids.

[0062] As used herein, the term “protein” refers to a polypeptide, a polypeptide dimer, or a polypeptide polymer. A polypeptide dimer may be a heterodimer or a homodimer. A polypeptide polymer may be a homopolymer or a heteropolymer. A heteropolymer can contain two or more copies of any given single-chain polypeptide. For example, immunoglobulins are heteropolymers containing two heavy chains and two light chains. The two heavy chains may be the same or different, and the two light chains may be the same or different. Single-chain polypeptide chains in a dimer or polymer can be linked by one or more covalent bonds (e.g., disulfide bonds), by non-covalent interactions, or by a combination thereof.

[0063] With respect to two or more nucleic acid sequences or polypeptide sequences, the term “identical” or “identity” percentage means that two or more sequences or subsequences are identical, or that two or more sequences or subsequences are identical to a certain percentage (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or more) of nucleic acid residues or amino acid residues when compared and aligned to maximize the match over a specific region, within a comparison range or designated region, and measured using a sequence comparison algorithm, or by manual alignment and visual inspection. For sequence comparison, typically one sequence is used as a reference sequence and compared with the candidate sequences. Alignment can be performed using various methods available to those skilled in the art (e.g., visual alignment, or alignment using publicly available software with known algorithms) to achieve the greatest possible alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to obtain maximum alignment can be determined by those skilled in the art. For the purpose of this application, the BLAST standard protein of the BLASTP algorithm is used to align two protein sequences using default parameters for polypeptide sequence comparison.

[0064] The expressions "corresponding to," "determined by reference to," or "numbered by reference to," used in relation to identifying a given nucleotide or amino acid residue in a nucleic acid or polypeptide sequence, refer to the position of a residue in a particular reference sequence when the given amino acid sequence is maximally aligned and compared to that reference sequence. The sequence aligned to the reference sequence does not need to be the same length as the reference sequence.

[0065] The "EU numbering system" is generally used in this technical field to refer to residues within the constant region of the antibody heavy chain. The EU numbering system for Sequence ID No. 8 is shown below. [Table A]

[0066] The terms “subject,” “individual,” and “patient,” as used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammalian species. In some embodiments, the subject is human.

[0067] The terms “to treat” and “treatment” refer to methods or steps taken to reduce, improve or alleviate the number, severity, side effects, and / or frequency of at least one symptom or pathological consequence of a disease, disorder, or illness in a subject. Treatment can be preventive in that it prevents or partially prevents the disease, or the symptoms or conditions of the disease. Prevention includes preventing the onset or recurrence of the disease in subjects who are susceptible to the disease but have not yet been diagnosed with the disease. Prevention also includes preventing the onset or recurrence of symptoms or pathological consequences of the disease in subjects who are susceptible to the symptoms or pathological consequences of the disease but have not yet been diagnosed with the symptoms or pathological consequences of the disease. Treatment can also be preventive in that it delays the onset of the disease or delays the symptoms or conditions of the disease. Delaying the onset of the disease or delaying the symptoms or pathological consequences of the disease means delaying, hindering, slowing, postponing, stabilizing, suppressing, and / or postponing the onset of the disease or the onset of symptoms or pathological consequences of the disease. This delay may vary in duration depending on the history of the disease being treated and / or the individual being treated. Treatment may include suppressing a disease, disorder, or illness, e.g., preventing its progression, and may also include alleviating a disease, disorder, or illness, e.g., causing the disease, disorder, and / or illness to regress. Treatment may also mean extending survival time compared to the predicted survival time without treatment. Treatment may aim to cure in the sense of partial or complete cure of a disease, disorder, symptom, or side effects caused by a disease, disorder, or illness. The term “treatment” may encompass (a) to (c) the following: (a) preventing the onset of a disease in an object susceptible to the disease but not yet diagnosed with it; (b) inhibiting the disease, i.e., preventing its onset; and (c) reducing the disease, i.e., reducing or improving the disease and / or its symptoms or condition. Treatment may refer to treatment aimed at curing only, treatment aimed at prevention only, or treatment aimed at both curing and prevention.Those who require treatment (or are subject to treatment) may include individuals who have been previously diagnosed with a disease, disorder, or illness, or who have been identified as being at risk of developing a disease, disorder, or illness. Treating a disease, disorder, or illness may include improving at least one symptom of a particular disease, disorder, or illness, even if it does not affect the underlying pathophysiology.

[0068] The term "pharmaceutically acceptable additive" refers to a non-active medicinal ingredient (e.g., buffers, carriers, or preservatives) that is biologically or pharmacologically compatible for use in humans or animals.

[0069] With regard to administration, the “effective dose” of a drug (e.g., a protein that targets amyloid-beta or a pharmaceutical preparation containing a protein that targets amyloid-beta) refers to the amount that is effective in obtaining the desired result (e.g., a therapeutic or prophylactic result) in the required number / amount of doses and for the required period of time.

[0070] The “therapeutic effective dose” of a drug (e.g., a protein that targets amyloid-beta or a pharmaceutical preparation containing amyloid-beta-targeting proteins) refers to the amount effective in obtaining the desired therapeutic outcome (e.g., the outcome of treating a disease, illness, or disorder, and / or the pharmacokinetic or pharmacodynamic effect of the treatment) in the required dosage and for the required duration. The therapeutic effective dose may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the population of cells being administered.

[0071] "Dosage," "unit dose," or "administration" refers to physically separate units suitable for use in the subject, each unit containing a predetermined amount of active pharmaceutical ingredient and / or pharmaceutical composition.

[0072] The term "administration" refers to a method of delivering a drug, compound, or composition to a desired site of action. Such methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, a composition described herein is administered intravenously.

[0073] II. Proteins that target amyloid-beta Linking the transferrin receptor binding domain of a molecule can increase the transport of molecules across the blood-brain barrier (BBB). For some molecules, such as antisense oligonucleotides, rapid delivery to the brain is preferred. For other compounds, the initial rate of delivery is less important than persistence. TfR-binding polypeptides with higher affinity for TfR (e.g., binding affinity less than 900 nM) typically have a higher initial rate of delivery to the brain, followed by rapid efflux. In contrast, TfR-binding polypeptides with lower affinity for TfR typically have a lower initial rate of delivery to the brain, but exhibit greater persistence in the brain compared to high-affinity TfR-binding polypeptides. Surprisingly, by linking a TfR-binding polypeptide with an affinity for TfR of approximately 900 nM to 10,000 nM (e.g., approximately 900 nM to 2,500 nM or approximately 900 nM to 1,300 nM) to an amyloid-beta-binding polypeptide, the initial delivery rate (Cmax) of the amyloid-beta-binding polypeptide to the brain is higher than that of amyloid-beta-binding polypeptide linked to a TfR-binding polypeptide with a higher affinity for TfR.

[0074] Disclosed are amyloid-beta-targeting proteins and compositions that can cross the blood-brain barrier and bind to amyloid-beta plaques. The amyloid-beta-targeting proteins include an amyloid-beta binding domain, a TfR binding domain that specifically binds to TfR with an affinity of about 900 nM to about 10,000 nM (e.g., about 900 nM to about 2,500 nM or about 900 nM to about 1,300 nM), and an FcγR binding domain. The amyloid-beta-targeting proteins of this disclosure are transported across the blood-brain barrier (BBB). The amyloid-beta-targeting proteins possess amyloid-beta binding properties and immunoeffector functional properties. Therefore, the amyloid-beta-targeting proteins can be administered to subjects to treat amyloid plaques. Contrary to expectations, when amyloid-beta binding polypeptides are linked to TfR-binding domains that specifically bind to TfR with affinity of approximately 900 nM to 10,000 nM (e.g., approximately 900 nM to 2,500 nM or approximately 900 nM to 1,300 nM), the delivery of the protein to the brain increases, and the degree of this increase is greater than that for TfR-binding domains with higher TfR affinity. Furthermore, when linked to amyloid-beta binding polypeptides, TfR-binding domains that specifically bind to TfR with affinity of approximately 900 nM to 10,000 nM (e.g., approximately 900 nM to 2,500 nM or approximately 900 nM to 1,300 nM) increase the brain retention of amyloid-beta targeting proteins. The combination of high initial brain delivery rates and high brain retention is useful for therapies designed to target amyloid-beta plaques and amyloid-beta fibrils.

[0075] This invention discloses a protein that targets amyloid-beta, a composition containing a protein that targets amyloid-beta, a nucleic acid encoding a protein that targets amyloid-beta, and a method for producing a protein that targets amyloid-beta.

[0076] A method for treating amyloid-beta-mediated diseases or illnesses (including neurodegenerative diseases such as Alzheimer's disease) using proteins that target amyloid-beta.

[0077] A. Amyloid-beta (amyloid-beta) binding region Amyloid-beta (amyloid β) is a peptide with a length of 36-43 amino acids that is the main component of amyloid plaques (extracellular deposits) found in the brains of people with Alzheimer's disease. The plaques are composed of tangled amyloid-beta oligomers and regularly arranged aggregates called amyloid fibrils. The soluble oligomer form of amyloid-beta is thought to be the causative substance of Alzheimer's disease.

[0078] The amyloid-beta binding region comprises a polypeptide or region or domain, or a polypeptide or protein, that specifically binds to amyloid-beta. The amyloid-beta binding region can specifically bind to oligomeric amyloid-beta and / or fibrillary amyloid-beta. In some embodiments, the amyloid-beta binding region binds to oligomeric amyloid-beta. In some embodiments, the amyloid-beta binding region binds to fibrillary amyloid-beta. In some embodiments, the amyloid-beta binding region binds to both oligomeric amyloid-beta and fibrillary amyloid-beta.

[0079] Proteins that target amyloid-beta can be monovalent or polyvalent with respect to amyloid-beta binding. That is, a protein that targets amyloid-beta may contain a single amyloid-beta binding region, or it may contain multiple amyloid-beta binding regions. In some embodiments, a protein that targets amyloid-beta includes two or more amyloid-beta binding regions. These two or more amyloid-beta binding regions may be the same or different. In some embodiments, a protein that targets amyloid-beta includes two amyloid-beta binding regions.

[0080] The amyloid-beta binding domain can be, but is not limited to, a peptide, a modified peptide, an anti-amyloid-beta antibody, or an amyloid-beta binding fragment of an anti-amyloid-beta antibody.

[0081] Anti-amyloid-beta antibodies are known in the art and are available from various commercial sources. Anti-amyloid-beta may also be antibodies against amyloid-beta derived from non-human species, insofar as the antibody binds to human amyloid-beta. In some embodiments, the anti-amyloid-beta antibody or the amyloid-beta binding fragment of the anti-amyloid-beta antibody binds to oligomeric amyloid-beta and fibrillary amyloid-beta.

[0082] The amyloid-beta binding fragment of an anti-amyloid-beta antibody may be, but is not limited to, an F(ab)2 fragment, a Fab fragment, or a single-chain variable fragment (scFv).

[0083] In some embodiments, the amyloid-beta binding region includes the antigen-binding domain of an anti-amyloid-beta antibody. In some embodiments, the amyloid-beta binding region includes the Fab region of an anti-amyloid-beta antibody, two Fab regions of an anti-amyloid-beta antibody, the F(ab)2 region of an anti-amyloid-beta antibody, or at least one scFv derived from an anti-amyloid-beta antibody. In some embodiments, the amyloid-beta binding region includes the antigen-binding domain of aducanumab. In some embodiments, the amyloid-beta binding region includes the Fab region of aducanumab, two Fab regions of aducanumab, the F(ab)2 region of aducanumab, or at least one scFv derived from aducanumab.

[0084] In some embodiments, the amyloid-beta binding region includes a heavy chain variable region (VH) and a light chain variable region (VL), where (a) VH includes a first heavy chain complementarity determination region (VHCDR1) having the amino acid sequence of SEQ ID NO: 1, a second heavy chain complementarity determination region (VHCDR2) having the amino acid sequence of SEQ ID NO: 2, and a third heavy chain complementarity determination region (VHCDR3) having the amino acid sequence of SEQ ID NO: 3. (b) The VL includes a first light chain complementarity determination region (VLCDR1) having the amino acid sequence of SEQ ID NO: 4, a second light chain complementarity determination region (VLCDR2) having the amino acid sequence of SEQ ID NO: 5, and a third light chain complementarity determination region (VLCDR3) having the amino acid sequence of SEQ ID NO: 6. VH and VL may be part of Fab, F(ab)2, or scFv.

[0085] In some embodiments, the amyloid-beta binding region includes VH and VL, where (a) VH comprises an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-120 of SEQ ID NO: 8 or 10, and VH comprises VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3. (a) VL comprises an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-109 of SEQ ID NO: 7, and VL comprises VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. VH and VL may be part of Fab, F(ab)2, or scFv.

[0086] In some embodiments, the amyloid-beta binding region comprises VH and VL, where VH comprises the amino acid sequence of amino acids 1-120 of SEQ ID NO: 8 or 10, and VL comprises the amino acid sequence of amino acids 1-109 of SEQ ID NO: 7. VH and VL may be part of Fab, F(ab)2, or scFv.

[0087] In some embodiments, the amyloid-beta binding region includes a heavy chain Fab region and a light chain Fab region, where (a) The heavy chain Fab region comprises an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-227 of SEQ ID NO: 8 or 10, and includes VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3. (a) The light chain Fab region includes an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ ID NO: 7, and includes VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0088] In some embodiments, the amyloid-beta binding region includes an F(ab)2 region having two heavy chain Fab regions and two light chain Fab regions, where (a) Each heavy chain Fab region contains an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-227 of SEQ ID NO: 8 or 10, and includes VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3. (b) Each light chain Fab region contains an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ ID NO: 7, and includes VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0089] In some embodiments, the amyloid-beta binding region includes a heavy chain Fab region and a light chain Fab region, where the heavy chain Fab region includes the amino acid sequence of amino acids 1-227 of SEQ ID NO: 8 or 10, and the light chain Fab region includes the amino acid sequence of SEQ ID NO: 7.

[0090] In some embodiments, the amyloid-beta binding region comprises an F(ab)2 region having two heavy-chain Fab regions and two light-chain Fab regions, where each heavy-chain Fab region contains the amino acid sequence of amino acids 1-227 of SEQ ID NO: 8 or 10, and each light-chain Fab region contains the amino acid sequence of SEQ ID NO: 7.

[0091] B. Transferrin receptor (TfR) binding domain A TfR-binding domain is a molecule such as a polypeptide, or a larger polypeptide, protein region, or domain, that specifically binds to TfR, such as human TfR. The TfR-binding domain of amyloid-beta targeting proteins specifically binds to TfR and facilitates the transport of amyloid-beta targeting proteins across the blood-brain barrier (e.g., by receptor-mediated transcytosis) or increases the penetration of amyloid-beta targeting proteins into the brain.

[0092] The transferrin receptor (TfR), also known as differentiation antigen 71 (CD71), binds to transferrin (Tf) and plays a crucial role in cellular iron uptake through interaction with iron-binding transferrin. TfR is highly expressed by brain capillary endothelial cells (BCECs), which form the blood-brain barrier (BBB). TfR is a 90 kDa type II transmembrane glycoprotein consisting of 760 amino acids, and it exists on the cell surface as a dimer (180 kDa) linked by disulfide bonds. The TfR1 monomer consists of a large extracellular C-terminal domain of 671 amino acids containing the Tf-binding site, a transmembrane domain (28 amino acids), and an intracellular N-terminal domain (61 amino acids). The C-terminal extracellular domain contains three N-linked glycosylation sites on asparagine residues 251, 317, and 727, as well as one O-linked glycosylation site on threonine 104, all of which are necessary for the proper functioning of the receptor.

[0093] In some embodiments, the TfR binding region binds to the apical domain of TfR, which comprises residues 189–383 of TfR. In some embodiments, the TfR binding region binds to an epitope including position 208 of the full-length human transferrin receptor sequence. In some embodiments, the TfR binding region binds to the apical domain of TfR in an epitope including positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and / or 294 of the full-length human TfR sequence (SEQ ID NO: 15).

[0094] In some embodiments, binding of the TfR-binding domain to TfR does not inhibit the binding of transferrin to TfR. In certain embodiments, binding of amyloid-beta-targeting proteins to TfR does not inhibit the binding of transferrin to TfR. In some embodiments, the inhibition of transferrin binding to TfR is less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%). In some embodiments, the inhibition of transferrin binding to TfR is less than about 20% (e.g., less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%).

[0095] In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 10,000 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 8,500 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 6,500 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 5,000 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 3,500 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 2,500 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 2000 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 1600 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 1300 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 1000 nM to approximately 1200 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with affinities of approximately 1400 nM, 1500 nM, 1600 nM, 1800 nM, 2000 nM, 2500 nM, 3000 nM, 3500 nM, 4000 nM, 4500 nM, 5000 nM, 5500 nM, 6000 nM, 6500 nM, 7000 nM, 7500 nM, 8000 nM, 8500 nM, 9000 nM, or 10,000 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with affinities of approximately 900 nM, 950 nM, 1000 nM, 1050 nM, 1100 nM, 1150 nM, or 1200 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of 1100 ± 200 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of 1100 ± 100 nM.In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of 1100 ± 50 nM.

[0096] In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 10,000 nM. In some embodiments, the TfR binding domain specifically binds to human TfR with an affinity of approximately 900 nM to approximately 8,500 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 6,500 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 5,000 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 3,500 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 2,500 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 2000 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 1600 nM. In some embodiments, the amyloid-beta targeting protein specifically binds to human TfR with an affinity of approximately 900 nM to approximately 1300 nM. In some embodiments, amyloid-beta targeting proteins specifically bind to human TfR with affinities of approximately 1400 nM, 1500 nM, 1600 nM, 1800 nM, 2000 nM, 2500 nM, 3000 nM, 3500 nM, 4000 nM, 4500 nM, 5000 nM, 5500 nM, 6000 nM, 6500 nM, 7000 nM, 7500 nM, 8000 nM, 8500 nM, 9000 nM, or 10,000 nM. In some embodiments, amyloid-beta targeting proteins specifically bind to human TfR with affinities of approximately 1000 nM to 1200 nM. In some embodiments, amyloid-beta targeting proteins specifically bind to human TfR with affinities of approximately 900 nM, 950 nM, 1000 nM, 1050 nM, 1100 nM, 1150 nM, or 1200 nM.In some embodiments, the amyloid-beta-targeting protein specifically binds to human TfR with an affinity of 1100 ± 200 nM. In some embodiments, the amyloid-beta-targeting protein specifically binds to human TfR with an affinity of 1100 ± 100 nM. In some embodiments, the amyloid-beta-targeting protein specifically binds to human TfR with an affinity of 1100 ± 50 nM. In some embodiments, the amyloid-beta-targeting protein specifically binds to human TfR with an affinity of 1100 nM.

[0097] Analysis of binding affinity, binding rate, and cross-reactivity between the TfR binding region and TfR can be performed using methods available in the art for analyzing binding affinity, binding rate, and cross-reactivity between two polypeptides. Such methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore® (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to measure binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art. In some embodiments, surface plasmon resonance (SPR) is used to measure binding affinity, binding rate, and / or cross-reactivity. In some embodiments, binding affinity, binding rate, and / or cross-reactivity are measured using a binding equilibrium exclusion method. In some embodiments, binding affinity, binding rate, and / or cross-reactivity are measured using a biolayer interference assay. In some embodiments, binding affinity is measured by surface plasmon resonance (optionally using a Biacore® instrument). In this method, a TfR-binding polypeptide is captured on a sensor chip, and a serial dilution of TfR is injected into the sensor chip at a predetermined flow rate (e.g., 30 μL / min) and temperature (e.g., room temperature). The sample is analyzed at predetermined association and dissociation times (e.g., 45 seconds and 180 seconds, respectively), and then the sensor chip is regenerated. The binding reaction can be corrected by subtracting the measured reaction from a control (e.g., using unrelated IgG at a similar density), and then the steady-state affinity can be determined by using software to fit the equilibrium reaction to the concentration.

[0098] In some embodiments, the amyloid-beta targeting protein is monovalent with respect to the TfR binding domain, i.e., the amyloid-beta targeting protein contains a single TfR binding domain.

[0099] The TfR binding region may, but is not limited to, a peptide, a modified peptide, an anti-TfR antibody, a TfR-binding fragment of an anti-TfR antibody, or a region of an antibody modified to bind to human TfR.

[0100] Anti-TfR antibodies are known in the art and are available from various commercial sources. Anti-TfR antibodies may also be antibodies against TfR derived from non-human species, insofar as they bind to human TfR with the aforementioned affinity (e.g., affinity of about 900 nM to about 10,000 nM). In some embodiments, the anti-TfR antibody or the TfR-binding fragment of the anti-TfR antibody binds to the apical domain of TfR. In some embodiments, the binding of the anti-TfR antibody or the TfR-binding fragment of the anti-TfR antibody to TfR does not inhibit the binding of transferrin to TfR. Exemplary anti-TfR antibodies include B3 / 25, RBC4, 7579, E2.3, A27.15, D65.30, D2C, ch128.1Av, ch128.1 / IgG3, ch128.1 / IgG1, and hu128.1 (Candelaria et al. Front.). Examples of exemplary anti-TfR antibodies include, but are not limited to, Immunol. 12 (17 March 2021), 2021), Ri7, and 8D3 (Weber et al. Cell Reports 22:149-162, 2018). Exemplary anti-TfR antibodies are also described in U.S. Patent Publications US2018282408A1, US2020071413A1, US20210138083, US20190092870, and US20130028891. Anti-TfR antibodies can be modified to have an affinity for TfR of approximately 900 nM to approximately 10,000 nM.

[0101] The TfR-binding fragment of an anti-hTfR antibody may be, but is not limited to, a Fab fragment, a single-chain variable fragment (scFv), a monovalent anti-TfR antibody, or a bivalent antibody having a single TfR-binding domain.

[0102] In some embodiments, the TfR binding region includes the antigen-binding domain of the anti-TfR antibody. In some embodiments, the TfR binding region includes the Fab region of the anti-TfR antibody or an scFv derived from the anti-TfR antibody.

[0103] TfR-binding domains can be obtained from proteins known to bind to TfR. Such proteins include, for example, P. vivax reticulocyte-binding protein 2b (PvRBP2b), or viral proteins such as arenavirus proteins (e.g., Machupovirus, Sabiavirus, Juninvirus, Guanaritovirus, or Chaparevirus) which are known to bind to TfR.

[0104] In some embodiments, the TfR binding region comprises an engineered polypeptide. The engineered polypeptide can be a polypeptide (e.g., an antibody Fc polypeptide) or the antigen-binding region of an anti-TfR antibody, which has been modified to alter the affinity of the polypeptide or the antigen-binding region of the anti-TfR antibody to Tfr. Engineered peptides can be identified or generated using methods available in the art to identify or generate peptides that have affinity for known targets (e.g., TfR). Such methods include, but are not limited to, phage displays, yeast displays (e.g., yeast surface displays), and directed evolution methods, as well as combinations thereof.

[0105] A method for manipulating a CH2 domain or CH3 domain to have transferrin receptor binding ability is described in U.S. Patent Publication US20200223935 (incorporated herein by reference). A nucleic acid encoding a polypeptide (e.g., a CH2 domain or a CH3 domain) can be modified to encode 1 to 10 or more amino acid mutations. The encoded mutations may be located at predetermined sites, randomly within selected predetermined sites, or they may be randomly distributed. The encoded mutations may be random, partially random, or biased with respect to amino acid mutations. The encoded modified polypeptide is then expressed using any number of systems, such as display systems. Display systems may be, but are not limited to, viral display systems, cell surface display systems such as yeast display systems, mRNA display systems, or polysome display systems. The modified polypeptide can then be screened using known methods to identify transferrin receptor binding substances, which can then be further characterized to determine their binding affinity. Polypeptides identified as having affinity for TfR can be subjected to one or more additional mutation, expression, display, and selection steps until a polypeptide with the desired affinity for TfR is identified.

[0106] In some embodiments, the manipulated polypeptide comprises an antibody Fc polypeptide modified to bind to transferrin. The Fc polypeptide modified to bind to TfR may contain a CH2 domain or a CH3 domain modified to bind to TfR. The CH2 or CH3 domain modified to bind to TfR may be derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the TfR binding region comprises a CH3 domain derived from IgG1 modified to bind to transferrin. In some embodiments, the TfR binding region comprises a CH3 domain derived from IgG1 modified to bind to transferrin with an affinity of about 900 nM to about 10,000 nM (e.g., about 900 nM to about 2,500 nM or about 900 nM to about 1,300 nM).

[0107] In some embodiments, the engineered polypeptide includes an antibody CH2 domain modified to bind to TfR, or an antibody CH3 domain modified to bind to TfR. The CH2 or CH3 domain modified to bind to TfR may be derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the TfR binding region includes a CH3 domain derived from IgG1 modified to bind to transferrin. In some embodiments, the TfR binding region includes a CH3 domain derived from IgG1 modified to bind to transferrin with an affinity of about 900 nM to about 10,000 nM (e.g., about 900 nM to about 2,500 nM or about 900 nM to about 1,300 nM). CH3 domains of other immunoglobulin isotypes (e.g., IgM, IgA, IgE, IgD, etc.) can be similarly engineered by identifying the amino acids of those domains corresponding to the amino acid positions described herein. It is also possible to modify the corresponding domains of immunoglobulins derived from other species, such as non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, and chickens.

[0108] In some embodiments, the TfR binding region comprises a modified CH3 domain having amino acid substitutions at at least 6, 7, 8, 9, or 10 positions selected from 380, 384, 386, 387, 388, 398, 390, 413, 415, 416, and 421 according to the EU numbering system. In some embodiments, the TfR binding region comprises a modified CH3 domain having tyrosine or phenylalanine at position 384, threonine or asparagine at position 386, aspartic acid at position 387, tryptophan at position 388, serine, threonine, or valine at position 389, serine or asparagine at position 390, threonine or serine at position 413, serine or glutamine at position 415, glutamic acid at position 416, and phenylalanine or tyrosine at position 421 according to the EU numbering system. In some embodiments, the TfR binding region comprises a modified CH3 domain having tyrosine (Y) at position 384, threonine (T) at position 386, glutamic acid (E) at position 387, tryptophan (W) at position 388, valine (V) at position 389, threonine (T) at position 413, glutamic acid (E) at position 415, glutamic acid (E) at position 416, and phenylalanine (F) at position 421 (according to EU numbering).

[0109] In some embodiments, the TfR binding region includes a modified CH3 domain having any combination of substitutions shown in Table 1. In some embodiments, the TfR binding region includes a modified CH3 domain having any combination of substitutions shown in Table 1 and having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 (according to EU numbering) of any of SEQ ID NOs. 8, 10, and 17-31. [Table 1]

[0110] In some embodiments, the TfR binding region comprises a modified CH3 domain polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering).

[0111] In some embodiments, the TfR binding region includes an amino acid sequence of amino acids 348-453 or 348-454 of any of SEQ ID NOs. 8, 10, or 17-31.

[0112] In some embodiments, the TfR binding region comprises a modified Fc polypeptide having an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and at least 99% identity to amino acids 228-454 of any of SEQ ID NOs. 8, 10, or 17-31, where the polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1. In some embodiments, the TfR binding region comprises a modified Fc polypeptide having an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and at least 99% identity to amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering).

[0113] In some embodiments, the TfR binding region comprises a modified Fc polypeptide having an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and at least 99% identity to amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the polypeptide has A at position 234, A at position 235, optionally G at position 239, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering).

[0114] In some embodiments, the TfR binding region includes an amino acid sequence of amino acids 228-453 or 228-454 of SEQ ID NO: 8, 10, or 17-31.

[0115] C.Fcγ receptor (FcγR) binding domain The FcγR binding domain comprises a polypeptide or region or domain or polypeptide or protein that can specifically bind to FcγR and induce the activity of an effector function. Examples of effector functions include, but are not limited to, ADCP, ADCC, and CDC. Effector functions are typically mediated by the binding of FcγR to the Fc domain of an immunoglobulin such as IgG. The amyloid-beta targeting proteins of this disclosure can mediate at least one effector function. The effector function can assist in the elimination of amyloid plaques in the target brain. In some embodiments, the amyloid-beta targeting proteins of this disclosure are configured such that the binding of the amyloid-beta targeting protein to TfR reduces the binding of the FcγR binding domain to FcγR. In some embodiments, the amyloid-beta targeting proteins of this disclosure mediate effector function (binding to FcγR) when binding to amyloid-beta, but do not mediate effector function or have reduced effectiveness in mediating effector function (do not bind to FcγR or have reduced binding to FcγR) when binding to TfR.

[0116] Fc receptors are proteins present on the surface of immune cells that contribute to the protective function of the immune system. Fc gamma receptors (FcγR) recognize IgG-coated targets such as opsonized antigens or immune complexes (ICs). FcγR induces antibody-dependent cell phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cell-mediated cytotoxicity (CDC).

[0117] The FcγR binding region may contain an Fc polypeptide derived from an immunoglobulin. The Fc polypeptide may contain all or part of the CH2 and CH3 domains of the immunoglobulin heavy chain. In some embodiments, the FcγR binding region contains an Fc polypeptide derived from IgG1, IgG2, IgG3, or IgG4, such as human IgG1, IgG2, IgG3, or IgG4. The Fc polypeptide may contain all or part of the CH2 and CH3 domains of the immunoglobulin heavy chain. Numerous mutations (e.g., substitutions, deletions, or insertions) are known in the art to increase or decrease the binding of an Fc polypeptide to FcγR. In some embodiments, the FcγR binding region contains an Fc polypeptide that does not contain mutations that reduce FcγR binding. The FcγR binding region may contain an Fc polypeptide having one or more mutations that increase FcγR binding or increase effector function.

[0118] In some embodiments, the FcγR binding region contains approximately 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the Fc polypeptide relative to the wild-type Fc polypeptide (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0119] In some embodiments, the FcγR binding region comprises an Fc polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 228-454 of SEQ ID NO: 9, and does not contain a TfR binding site.

[0120] In some embodiments, the FcγR binding region comprises an Fc polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 228-454 of SEQ ID NO: 9, and does not include a TfR binding site or any modification that reduces FcγR binding.

[0121] In some embodiments, the FcγR binding region comprises an Fc polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 228-454 of SEQ ID NO: 9, having S at position 336, A at position 368, and V at position 407, and not including a TfR binding site or any modification that reduces FcγR binding.

[0122] In some embodiments, the FcγR binding region comprises an Fc polypeptide, the Fc polypeptide comprising an amino acid sequence having 100% identity with amino acids 228-453 or 228-454 of SEQ ID NO: 9.

[0123] Methods for analyzing the binding affinity, binding rate, and cross-reactivity between the FcγR binding region and FcγR are known in the art. Such methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore® (GE Healthcare, Piscataway, NJ)), binding equilibrium exclusion (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to measure binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art. In some embodiments, surface plasmon resonance (SPR) is used to measure binding affinity, binding rate, and / or cross-reactivity. In some embodiments, binding equilibrium exclusion is used to measure binding affinity, binding rate, and / or cross-reactivity. In some embodiments, a biolayer interference assay is used to measure binding affinity, binding rate, and / or cross-reactivity.

[0124] To improve effector function, it may be desirable to introduce modifications to Fc polypeptides that include an FcγR binding domain. One method to increase effector function is to produce non-fucosylated or fucose-deficient modified Fc polypeptides. One approach to generating fucose-deficient Fc polypeptides is to use a fucose analog such as 2-fluorofucose (2-FF). Fucose analogs can deplete or reduce available GDP-fucose (a substrate required by fucosyltransferase to incorporate fucose into proteins). Another approach to generating fucose-deficient Fc polypeptides is to use an alpha-1,6-fucosyltransferase (FUT8) knockout cell line for Fc polypeptide expression. A non-limiting example of a suitable FUT8 knockout cell line is the Chinese hamster ovary cell (CHO) FUT8 knockout cell line available from Lonza Biologies. Furthermore, as described in Mori et al. (Biotechnol. Bioeng. (2004) 88:901-908 (the entire work is incorporated herein by reference)), CHO cell lines can be converted for the production of fucose-deficient proteins using FUT8 small interfering RNA (siRNA) (e.g., by constitutive expression of FUT8 siRNA).

[0125] D.Fc dimer In some embodiments, the TfR binding region and the Fcγ binding region are provided by the Fc dimer polypeptide. The TfR binding function and the FcγR binding function can be provided by a modified Fc dimer, where the Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the TfR binding region is affixed to the first Fc polypeptide, and the FcγR binding region is affixed to the first and second Fc polypeptides. In some embodiments, the TfR binding region and the Fcγ binding region are affixed to the first Fc polypeptide. In some embodiments, the TfR binding region is affixed to the first Fc polypeptide, and the FcγR binding region is affixed to the second Fc polypeptide.

[0126] In some embodiments, the first Fc polypeptide includes a TfR binding region and does not contain mutations that reduce effector function (e.g., FcγR binding, ADPC, ADCC, or CDC).

[0127] In some embodiments, the first Fc polypeptide includes a TfR binding region and contains one or more mutations that reduce effector function (e.g., FcγR binding, ADPC, ADCC, or CDC). In some embodiments, the first Fc polypeptide includes a CH3 domain modified to bind to the TfR binding region and one or more mutations that reduce effector function.

[0128] Fc polypeptides with reduced FcγR binding refer to modified Fc polypeptides containing mutations in the CH2 domain and / or CH3 domain. Such mutations result in Fc polypeptides with reduced affinity for FcγR. Fc polypeptides with reduced FcγR binding can have an affinity for FcγR that is reduced by about 10% to about 90% compared to wild-type Fc polypeptides or Fc polypeptides that do not contain mutations that reduce FcγR binding. In some embodiments, mutations that reduce FcγR binding or reduce effector function reduce FcγR binding or reduce effector function by about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, or about 90% or more. FcγR binding can be measured, for example, using surface plasmon resonance (SPR) spectroscopy (e.g., the Biacore® system). Alternatively, FcγR binding can be measured using functional assays, such as ADCP assays or ADCC assays (e.g., in vivo or in vitro assays of cell death). A decrease in FcγR binding can be measured when modified Fc polypeptides, Fc dimers, or amyloid-beta targeting proteins bind to TfR and / or amyloid-beta. In some embodiments, FcγR binding of Fc polypeptides, Fc dimers, or amyloid-beta targeting proteins may be significantly reduced when bound to TfR compared to when not bound to TfR.

[0129] In some embodiments, the second Fc peptide does not contain a TfR binding domain and does not include modifications that reduce FcγR binding.

[0130] In some embodiments, the Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprising a CH3 domain modified to bind to TfR and a CH2 domain, wherein the CH2 domain does not contain mutations that reduce effector function, and the second Fc polypeptide comprises a CH3 domain and a CH2 domain, wherein the second Fc polypeptide does not contain a TfR binding region.

[0131] In some embodiments, the Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprising a CH3 domain modified to bind to TfR and a CH2 domain, wherein the CH2 domain does not contain mutations that reduce effector function, and the second Fc polypeptide comprises a CH3 domain and a CH2 domain, wherein the second Fc polypeptide does not contain a TfR binding region and does not contain modifications that reduce FcγR binding.

[0132] In some embodiments, the Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprising a CH3 domain modified to bind to TfR and a CH2 domain, wherein the CH2 domain does not contain any mutations that reduce effector function, and the second Fc polypeptide comprises a CH3 domain and a CH2 domain, wherein the second Fc polypeptide does not contain a TfR binding region and the CH2 domain contains one or more mutations that reduce effector function.

[0133] In some embodiments, the first Fc polypeptide comprises a CH3 domain modified to bind to a TfR binding region and one or more mutations that reduce effector function, while the second Fc polypeptide does not include modifications that reduce effector function (e.g., FcγR binding). In some embodiments, the Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises a CH3 domain modified to bind to TfR and a CH2 domain modified to reduce effector function (e.g., FcγR binding, ADPC, ADCC, or CDC), while the second Fc polypeptide comprises the CH3 domain and the CH2 domain, where the second Fc polypeptide does not include a TfR binding region and does not include modifications that reduce FcγR binding. Antibody constructs having such Fc dimer configurations are described in WO2019140050. Amyloid-beta targeting proteins having Fc dimers of this configuration either do not bind to FcγR and do not mediate effector function, or, upon binding to TfR, FcγR binding is reduced or effector function is reduced, but when not bound to TfR, they can bind to FcγR and mediate effector function. Reduced binding to FcγR and reduced effector function upon binding to TfR lead to reduced reticulocyte depletion. Furthermore, such dimers confer effector function when amyloid-beta targeting proteins bind to amyloid-beta. Effector function upon binding to amyloid-beta (e.g., amyloid plaques or amyloid fibrils) can promote the reduction of amyloid plaques or amyloid fibrils in the target.

[0134] In some embodiments, the amyloid-beta targeting proteins of the Disclosure do not cause a substantial decrease in reticulocytes (e.g., a decrease in bone marrow reticulocytes or circulating reticulocytes). In some embodiments, the decrease in bone marrow reticulocytes or circulating reticulocytes that occurs when an amyloid-beta targeting protein of the Disclosure is administered to a subject is less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, less than 5%, less than 3%, less than 2%, or less than 1%, compared to the number of bone marrow reticulocytes or circulating reticulocytes in the subject before administration of the amyloid-beta targeting protein, compared to a control subject administered with a control protein, or compared to a given control.

[0135] Mutations in Fc polypeptides that regulate effector function include substitutions at position 234 (e.g., L234A), 235 (e.g., L235A), and 329 (e.g., P329G) (IgG1 Fc), substitutions at position 228 (e.g., S228P) and 235 (e.g., L235E) (IgG4 Fc), substitutions at position 234 (e.g., L234A) and 237 (e.g., G237A) (IgG1 Fc), substitutions at position 234 (e.g., L234A), 235 (e.g., L235A), and 237 (e.g., G237A) (IgG1 Fc), and substitutions at position 234 (e.g., V234A) and 237 (e.g., G237A) (IgG2 Substitutions include, but are not limited to, substitutions at positions Fc), 235 (e.g., L235A), 237 (e.g., G237A), and 318 (e.g., E318A) (IgG4 Fc), as well as substitutions at positions 228 (e.g., S228P) and 236 (e.g., L236E) (IgG4 Fc) (according to EU numbering).

[0136] In some embodiments, the first Fc polypeptide comprises one or more substitutions of L234A, L235A, or P329G. In some embodiments, the first Fc polypeptide comprises substitutions of L234A and L235A. In some embodiments, the first Fc polypeptide comprises a substitution of P329G. In some embodiments, the first Fc polypeptide comprises substitutions of L234A, L235A, and P329G.

[0137] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide containing a TfR binding region and amino acid modifications L234A and L235A according to the EU numbering system, and (b) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces FcγR binding.

[0138] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide containing a TfR binding region and including amino acid modifications L234A, L235A, and P329G according to the EU numbering system, and (b) a second Fc polypeptide that does not include a TfR binding site or modifications that reduce FcγR binding.

[0139] Fc dimers can be obtained as a single chain or as two polypeptides. The first or second Fc polypeptide in the Fc dimer may have one or more mutations that promote heterodimerization of the two Fc polypeptides. Any Fc mutation known in the art that promotes, facilitates, or enhances heterodimerization can be used in the Fc polypeptides of this disclosure. Such mutations include, but are not limited to, knob and hole mutations. In some embodiments, the first Fc polypeptide comprises a knob mutation and the second Fc polypeptide comprises a hole mutation. In some embodiments, the first Fc polypeptide comprises a hole mutation and the second Fc polypeptide comprises a knob mutation. The knob mutation may include the substitution of T366W (according to the EU numbering system). The hole mutation may include the substitution of T366S, T368A, and Y407V (according to the EU numbering system). In some embodiments, the first Fc polypeptide includes the substitution of T366W, and the second Fc polypeptide includes the substitutions of T366S, T368A, and Y407V.

[0140] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide comprising a TfR binding site that specifically binds to TfR, amino acid modifications L234A and L235A, and hole mutations T366S, L368A, and Y407V (according to the EU numbering system), and (b) a second Fc polypeptide comprising the knob mutation T366W (according to the EU numbering system), without a TfR binding site, and without modifications that reduce FcγR binding.

[0141] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide comprising a TfR binding site that specifically binds to TfR, amino acid modifications L234A, L235A, and P329G, and hole mutations T366S, L368A, and Y407V (according to the EU numbering system), and (b) a second Fc polypeptide comprising the knob mutation T366W (according to the EU numbering system) and not comprising a TfR binding site or modifications that reduce FcγR binding.

[0142] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide comprising a TfR binding site that specifically binds to TfR, amino acid modifications L234A and L235A, and a knob mutation T366W (according to the EU numbering system), and (b) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V (according to the EU numbering system), and not comprising a TfR binding site or modifications that reduce FcγR binding.

[0143] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide comprising a TfR binding site that specifically binds to TfR, amino acid modifications L234A, L235A, and P329G, and a knob mutation T366W (according to the EU numbering system), and (b) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V (according to the EU numbering system), and not comprising a TfR binding site or modifications that reduce FcγR binding.

[0144] In some embodiments, the Fc dimer is a first Fc polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to amino acids 228-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the amino acids are at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 as shown in Table 1 (according to EU numbering). (b) a first Fc polypeptide having the amino acids, and a second Fc polypeptide having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 228-454 of SEQ ID NO: 9, wherein the second Fc polypeptide has S at position 366, A at position 368, and V at position 407, and does not include a TfR binding site or any modification that reduces FcγR binding.

[0145] In some embodiments, the Fc dimer comprises a first Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 225-453 or 228-454 of any of SEQ ID NOs. 8, 10, and 17-31, and a second Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-453 or 228-454 of SEQ ID NOs. In some embodiments, the first Fc polypeptide contains (a) A at position 234 and A at position 235, (b) G at position 329, (c) A at position 234 and A at position 235, and G at position 329, (d) W at position 366, (e) A at position 234 and A at position 235, and W at position 366, (f) G at position 329 and W at position 366, or (g) A at position 234 and A at position 235, G at position 329 and W at position 366.

[0146] In some embodiments, the Fc dimer is a first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-454 of SEQ ID NO: 8, wherein A is at position 234, A is at position 235, G is at position 329, W is at position 366, Y is at position 384, T is at position 386, E is at position 387, W is at position 388, V is at position 389, T is at position 413, E is at position 415, E is at position 416, and (b) a first Fc polypeptide having F (according to the EU numbering system) at position 421, and a second Fc polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 228-454 of SEQ ID NO: 9, having S at position 366, A at position 368, and V at position 407, and not including a TfR binding site or any modification that reduces FcγR binding.

[0147] In some embodiments, the Fc dimer comprises a first Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-454 of SEQ ID NO: 8, and a second Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-454 of SEQ ID NO: 9. In some embodiments, the Fc dimer comprises a first Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-453 of SEQ ID NO: 8, and a second Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-453 of SEQ ID NO: 9.

[0148] In some embodiments, the Fc dimer is a first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-454 of SEQ ID NO: 10, wherein A is at position 234, A is at position 235, W is at position 366, Y is at position 384, T is at position 386, E is at position 387, W is at position 388, V is at position 389, T is at position 413, E is at position 415, E is at position 416, and 421 (b) a first Fc polypeptide having F (according to the EU numbering system), and a second Fc polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acids 228-454 of SEQ ID NO: 9, having S at position 366, A at position 368, and V at position 407, and not including a TfR binding site or any modification that reduces FcγR binding.

[0149] In some embodiments, the Fc dimer comprises a first Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-454 of SEQ ID NO: 10, and a second Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-454 of SEQ ID NO: 9. In some embodiments, the Fc dimer comprises a first Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-453 of SEQ ID NO: 10, and a second Fc polypeptide containing an amino acid sequence having 100% identity with amino acids 228-453 of SEQ ID NO: 9.

[0150] Any of the Fc dimers of this disclosure may have one or more mutations that increase or decrease the FcRn binding site or FcRn binding. The term "FcRn" refers to the neonatal Fc receptor. Binding of Fc polypeptide to FcRn reduces the efflux of Fc polypeptide and increases the serum half-life of Fc polypeptide. Any of the Fc dimers of this disclosure may have one or more mutations that increase the half-life or serum half-life (e.g., stability) of amyloid-beta-targeting proteins. Such mutations may be any of the mutations known in the art to increase the half-life or serum half-life of IgG. Mutations known to increase the half-life of IgG include, but are not limited to, mutations at one or more of the following locations: T250, M252, S254, T256, T307, E380, M428, and N434 (according to EU numbering). In some embodiments, the first Fc polypeptide and / or the second polypeptide in the Fc dimer may include (a) one or more substitutions of M252Y, S254T, and T256E, (b) a substitution of M428L and / or N434S, (c) one or more substitutions of T307A, E380A, and N434A, (d) a substitution of T250Q and / or M428L, (e) a substitution of M428L and / or N434S, and / or (f) a substitution of N434S and / or N434A. Mutations that modulate FcRn binding may be present in the first PC polypeptide of the Fc dimer, in the second Fc polypeptide of the Fc dimer, or in the polypeptides of both Fc dimers. Mutations that increase the half-life or serum half-life can be present in the first Fc polypeptide of the Fc dimer, in the second Fc polypeptide of the Fc dimer, or in the polypeptides of both Fc dimers.

[0151] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A and optionally P329G, and amino acid modification N434S with or without M428L (according to EU numbering), and (b) a second Fc polypeptide comprising neither a TfR binding site nor any modification that reduces FcγR binding.

[0152] In some embodiments, the Fc dimer comprises (a) a first Fc polypeptide comprising a TfR binding region, amino acid modifications L234A and L235A and optionally P329G, and amino acid modification N434S with or without M428L (according to EU numbering), and (b) a second Fc polypeptide comprising amino acid modification N434S with or without M428L, and without a TfR binding site or modifications that reduce FcγR binding.

[0153] In some embodiments, the Fc dimers or Fc polypeptides described herein further comprise a partial hinge region or a complete hinge region. The hinge region may be derived from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, such as the IgG1 hinge region (e.g., the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 16)). In further embodiments, an Fc polypeptide, which may comprise a hinge region or a partial hinge region, is further fused to a polypeptide comprising an amyloid-beta binding region.

[0154] In some embodiments, the Fc dimer or Fc polypeptide described herein is fused to a polypeptide containing an amyloid-beta binding region via a linker. The linker may be, but is not limited to, a peptide linker (e.g., a hinge region). The peptide linker may be configured such that the variable region and the Fc polypeptide or Fc dimer are rotatable relative to each other and / or resistant to digestion by proteases. In some embodiments, the linker may be a mobile linker containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters. For example, the linker may be a Gly-Ser repeat or (Gly) m (Ser) n It can have repetition, such as in a repetitional form.

[0155] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region including a heavy chain Fab region and a light chain Fab region, a TfR binding region including a first Fc polypeptide, and an FcγR binding region including a second Fc polypeptide, where the first and second Fc polypeptides are dimerized. The first or second Fc polypeptide may be linked to the heavy chain Fab region.

[0156] In some embodiments, a protein targeting amyloid-beta comprises two amyloid-beta binding regions, each containing a heavy-chain Fab region and a light-chain Fab region; a TfR binding region containing a first Fc polypeptide; and an FcγR binding region containing a second Fc polypeptide, where the first and second Fc polypeptides are dimerized, with the first Fc polypeptide linked to one heavy-chain Fab region and the second Fc polypeptide linked to the other heavy-chain Fab region. As those skilled in the art will recognize, this configuration is analogous to immunoglobulins.

[0157] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a TfR-binding domain that specifically binds to TfR with an affinity of approximately 900 nM to approximately 10,000 nM (for example, approximately 900 nM to approximately 2,500 nM or approximately 900 nM to approximately 1,300 nM), (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0158] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a TfR-binding domain that specifically binds to TfR with an affinity of approximately 900 nM to approximately 10,000 nM (for example, approximately 900 nM to approximately 2,500 nM or approximately 900 nM to approximately 1,300 nM), (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0159] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a TfR-binding domain that specifically binds to TfR with an affinity of approximately 900 nM to approximately 10,000 nM (for example, approximately 900 nM to approximately 2,500 nM or approximately 900 nM to approximately 1,300 nM), (b) A second Fc polypeptide comprising an FcγR binding region and a second Fc polypeptide not comprising a TfR binding region, The first Fc polypeptide contains one or more amino acid substitutions that reduce binding to FcγR. The second Fc polypeptide does not contain any modifications that reduce FcγR binding.

[0160] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0161] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0162] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0163] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0164] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0165] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0166] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has one or more amino acid substitutions that reduce binding to FcγR, (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0167] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), and has one or more amino acid substitutions that reduce binding to FcγR, (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0168] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has one or more amino acid substitutions that reduce binding to FcγR, (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0169] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), and has one or more amino acid substitutions that reduce binding to FcγR, (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0170] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has one or more amino acid substitutions that reduce binding to FcγR, (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0171] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), and has one or more amino acid substitutions that reduce binding to FcγR, (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0172] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has A at position 234 and A at position 235, (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0173] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0174] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has A at position 234, A at position 235, and G at position 329, (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0175] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, G at position 329, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide containing an FcγR binding region, comprising

[0176] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has A at position 234 and A at position 235, (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0177] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0178] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has A at position 234, A at position 235, and G at position 329, (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0179] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, G at position 329, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an FcγR binding domain and a second Fc polypeptide not comprising a TfR binding domain.

[0180] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has A at position 234 and A at position 235, (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0181] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0182] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and has A at position 234, A at position 235, and G at position 329, (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0183] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, G at position 329, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an FcγR binding region, which does not include a TfR binding region and does not include any modifications that reduce FcγR binding.

[0184] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1, and A at position 234, A at position 235, G optionally at position 329, and W at position 366, (b) A second Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-453 or 228-454 of SEQ ID NO: 9, wherein the second Fc polypeptide has S at position 366, A at position 368, and V at position 407 (according to EU numbering), comprises an FcγR binding region, and does not contain a TfR binding region.

[0185] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, optionally G at position 329, W at position 366, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (b) A second Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-453 or 228-454 of SEQ ID NO: 9, wherein the second Fc polypeptide has S at position 366, A at position 368, and V at position 407 (according to EU numbering), comprises an FcγR binding region, and does not contain a TfR binding region.

[0186] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence of amino acids 348-453 or amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, (b) A second Fc polypeptide comprising the amino acid sequence of amino acids 348-453 or amino acids 348-454 of SEQ ID NO: 9.

[0187] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising the amino acid sequence of amino acids 348-453 or amino acids 348-454 of SEQ ID NO: 8, (b) A second Fc polypeptide comprising the amino acid sequence of amino acids 348-453 or amino acids 348-454 of SEQ ID NO: 9.

[0188] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising the amino acid sequence of amino acids 348-453 or amino acids 348-454 of SEQ ID NO: 10, (b) A second Fc polypeptide comprising the amino acid sequence of amino acids 348-453 or amino acids 348-454 of SEQ ID NO: 9.

[0189] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising an amino acid sequence of amino acids 228-453 or amino acids 228-454 of any of SEQ ID NOs. 8, 10, and 1-31, (b) A second Fc polypeptide comprising the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 9.

[0190] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8, (b) A second Fc polypeptide comprising the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 9.

[0191] In some embodiments, the amyloid-beta targeting protein comprises an amyloid-beta binding region linked to an Fc dimer, where the Fc dimer is (a) A first Fc polypeptide comprising the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 10, (b) A second Fc polypeptide comprising the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 9.

[0192] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each contain a VL, the VL comprising VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. (b) The first heavy chain polypeptide is (i) VH comprising VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A first Fc polypeptide comprising a TfR-binding domain that specifically binds to TfR with an affinity of approximately 900 nM to approximately 10,000 nM (for example, approximately 900 nM to approximately 2,500 nM or approximately 900 nM to approximately 1,300 nM), (c) The second heavy chain polypeptide is (i) VH comprising VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A second Fc polypeptide containing an FcγR binding region, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0193] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each contain a VL, the VL containing an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-109 of SEQ ID NO: 7, VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. (b) The first heavy chain polypeptide is (i) A VH having an amino acid sequence that has at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with any of the amino acids 1 to 120 of SEQ ID NOs. 8, 10, and 17-31, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO. 1, VHCDR2 having the amino acid sequence of SEQ ID NO. 2, and VHCDR3 having the amino acid sequence of SEQ ID NO. 3, (ii) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with respect to amino acids 348-454 of any of SEQ ID NOs. 8, 10, and 17-31, wherein the first Fc polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to the EU numbering system) as shown in Table 1, (iii) Depending on the case, the CH3 domain polypeptide may contain one or more of the following: A at position 234, A at position 235, G at position 329, and W at position 366. (c) The second heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A second Fc polypeptide comprising an FcγR binding region, wherein the second Fc polypeptide may not include a TfR binding region, and the second Fc polypeptide may comprise an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228-453 or 228-454 of SEQ ID NO: 9, and the second Fc polypeptide may have S at position 366, A at position 368, and V at position 407 (according to EU numbering), The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0194] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each contain a VL, the VL containing an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-109 of SEQ ID NO: 7, VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. (b) The first heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (c) The second heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A second Fc polypeptide containing an FcγR binding region, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0195] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each contain a VL, the VL containing an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-109 of SEQ ID NO: 7, VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. (b) The first heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A first Fc polypeptide comprising a modified CH3 domain polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 348-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F (according to EU numbering) at position 421, and has one or more amino acid substitutions that reduce binding to FcγR, (c) The second heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A second Fc polypeptide comprising an FcγR binding region and a second Fc polypeptide that does not include a TfR binding region, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0196] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each contain a VL, the VL containing an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-109 of SEQ ID NO: 7, VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. (b) The first heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, optionally G at position 329, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (c) The second heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A second Fc polypeptide comprising an FcγR binding region and a second Fc polypeptide that does not include a TfR binding region, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0197] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each contain a VL, the VL containing an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1-109 of SEQ ID NO: 7, VLCDR1 having the amino acid sequence of SEQ ID NO: 4, VLCDR2 having the amino acid sequence of SEQ ID NO: 5, and VLCDR3 having the amino acid sequence of SEQ ID NO: 6. (b) The first heavy chain polypeptide is (i) A VH having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH having VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A first Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228-453 or amino acids 228-454 of SEQ ID NO: 8 or 10, wherein the first Fc polypeptide has A at position 234, A at position 235, optionally G at position 329, W at position 366, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, T at position 413, E at position 415, E at position 416, and F at position 421 (according to EU numbering), (c) The second heavy chain polypeptide is (i) A VH comprising an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 1 to 120 of SEQ ID NO: 8 or 10, the VH comprising a VHCDR1 having the amino acid sequence of SEQ ID NO: 1, a VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and a VHCDR3 having the amino acid sequence of SEQ ID NO: 3, (ii) A second Fc polypeptide comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 228 to 453 or amino acids 228 to 454 of SEQ ID NO: 9, the second Fc polypeptide having S at position 366, A at position 368, and V at position 407 (according to the EU numbering system), comprising an FcγR binding region and not comprising a TfR binding region, The first light chain polypeptide forms a dimer with the first heavy chain to form an amyloid beta binding domain, the second light chain polypeptide forms a dimer with the second heavy chain to form an amyloid beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0198] In some embodiments, the protein targeting amyloid beta comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each comprise the amino acid sequence of SEQ ID NO: 7, (b) The first heavy chain polypeptide comprises the amino acid sequence of amino acids 1 to 453 or amino acids 1 to 454 of any one of SEQ ID NO: 8, 10, and 17 to 31, (c) The second heavy chain polypeptide comprises the amino acid sequence of amino acids 1 to 453 or amino acids 1 to 454 of SEQ ID NO: 9, The first light chain polypeptide forms a dimer with the first heavy chain to form an amyloid beta binding domain, the second light chain polypeptide forms a dimer with the second heavy chain to form an amyloid beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0199] In some embodiments, the protein targeting amyloid beta comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, wherein (a) the first and second light chain polypeptides each comprise the amino acid sequence of SEQ ID NO: 7, (b) the first heavy chain polypeptide comprises the amino acid sequence of amino acids 1 to 453 or 1 to 454 of SEQ ID NO: 8, (c) the second heavy chain polypeptide comprises the amino acid sequence of amino acids 1 to 453 or 1 to 454 of SEQ ID NO: 9, The first light chain polypeptide forms a dimer with the first heavy chain to form an amyloid beta binding domain, the second light chain polypeptide forms a dimer with the second heavy chain to form an amyloid beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0200] In some embodiments, the protein targeting amyloid beta comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, wherein (a) the first and second light chain polypeptides each comprise the amino acid sequence of SEQ ID NO: 7, (b) the first heavy chain polypeptide comprises the amino acid sequence of amino acids 1 to 453 or 1 to 454 of SEQ ID NO: 10, (c) the second heavy chain polypeptide comprises the amino acid sequence of amino acids 1 to 453 or 1 to 454 of SEQ ID NO: 9, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0201] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each consist of the amino acid sequence of SEQ ID NO: 7, (b) The first heavy chain polypeptide consists of an amino acid sequence of amino acids 1-453 or amino acids 1-454 of any of SEQ ID NOs. 8, 10, and 17-31, (c) The second heavy chain polypeptide consists of the amino acid sequence of amino acids 1-453 or amino acids 1-454 of SEQ ID NO: 9, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0202] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each consist of the amino acid sequence of SEQ ID NO: 7, (b) The first heavy chain polypeptide consists of the amino acid sequence of amino acids 1-453 or amino acids 1-454 of SEQ ID NO: 8, (c) The second heavy chain polypeptide consists of the amino acid sequence of amino acids 1-453 or amino acids 1-454 of SEQ ID NO: 9, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0203] In some embodiments, the amyloid-beta targeting protein comprises a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide, and a second heavy chain polypeptide, where (a) The first and second light chain polypeptides each consist of the amino acid sequence of SEQ ID NO: 7, (b) The first heavy chain polypeptide consists of the amino acid sequence of amino acids 1-453 or amino acids 1-454 of SEQ ID NO: 10, (c) The second heavy chain polypeptide consists of the amino acid sequence of amino acids 1-453 or amino acids 1-454 of SEQ ID NO: 9, The first light chain polypeptide dimerizes with the first heavy chain to form an amyloid-beta binding domain, the second light chain polypeptide dimerizes with the second heavy chain to form an amyloid-beta binding domain, and the first and second Fc polypeptides form an Fc dimer.

[0204] In further embodiments, the amyloid-beta targeting protein may be fused with a peptide or protein useful in protein purification. Such peptides include, but are not limited to, polyhistidines, epitope tags (e.g., FLAG, c-Myc, and hemagglutinin tags), glutathione S-transferase (GST), thioredoxin, protein A, protein G, and maltose-binding proteins (MBPs). The peptide or protein to which the protein is fused may contain protease cleavage sites, such as cleavage sites for factor Xa or thrombin. In certain embodiments, the ligation is cleavable by enzymes present in the central nervous system.

[0205] III. Nucleic acids, vectors, and host cells The amyloid-beta targeting proteins described herein can be prepared using recombinant methods. Therefore, isolated nucleic acids containing sequences encoding any of the amyloid-beta targeting proteins or portions thereof described herein can be readily produced by methods available in the art. Host cells into which nucleic acids are introduced, which can be used to replicate nucleic acids encoding polypeptides and / or to express polypeptides, are also available in the art. The host cells can be, but are not limited to, prokaryotic or eukaryotic cells. Eukaryotes can be, but are not limited to, yeast cells, insect cells, or mammalian cells (e.g., human cells).

[0206] The nucleic acids encoding the amyloid-beta targeting protein or a portion thereof can be DNA, RNA, cDNA, mRNA, single-stranded, double-stranded, linear, or circular.

[0207] In some embodiments, the nucleic acid encoding a protein targeting amyloid beta comprises a nucleic acid sequence encoding a polypeptide having at least 85% identity, at least 90% identity, or at least 95% identity to any of SEQ ID NOs: 8, 10, and 17-31, wherein the encoded polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (according to EU numbering) as shown in Table 1. In some embodiments, the encoding polypeptide further comprises (a) alanine at position 234 and alanine at position 235, or (b) alanine at position 234, alanine at position 235, and glycine at position 329, or (c) alanine at position 234, alanine at position 235, glycine at position 329, and tryptophan at position 366.

[0208] In some embodiments, the nucleic acid encoding a protein targeting amyloid beta comprises a nucleic acid sequence encoding a polypeptide having at least 85% identity, at least 90% identity, or at least 95% identity to SEQ ID NO: 8 or 10, wherein the polypeptide encoded herein comprises (a) alanine at position 234, alanine at position 235, glycine at position 329, tryptophan at position 366, tyrosine at position 384, threonine at position 386, glutamic acid at position 387, and tryptophan at position 388. (b) containing valine at position 389, threonine at position 413, glutamic acid at position 415, glutamic acid at position 416, and phenylalanine at position 421, or (b) containing alanine at position 234, alanine at position 235, tryptophan at position 366, tyrosine at position 384, threonine at position 386, glutamic acid at position 387, tryptophan at position 388, valine at position 389, threonine at position 413, glutamic acid at position 415, glutamic acid at position 416, and phenylalanine at position 421 (according to EU numbering).

[0209] A protein targeting amyloid-beta may contain two or more (e.g., three) polypeptides, each of which may be encoded by a distinct nucleic acid sequence. These distinct nucleic acid sequences may reside in the same plasmid or vector, or in different plasmids or vectors. If present in the same plasmid or vector, the distinct nucleic acid sequences may be expressed from a single promoter or different promoters. Methods for expressing nucleic acids encoding distinct polypeptides from a single promoter are known in the art and, without limitation, include the use of 2A elements and internal ribosome entry sites.

[0210] A nucleic acid encoding a protein or part thereof that targets amyloid beta can be incorporated into a plasmid or vector. The plasmid or vector can be used to replicate the nucleic acid or to promote its expression. The plasmid or vector may be, but is not limited to, a viral vector, a phagemid, a yeast chromosome vector, or a non-episomal mammalian vector.

[0211] In some embodiments, nucleic acids encoding an amyloid-beta-targeting protein or a portion thereof are operably ligated to one or more regulatory sequences in an expression construct. The expression construct may correspond to the expression of a polypeptide in a system that produces an amyloid-beta-targeting protein. Such systems may be, but are not limited to, mammalian cell expression systems, insect cell expression systems, yeast cell expression systems, or bacterial cell expression systems.

[0212] Expression media for the production of recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the following types. Plasmids derived from pBR322, pEMBL, pEX, pBTac, and pETC for expression in prokaryotic cells such as E. coli. Vectors derived from pcDNAI / amp, pcDNAEneo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express recombinant polypeptides by using a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393, and pVL941), pAcUW (such as pAcUW1), and pBlueBac. Further expression systems include expression systems of adenovirus, adeno-associated virus, and other viruses.

[0213] An expression vector, or a plasmid or vector containing a nucleic acid, for expressing a protein or a part thereof targeting amyloid beta can be transformed, transfected, or transduced into a host cell. The host cell can be a mammalian cell, yeast cell, insect cell, prokaryotic cell, Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK) cell, NSO cell, YO cell, HEK293 cell, COS cell, Vero cell, or HeLa cell, but is not limited thereto. The host cell containing the expression vector can be cultured under appropriate conditions that enable the expression of the protein or a part thereof targeting amyloid beta.

[0214] Amyloid-beta targeting proteins can be produced by culturing host cells containing one or more nucleic acids encoding amyloid-beta targeting proteins, expressing the amyloid-beta targeting proteins, and isolating the expressed amyloid-beta targeting proteins from the culture. In some embodiments, the cells include (a) a first nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to any of SEQ ID NOs: 8, 10, or 17-31; (b) a second nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to SEQ ID NOs: 9; and (c) a third nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to SEQ ID NOs: 7.

[0215] IV. Formulations Any of the amyloid-beta targeting proteins of this disclosure may be prepared, provided, or formulated as a salt, mixed salt, or free acid. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts.

[0216] Any of the amyloid-beta targeting proteins of this disclosure may be provided or formulated in the form of a pharmaceutical composition. A pharmaceutical composition or drug comprises at least one pharmaceutically effective amount of an amyloid-beta targeting protein of this disclosure and, optionally, one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic agent (e.g., an amyloid-beta targeting protein)) that is intentionally included in the pharmaceutical composition. An excipient does not produce, or is not intended to produce, a therapeutic effect at the intended dose. An excipient may act to (a) assist in the processing of the API during manufacturing, (b) protect, assist, or improve the stability, bioavailability, or patient tolerability of the API, (c) assist in product identification, and / or (d) improve the overall safety, efficacy, or other properties of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0217] Examples of additives include, but are not limited to, absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, fluidizing agents, wetting agents, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, media, water repellents, and wetting agents.

[0218] The carrier may be, but is not limited to, a solvent or dispersion medium containing, for example, water, physiological saline, phosphate-buffered saline, Ringer's solution, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The carrier may also contain auxiliary agents or additives such as preservatives, wetting agents, emulsifiers, and dispersants. The carrier may also contain isotonic agents such as sugars, polyhydric alcohols, and sodium chloride.

[0219] The pharmaceutical composition may contain other further components commonly used in pharmaceutical compositions. Such further components may include, but are not limited to, antipruritic agents, astringents, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.).

[0220] Pharmacopoeia means that a property and / or substance is acceptable to a subject from a pharmacological / toxicological standpoint. The expression "pharmacopoeia" means that a molecule, composition, or property is physiologically acceptable to administration to a subject and does not typically produce allergic reactions or other harmful or toxic reactions. In some embodiments, a pharmacopoeia is approved by a federal or state regulatory authority, or is listed in the United States Pharmacopeia, or is listed in another generally recognized pharmacopoeia for use in animals, more specifically in humans.

[0221] In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. The additional active pharmaceutical ingredients may, but are not limited to, small molecule drugs.

[0222] A protein that targets amyloid beta or a pharmaceutical composition containing a protein that targets amyloid beta can be prepared as a liquid formulation or as a solid formulation (a powder or lyophilized formulation, for example, including a lyophilized cake or powder).

[0223] In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

[0224] As disclosed above, amyloid-beta targeting proteins or pharmaceutical compositions comprising amyloid-beta targeting proteins can be formulated for parenteral administration by injection (e.g., bolus injection or continuous infusion). For injection, amyloid-beta targeting proteins can be prepared into formulations by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent (e.g., vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol) along with commonly used additives as needed (e.g., solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives). In some embodiments, polypeptides can be formulated in aqueous solutions, preferably physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically compatible buffer solutions. The formulations for injection can be provided in unit dosage forms (e.g., ampoules or multi-dose containers) with added preservatives. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0225] Pharmaceutical compositions intended for in vivo administration are typically sterilized. Sterilization can be carried out by methods known in the art (e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation).

[0226] The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is well within the scope of the art of those skilled in the art.

[0227] A. Kit In some embodiments, a kit comprising amyloid-beta targeting proteins described herein is provided. In some embodiments, the kit is intended for use in the prevention or treatment of amyloid-beta-related neurological disorders.

[0228] The amyloid-beta targeting proteins and pharmaceutical compositions comprising amyloid-beta targeting proteins disclosed herein may be packaged in or included in kits, containers, packs, or dispensers. The amyloid-beta targeting proteins and pharmaceutical compositions comprising amyloid-beta targeting proteins may be filled in pre-filled syringes or vials. Any of the amyloid-beta targeting proteins or pharmaceutical compositions comprising amyloid-beta targeting proteins described herein may be formulated or packaged in single-dose or multi-dose form. Any of the amyloid-beta targeting proteins or pharmaceutical compositions comprising amyloid-beta targeting proteins identified herein may be formulated for repeated dosing.

[0229] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a transferrin receptor-binding polypeptide described herein and further comprises one or more additional therapeutic agents for the treatment of neurological disorders.

[0230] The kit or package may further include instructions for use. The instructions include documentation describing the relevant substances or methods relating to the kit. The instructions may include one or more of the following: background information, a list of ingredients and information on how to obtain them (e.g., purchase information), a simple or detailed protocol for using the kit, troubleshooting guidance, references, technical support, indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the drug, and any other relevant documentation. The instructions may be attached to the kit or provided as a separate component, either in paper or electronic form. The instructions may include notices in the format prescribed by the government agency that has jurisdiction over the manufacture, use, or sale of the drug or biological product, and such notices may reflect the government agency's approval of the manufacture, use, or sale for human administration.

[0231] V. Treatment The amyloid-beta-targeting proteins of this disclosure can be used to deliver compounds having amyloid-beta binding ability and effector function into the target brain, i.e., across the blood-brain barrier.

[0232] Amyloid-beta-targeted proteins of this disclosure can be administered to a subject to treat diseases or illnesses at least partially mediated by amyloid-beta or amyloid-beta plaques. Following administration to a subject, the amyloid-beta-targeted proteins of this disclosure cross the BBB to the brain, where they bind to amyloid plaques, vascular amyloid-beta, or diffuse amyloid-beta deposits. The binding of amyloid-beta-targeted proteins to amyloid plaques, vascular amyloid-beta, or diffuse amyloid-beta can facilitate immune responses, such as ADCP, against amyloid plaques, vascular amyloid-beta, or diffuse amyloid-beta.

[0233] Disclosed are methods for treating subjects suffering from a disease or illness at least partially mediated by amyloid beta or amyloid beta plaques, the methods comprising administering to the subject a protein that targets amyloid beta or a composition comprising a protein that targets amyloid beta. In some embodiments, the disease or illness at least partially mediated by amyloid beta or amyloid beta plaques includes neurodegenerative diseases or neurodegenerative conditions. Neurodegenerative diseases may be, but are not limited to, Alzheimer's disease.

[0234] Disclosed is a method for reducing amyloid plaques in the brain of a subject, the method comprising administering to the subject a protein that targets amyloid beta or a composition comprising a protein that targets amyloid beta. Reducing amyloid plaques may include increasing the phagocytosis of amyloid beta in the subject and / or increasing the recruitment of microglia to amyloid beta-positive plaques. In some embodiments, the subject has, has been diagnosed with, or is at high risk of developing a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0235] Disclosed is a method for reducing amyloid plaques in the brain of a subject, the method comprising administering to the subject a protein that targets amyloid beta or a composition comprising a protein that targets amyloid beta. In some embodiments, the subject has a neurodegenerative disease, has been diagnosed with a neurodegenerative disease, or is at high risk of developing a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0236] Disclosed are methods for treating cognitive impairment, memory loss, and / or dementia in subjects, the methods comprising administering to the subjects a protein that targets amyloid-beta or a composition comprising a protein that targets amyloid-beta. In some embodiments, the subjects have a neurodegenerative disease, have been diagnosed with a neurodegenerative disease, or are at high risk of developing a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0237] Disclosed are methods for treating the loss of neural connectivity in the brain of a subject, the method comprising administering to the subject a protein that targets amyloid-beta or a composition comprising a protein that targets amyloid-beta. In some embodiments, the subject has a neurodegenerative disease, has been diagnosed with a neurodegenerative disease, or is at high risk of developing a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0238] Disclosed are methods for delaying or preventing one or more symptoms or conditions at least partially related to the accumulation of amyloid plaques in the brain of a subject, the methods comprising administering to the subject a protein that targets amyloid beta or a composition comprising a protein that targets amyloid beta. In some embodiments, the subject has a neurodegenerative disease, has been diagnosed with a neurodegenerative disease, or is at high risk of developing a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0239] Disclosed are methods for reducing amyloid-related imaging abnormalities (ARIA) in subjects, the methods comprising administering to the subjects a protein that targets amyloid-beta or a composition comprising a protein that targets amyloid-beta. In some embodiments, the subjects have, have been diagnosed with, or are at high risk of developing a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the subjects need to receive or are scheduled to receive treatment for a neurodegenerative disease (e.g., Alzheimer's disease). In some embodiments, the ARIA is related to the treatment of a neurodegenerative disease (e.g., Alzheimer's disease). In some embodiments, the therapeutic agent for the neurodegenerative disease (e.g., Alzheimer's disease) is a therapeutic antibody.

[0240] In some embodiments, ARIA includes two types of MRI signal abnormalities: ARIA-E (edema / exudation) and ARIA-H (hemosiderin deposition / microbleeds). ARIA-E refers to extravasation of proteinaceous fluid resulting in interstitial vascular edema or groove exudation in the leptomeningeal / subpia space. These appear as high-intensity parenchymal or groove abnormalities in T2-weighted images and fluid attenuation inversion recovery (FLAIR) sequence images. ARIA-H refers to microbleeds (mH) or major hemorrhages observed as low-intensity hemosiderin deposition. These reflect iron accumulation after the breakdown of extravasated hemoglobin in gradient recall echo (GRE) / T2* images or through susceptibility-weighted imaging (SWI) sequence enhancement visualization. In some embodiments, the method reduces the incidence of ARIA-E events. In some embodiments, the method reduces the incidence of ARIA-H events. In other embodiments, the method reduces the incidence of both ARIA-E and ARIA-H events.

[0241] Proteins that target amyloid-beta can be administered parenterally, intravenously, intrathecally, epidurally, or intraventricularly. Administration can be by bolus injection, infusion, or a combination thereof. Infusions can be performed, for example, over a period of about 10 to 30 minutes, or over a period of at least 1 to 4 hours or longer.

[0242] Proteins that target amyloid-beta can be administered in combination with one or more additional therapeutic agents. These additional therapeutic agents may include, but are not limited to, drugs useful in treating Alzheimer's disease.

[0243] A. Alzheimer's disease Alzheimer's disease (AD) is a type of dementia identified primarily by clinical diagnosis and established by disease markers. AD is a continuum in which disease progression is divided into specific, operationally defined stages. The pathology of AD begins before the onset of clinical symptoms. Amyloid plaques, one marker of AD pathology, can occur 10 to 20 years before the onset of AD-associated dementia. Currently recognized stages of AD include pre-symptomatic, prodromal, mild, moderate, and severe, although these stages are neither discrete nor clearly distinguishable from one another. However, the stages and progression of AD can be monitored by measuring or evaluating amyloid-beta accumulation (CSF / PET), synaptic dysfunction (FDG-PET / fMRI), tau-mediated neuronal damage (CSF), brain structure (volumetric MRI), cognition, and clinical function. Systems for diagnosing and monitoring Alzheimer's disease (AD) include, but are not limited to, the new survey criteria for the diagnosis of AD by the International Working Group (IWG) (Dubois B et al. Lancet Neurol 2007;6(8):734-736), the IWG survey criteria (Dubois et al. Lancet Neurol 2010;9(11):1118-27), the NIA / AA criteria (Jack CR et al. Alzheimer's Dement 2011;7(3):257-62), and the DSM-5 criteria (American Psychiatric Association, DSM-5, 2013).

[0244] The amyloid-beta targeting proteins of this disclosure can be administered to a subject to treat an amyloid-beta-related disease or illness, or to reduce amyloid-beta plaques in the subject's brain. In some embodiments, treating an amyloid-beta-related disease or illness includes (a) alleviating or improving an amyloid-beta-related disease, (b) alleviating or improving one or more symptoms or conditions associated with an amyloid-beta-related disease, (c) reducing amyloid-beta plaques in the subject's brain, (d) preventing or delaying at least one symptom or condition in a subject who has or is at risk of developing an amyloid-beta-related disease, or (e) extending the survival time of a subject diagnosed with an amyloid-beta-related disease. In some embodiments, treating an amyloid-beta-related disease or illness includes reducing amyloid-related imaging abnormalities (AIRAs) in the subject. In some embodiments, the amyloid-beta-related disease or illness (amyloid-beta-related disease) is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease (AD). Conditions associated with amyloid-beta-related diseases include, but are not limited to, cognitive impairment, memory loss, dementia, or loss of neural connections in the brain.

[0245] Disclosed is a method for treating a subject (e.g., a human subject) who has a condition at least partially mediated by amyloid-beta or is at risk of developing a condition at least partially mediated by amyloid-beta, the method comprising administering an effective amount (e.g., a therapeutic effective amount or a prophylactic effective amount) of an amyloid-beta-targeting protein or a composition comprising an amyloid-beta-targeting protein to the subject. The administration of an amyloid-beta-targeting protein or a composition comprising an amyloid-beta-targeting protein to the subject may be combined with one or more steps of administering one or more additional (i.e., second, third, etc.) therapeutic or therapeutic agents.

[0246] In some embodiments, the method further comprises administering one or more additional therapeutic agents to a target. For example, in some embodiments for treating diseases of the brain or central nervous system, the method may include administering neuroprotective agents (e.g., anticholinergics, dopamine agonists, glutamatergics, histone deacetylase (HDAC) inhibitors, cannabinoids, caspase inhibitors, melatonin, anti-inflammatory agents, hormones (e.g., estrogen or progesterone), or vitamins) to a target. In some embodiments, the method may include administering agents used to treat cognitive or behavioral symptoms of neurological disorders (e.g., antidepressants, dopamine agonists, or antipsychotics) to a target. VI.Array [Table B-1] [Table B-2] [Table B-3] [Table B-4] [Table B-5] [Examples]

[0247] Examples are provided below to illustrate specific embodiments of the Disclosure. As will be apparent to those skilled in the art, the methods disclosed in the following examples are representative of methods that function well in the implementation of the Disclosure and can therefore be considered to constitute specific forms of implementation. However, as will be apparent to those skilled in the art in light of the Disclosure, many modifications can be made to the specific embodiments disclosed, and such modifications can still be made to obtain similar results without departing from the spirit and scope of the Disclosure.

[0248] Example 1. Proteins that target amyloid beta bind to oligomeric amyloid beta and fibrillary amyloid beta, but do not bind to monomeric amyloid beta. To prepare monomeric amyloid-beta peptides, 1-40 The powder (Anaspec, AS-24235) was reconstituted in hexafluoroisopropanol (HFIP) at a concentration of 1 mg / mL. Amyloid beta 1-40 Dispense into 50 μL / vial, dry each aliquot under N2 gas, and inflate in HFIP at -80°C with amyloid beta 1-40 It was saved as a film. In order to form monomers, amyloid beta 1-40 The HFIP film was dissolved in dimethyl sulfoxide (DMSO) and then diluted to the target concentration in assay buffer before use.

[0249] Oligomer amyloid beta 1-42 and fibrous amyloid beta 1-42 To prepare, amyloid beta in HFIP 1-42 The film (Anaspec, AS-64129) was resuspended in DMSO at a concentration of 5 mg / mL, and then diluted in PBS at a concentration of 1 mg / mL. Oligomer amyloid beta 1-42 To form amyloid beta 1-42 The sample was incubated at 37°C for 3 days. Fibrous amyloid beta 1-42 To form amyloid beta 1-42 The sample was incubated at 37°C for 7 days, and then centrifuged at 14,000 × g for 15 minutes at 4°C. Fibrous amyloid beta 1-42 The amyloid-beta was dissolved in PBS. Dynamic light scattering (DLS) was used to verify the quality of the amyloid-beta preparation.

[0250] The binding specificity of ATVs to amyloid beta was measured by ELISA (IBL, 27725). The following four different ATVs were analyzed. 1. ATV35.23.4 cisLALA: It has an anti-amyloid beta IgG amyloid beta Fab binding domain and an Fc dimer (ATV35.23.4 cisLALA (Also called amyloid beta), the Fc dimer comprises a first Fc polypeptide having a TfR binding region (affinity for TfR of approximately 600 nM) and substitutions of L324A and L235A that reduce FcγR binding. 2.ATV35.23.4 cisLALAPG : It has an anti-amyloid beta IgG amyloid beta Fab binding domain and an Fc dimer (ATV35.23.4 cisLALAPG (Also called amyloid beta), the Fc dimer comprises a first Fc polypeptide having a TfR binding domain (affinity for TfR of approximately 600 nM) and substitutions of L324A, L235A, and P329G that reduce FcγR binding. 3.ATV35.23.3 cisLALA (Amyloid-beta targeting proteins): These proteins have an anti-amyloid-beta IgG amyloid-beta Fab binding domain and an Fc dimer (ATV35.23.3 cisLALA (Also called amyloid beta), the Fc dimer comprises a first Fc polypeptide having a TfR binding domain (affinity for TfR of approximately 1100 nM) and substitutions of L324A and L235A that reduce FcγR binding. 4.ATV35.23.3 cisLALAPG (Amyloid-beta targeting proteins): These proteins have an anti-amyloid-beta IgG amyloid-beta Fab binding domain and an Fc dimer (ATV35.23.3 cisLALAPG (Also called amyloid beta), the Fc dimer comprises a first Fc polypeptide having a TfR binding domain (affinity for TfR of approximately 600 nM) and substitutions of L324A, L235A, and P329G that reduce FcγR binding.

[0251] The ATV had the configuration shown in Figure 3A (left diagram).

[0252] Anti-amyloid-beta IgG antibody was used as a control. Proteins targeting amyloid-beta contained the antibody-binding domain of the anti-amyloid-beta IgG antibody. 20 μL of 0.4 nM antibody was mixed with 100 μL of monomeric amyloid-beta, oligomeric amyloid-beta, or fibrillary amyloid-beta and incubated at 4°C for 60 minutes. 100 μL of each solution was added to pre-coated plates and incubated at 4°C for 60 minutes. After washing three times with PBS containing 0.05% Tween®-20 (PBST), each well was treated with 100 μL of horseradish peroxidase-conjugated goat anti-human antibody at room temperature for 1 hour. After washing three times with PBST, 100 μL of TMB (chromogen) was added to each well, followed by 100 μL of stop solution. ELISA signals were measured using a microplate luminometer (BioTek Neo2). The results (Figure 1) show that all the anti-amyloid beta antibodies tested were oligomeric amyloid beta antibodies. 1-42 and fibrous amyloid beta 1-42 It binds to monomer amyloid beta to an equal degree of specificity. 1-40 It was found that it did not bind to it.

[0253] Example 2. ATV35.23.3 with weaker affinity cisLALA Amyloid-beta molecules are found in ATV35.23.4 cisLALA : Higher exposure to the brain than amyloid-beta molecules TfR mu / hu KI mice were administered an intravenous dose of 25 mg / kg of anti-amyloid beta or amyloid beta-targeting protein (ATV35.23.3 and ATV35.23.4) (n=4-5 / group). Blood was collected at 30 minutes post-administration, and terminal blood and fresh brain (after perfusion) samples were flash-frozen on days 1, 2, 4, and 7 post-administration to determine human IgG concentrations in plasma and brain lysates. Whole blood was collected on days 1, 2, and 4 for reticulocyte count.

[0254] Plasma PK profiles show affinity-dependent TfR-mediated clearance, and consequently, TfR mu / huIn KI mice, a single IV dose of anti-amyloid beta or ATV:amyloid beta molecules resulted in faster clearance of ATV molecules from plasma (Figure 2A). Brain PK profiles showed approximately four times higher brain concentrations for ATV:amyloid beta molecules at 1 day post-administration, and significantly higher concentrations at 2 and 4 days post-administration (ATV35.23.3 only) (Figure 2B) (two-way ANOVA, Dunnett's post-hoc test). Surprisingly, ATV:amyloid beta molecules with weaker TfR affinity (ATV35.23.3) were also affected. cisLALA Amyloid beta (ATV35.23.4) is a variant with stronger TfR affinity. cisLALA It showed higher Cmax brain concentrations than amyloid-beta. This is inconsistent with the expected relationship between TfR affinity and uptake into the brain. Circulating reticulocytes in the blood of ATV-treated mice did not differ from the anti-amyloid-beta group at any time point (Figure 2C) (one-way ANOVA, Dunnett's post-hoc test). This suggests that cisLALA prevents reticulocyte loss due to effector function.

[0255] Furthermore, to demonstrate the effects of LALA mutations, TfR mu / hu KI mice were administered intravenously with a single dose of 10 mg / kg of anti-amyloid beta or ATV:amyloid beta molecule (n=5 / group).

[0256] In this experiment, the following three proteins that target amyloid beta were compared with anti-amyloid beta IgG antibodies. 1. ATV35.23.3:Anti-amyloid beta IgG has an amyloid beta Fab binding domain and an Fc dimer (also called ATV35.23.3:amyloid beta), and the Fc dimer contains a first Fc polypeptide having a TfR binding region (affinity for TfR of approximately 1100 nM). 2.ATV35.23.3 cisLALA (Amyloid-beta targeting proteins): These proteins have an anti-amyloid-beta IgG amyloid-beta Fab binding domain and an Fc dimer (ATV35.23.3 cisLALA(Also called amyloid beta), the Fc dimer comprises a first Fc polypeptide having a TfR binding domain (affinity for TfR of approximately 1100 nM) and substitutions of L324A and L235A that reduce FcγR binding. 3.ATV35.23.3 LALA (Amyloid-beta targeting proteins): These proteins have an anti-amyloid-beta IgG amyloid-beta Fab binding domain and an Fc dimer (ATV35.23.3 LALA (Also called amyloid beta), the Fc dimer comprises a first Fc polypeptide having a TfR binding region (affinity for TfR of approximately 1100 nM) and substitutions of L324A and L235A that reduce FcγR binding, and a second Fc polypeptide having substitutions of L324A and L235A that reduce FcγR binding.

[0257] Terminal blood and fresh brain (after perfusion) were collected 24 hours after administration and rapidly frozen to determine human IgG concentrations in plasma and brain lysates. Fresh bone marrow was collected from the femur, stained for Ter119 (erythrocyte lineage) and CD44, and the reticulocyte population was identified using flow cytometry. Terminal whole blood was also collected to measure reticulocytes.

[0258] As expected, terminal plasma revealed that all ATVs were similarly exposed to ATVs compared to anti-amyloid beta, with lower human IgG concentrations (Figure 2D), while brain samples showed higher ATV concentrations compared to anti-amyloid beta (Figure 2E). Compared to anti-amyloid beta, serum reticulocytes showed a clear decrease after treatment with effector-positive ATV35.23.3:amyloid beta with a wild-type (WT) Fc region, although a single cisLALA mutation (ATV35.23.3) was detected. cisLALA : Amyloid beta) or two LALA mutations (ATV: 35.23.3 LALAAmyloid-beta completely prevented the reduction of reticulocytes (Figure 2F). Similarly, bone marrow reticulocytes showed cytopenia only against effector-positive ATV35.23.3:amyloid-beta with wild-type (WT) Fc (Figure 2G). This suggests that its structure enables TfR-mediated reticulocyte cell death, while mutations in cisLALA and LALA appear to completely inhibit it.

[0259] Example 3. Comparison of brain uptake of ATV and C-terminal TfR Fab forms and reticulocytes in the blood. TfR mu / hu KI mice were intravenously administered a single dose of 10 mg / kg of anti-amyloid beta, ATV:amyloid beta (left panel in Figure 3A), or an anti-amyloid beta TfR Fab fusion molecule (right panel in Figure 3A) (n=5 / group). The molecule shown in the right panel was a fusion of anti-amyloid beta IgG and a Fab fragment that binds to wild-type (WT) Fc and TfR. The affinity of the two molecules (left and right panels) for TfR was similar. Terminal blood and fresh brain (after perfusion) were collected 24 hours after administration and rapidly frozen to determine the number of circulating reticular erythrocytes in whole blood and the concentration of human IgG in the brain. The molecules targeting TfR showed similar brain concentrations, as expected given their matching TfR affinity (Figure 3B). However, circulating reticular cells showed a significant decrease in anti-amyloid beta TfR Fab fusion molecules (one-way ANOVA, Dunnett's post-hoc test), but no decrease in ATV:amyloid beta (Figure 3C). This suggests that anti-amyloid beta TfR Fab (TfR C末端Fab Compared to amyloid-beta fusion molecules, cisLALA is suggested to better prevent the reduction of reticulocytes due to effector function and to possess high hemoprotective properties.

[0260] Example 4. App SAA KI TfR mu / hu PK profile and microglia mobilization to plaques in KI mice ATV35.23.3 cisLALA Amyloid beta is ATV35.23.4cisLALA Compared to amyloid beta, after a single dose, brain exposure improved, immunohistochemical staining of plaques increased, and plaques decreased.

[0261] App SAA KI;TfR mu / hu KI mice were administered IV with a single dose of 10 mg / kg of anti-amyloid beta or ATV:amyloid beta molecules (n=4-5 / group). Terminal blood and fresh brain (after perfusion) were collected and flash-frozen on days 2, 7, and 14 post-administration to determine human IgG concentrations in plasma and brain lysates. One fresh cerebral hemisphere per animal was fixed by immersion at 4°C for approximately 24 hours for immunohistochemistry. Three to four sagittal brain sections (40 μm) were selected from each animal, incubated in blocking buffer, and stained by incubation overnight at 4°C in primary / secondary antibodies (CD68, Biorad, MCA1957, amyloid beta, IBL America 18584, and donkey anti-human IgG, Jackson, 709-606-149). Next, the slides were washed and incubated with secondary antibodies (donkey anti-rat, Invitrogen, SA5-10027; donkey anti-rabbit, Invitrogen, A21206) and DAPI (5 μg / mL, Invitrogen, D1306). Afterward, they were washed and mounted on coverslips using Prolong Glass (Invitrogen, P36984). Slides were imaged at 20x magnification using a Zeiss Axioscan.Z1 slide scanner and processed using custom macros in Zeiss ZEN software to generate binary masks using dynamic thresholds for each target channel (amyloid beta, human IgG, CD68, and tissue regions using DAPI). Data analysis was performed using Microsoft Excel and GraphPad Prism 9.

[0262] TfR mu / hu Similar to the results in KI mice, App SAA KI TfR mu / huPlasma PK profiles in KI mice showed TfR-mediated clearance after a single IV dose of anti-amyloid beta or ATV:amyloid beta molecules (Figure 4A). Brain concentrations at 24 hours were approximately 4-5 times higher with ATV compared to anti-amyloid beta (Figure 4B). mu / hu As seen in KI mice, ATV35.23.3 cisLALA Amyloid beta is ATV35.23.4 cisLALA Compared to amyloid-beta, ATV resulted in higher brain concentrations (Figure 4B). Furthermore, both ATV and anti-amyloid-beta showed localization of human IgG signaling to plaques, and immunohistochemistry correlated well with overall brain exposure, showing higher concentrations for ATV 48 hours post-administration. Additionally, plaque staining tended to be higher with the less affinity ATV:amyloid-beta compared to the more affinity ATV:amyloid-beta (Figure 4C). This was consistent with brain ELISA data but unexpected given the established TfR affinity-brain concentration relationship. ATV increased the overlap of CD68 signaling within and around plaques by approximately twofold in untreated animals, demonstrating its effectiveness in recruiting CD68-positive microglia to plaques (Figure 4D). Microglia recruitment to plaques plays a crucial role for amyloid-beta therapeutics, enabling phagocytosis by the plaques. Functionally, this reduces amyloid-beta plaques from the brain. In fact, ATV35.23.3 cisLALA Amyloid beta was the most effective at reducing plaques, resulting in a reduction of approximately 34% two days after administration and a 49% reduction one week after administration, which was consistent with the highest brain exposure levels (Figure 4E).

[0263] Example 5. PK profile in wild-type (WT) C57BL / 6J mice In wild-type (WT) C57BL / 6J KI mice, a single dose of 10 mg / kg of ATV (amyloid beta molecule (ATV35.23.4) was administered. cisLALA : Amyloid beta, ATV35.23.3 cisLALA : Amyloid beta, ATV35.23.4 cisLALAPG : Amyloid beta, and ATV35.23.3 cisLALAPGHuman IgG (ATV) was administered intravenously (n=3 / group / time point). Human IgG concentrations in plasma were determined from living or terminal blood samples collected at multiple time points. Both ATV and amyloid-beta molecules showed similar clearance and PK profiles in wild-type mice. Here, these molecules lack TfR binding to mouse TfRs. This suggests that these molecules do not exhibit nonspecific binding when binding to the target is absent. The cisLALA and cisLALAPG molecules acted similarly in wild-type mice (Table 5A and Figure 5). [Table 5A]

[0264] Example 6. Ex vivo microglial phagocytosis was observed, and ATV35.23.3 was found to be effective against amyloid beta. cisLALA It has been revealed that amyloid-beta has an equivalent ability to promote the phagocytosis of amyloid-beta. To generate FAM (fluorescein)-labeled amyloid-beta fibrils, FAM-labeled β-amyloid (1-42) (0.5 mg, Anaspec AS-23525-05) was resuspended in 100 μL of DMSO and then diluted to 100 μM in 1 ml of PBS. The solution was incubated at 37°C for 24 hours with shaking. The FAM amyloid-beta fibrils were then transferred to a 1.5 mL ultracentrifuge tube and centrifuged at 100,000 × g for 30 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of PBS, followed by thorough pipetting. The ultracentrifugation process was repeated, followed by two further washes. Finally, the pellet was resuspended in 111 μL of PBS and stored in a freezer at -80°C.

[0265] The properties of amyloid-beta aggregates were confirmed by dynamic light scattering (DLS). Briefly, 30 μL of 1 mg / mL amyloid-beta aggregates was placed in a 384-well plate (black with a transparent bottom, Costar). A plate cover was used to protect the bottom of the plate from debris and scratches that could affect DLS readings. Next, the plate was sealed with film and centrifuged at 1000 rpm for 5 minutes to settle the solution to the bottom of the plate and remove air bubbles. Subsequently, measurements were performed using Dynamics v7 software at 25°C with a DynaPro Plate Reader III (Wyatt Technology). For the measurements, readings were taken for 5 seconds per well, and 10 measurements were averaged. From the DLS results, it was revealed that the generated FAM-amyloid-beta 1-42 fibers consisted of a heterogeneous mixture of monomers, oligomers, and fibers (Figure 6A and Table 6A). [Table 6A]

[0266] To perform an exobibomicroglia phagocytic experiment, naive TfR mu / huKI (3 months old, male) were perfused with 1×PBS, and their brains were subjected to single-cell isolation. Adult brain isolation kit (Miltenyi Biotec, 130-107-677) was used with gentleMACS® Octo Dissociator, and cells were isolated according to the kit's instructions. Excess debris and myelin were removed using the kit's Debris Removal solution, and the final cell pellet was resuspended in 200 μL of 0.5% BSA in dPBS (containing calcium and magnesium). Next, a small portion of cells from each sample were stained with Cd11b-BV421 (BioLegend 101251, 1:100), CD45-APC (BD Biosciences Cat 559864, 1:100), and Fc block (BioLegend 101320, 1:100) at 4°C for 15 minutes. Subsequently, the cells were washed and resuspended in FACS buffer (1% BSA + 1 mM EDTA in PBS) containing propidium iodide (PI) (Miltenyi Biotec, 130-093-233) to quantify the number of viable microglia per sample. Then, the cell fraction was mixed with CountBright Plus Absolute Counting Beads (Invitrogen, RefC36995) and placed in a BD FACSAria III sorter to quantify the total number of viable microglia per sample. Cells from the same animal were distributed to treatment groups so that each group contained 50,000 viable microglia, and treated with 10 nM IgG control, anti-amyloid beta, and ATV along with 100 nM FAM-labeled amyloid-beta fibrils. cisLALA : Amyloid beta, ATV: Amyloid beta, or ATV LALAThe samples were treated with amyloid-beta and incubated at 37°C for 45 minutes (Figure 6B). Control samples were treated with 10 nM of each antibody and 100 nM of FAM-amyloid-beta fibrils, but incubated at 4°C for 30 minutes instead. After these incubations, the treated samples were washed and stained with Cd11b-BV421 (BioLegend 101251, 1:100), CD45-APC (BD Pharmingen, 1:100), and Fc block (BioLegend 101320, 1:100) for 15 minutes at 4°C. The samples were then washed and resuspended in FACS buffer containing PI. In a sorter, 10,000 viable microglia per treated sample were measured along with the intensity of the FAM signal. To quantify the intensity of the FAM signal per viable microglial cell, analysis was performed using FlowJo 10.8.1.

[0267] The results revealed a significant increase in the proportion of microglia treated with anti-amyloid-beta antibody compared to control IgG (Figure 6C). Interestingly, ATV cisLALA The ability of amyloid-beta to promote phagocytosis of amyloid-beta was comparable to that of both anti-amyloid-beta and ATV:amyloid-beta (Figure 6C) (one-way ANOVA, correspondence analysis). In contrast, ATV with effector-reducing LALA mutations in both Fc chains was comparable. LALA Amyloid beta is ATV cisLALA Compared to both ATV:amyloid-beta molecules and ATV:amyloid-beta molecules, microglial phagocytosis was significantly reduced (Figure 6C) (one-way ANOVA, correspondence analysis). The mean fluorescence intensity of the FAM-amyloid-beta signal per viable microglia was higher than that of ATV. cisLALA Amyloid beta: This suggests that amyloid beta promotes the uptake of amyloid beta into microglia at a similar level compared to anti-amyloid beta and ATV: amyloid beta, while ATV has no effectors at all. LALA : The amount of amyloid beta phagocytosed was significantly reduced (Figure 6D). These results suggest that cisLALA mutations are associated with ATV cisLALA: It has been clarified that it does not reduce the ability to induce microglial phagocytosis of amyloid beta.

[0268] Example 7. APP SAA KI; TfR mu / hu Single-dose immunostaining and microglial mobilization in KI mice reveal the efficacy of cisLALA molecules APP SAA KI; TfR mu / hu KI mice were given a single dose of 3 mg / kg of control IgG, 3 mg / kg of ATV: amyloid beta, 2 mg / kg of ATV cisLALA : amyloid beta, 3 mg / kg of ATV LALA : amyloid beta, or 7 mg / kg of anti-amyloid beta were administered IV (n = 8 / group). At 7 days after administration, fresh brains were collected after perfusion. These dose levels achieved approximately equal brain concentrations of anti-amyloid beta and ATV: amyloid beta at the final time point. Furthermore, one perfused fresh brain hemisphere per animal was immersion-fixed for immunohistochemistry and image quantification as described above.

[0269] As a result, as expected, it was revealed that the in-brain distribution of human IgG per plaque area was equivalent as shown by immunostaining analysis (Figure 7A). Interestingly, despite having one copy of the LALA mutation, ATV cisLALA : amyloid beta was able to mobilize activated microglia to plaques, similar to both ATV: amyloid beta and anti-amyloid beta. On the other hand, ATV LALA : amyloid beta with two copies of the LALA mutation showed a decrease in ability (Figure 7B). The ability of each molecule to mobilize microglia to amyloid beta plaques of different sizes (30 - 125, 125 - 250, 250 - 500, and >500 μm 2 ) was also analyzed. The results showed that, similar to both anti-amyloid beta and ATV: amyloid beta, ATV cisLALAThis study revealed that amyloid beta has a consistent ability to recruit activated microglia to amyloid beta plaques of various sizes. By evaluating the total plaque area between groups, ATV cisLALA Amyloid-beta significantly reduced plaque area, similar to both anti-amyloid-beta and ATV:amyloid-beta (Figures 7C-D). This was evident even when analyzing results using amyloid-beta plaques of varying numbers and sizes. In summary, these results suggest that ATV... cisLALA It has been shown that amyloid beta retains its ability to mobilize microglia into plaques and maintains its ability to reduce plaques at a similar level to anti-amyloid beta. In contrast, ATV LALA Amyloid-beta is less effective than anti-amyloid-beta in terms of microglia recruitment and plaque reduction.

[0270] Example 8. cisLALA reduces TFR-mediated in vivo hematological disorders in non-human primates (NHPs). Cynomolgus monkeys were given 15 mg / kg or 50 mg / kg doses of anti-amyloid beta or ATV:amyloid beta molecules intravenously on days 1, 15, and 29 (e.g., every two weeks). Blood samples were collected for testing one week prior to administration, and on days 8, 15 (before the dose on day 15), 29 (before the dose on day 29), and 31.

[0271] Consistent with previous data, a rapid dose-dependent and TfR affinity-dependent decrease in circulating reticular cells was observed (Figure 8A). After 4 weeks of exposure, the effects on circulating RBCs and hemoglobin were minimal, and no differences were observed between cisLALA and cisLALAPG ATV:amyloid beta molecules (Figures 8B-C). These data suggest that multiple doses of ATV have a favorable safety profile in NHP (non-human primates).

[0272] Example 9. Single-dose ATV: PK of amyloid beta in cynomolgus monkeys Cynomolgus monkeys were administered a single dose of 6 mg / kg of anti-amyloid beta or ATV: amyloid beta molecule by intravenous injection. Blood was collected at various time points for measurement of human IgG concentration. ATV35.23.3 cisLALA : Amyloid beta and ATV35.23.3 cisLALAPG : The area under the curve (AUC) and clearance of amyloid beta were similar to those in the anti-amyloid beta control group in non-human primates (Figure 9). ATV35.23.4 cisLALA : Amyloid beta and ATV35.23.4 cisLALAPG : The clearance of amyloid beta was slightly faster than that of anti-amyloid beta.

[0273] Example 10.5XFAD; PK profile of ATV with cisLALA or cisLALAPG mutations and microglial recruitment to plaques in TfRmu / huKI mice 5XFAD;TfR mu / hu KI mice were administered four doses (q3d, i.e., days 0, 3, 6, 9) of ATV35.23.4 cisLALA 、ATV35.23.4 cisLALAPGAlternatively, control IgG was administered intraperitoneally (IP) (n=15 / group). Live plasma was collected from n=3 mice per group at each time point. Terminal blood and fresh brain (after perfusion) were collected from all animals 12 days post-administration and rapidly frozen to determine human IgG concentrations in plasma and brain lysates. One fresh cerebral hemisphere per animal was fixed by immersion at 4°C for approximately 24 hours for immunohistochemistry. Three to four sagittal brain sections (40 μm) were selected from each animal, incubated in blocking buffer, and stained by incubation overnight at 4°C in primary / secondary antibodies (CD68, Biorad, MCA1957, amyloid beta, IBL America 18584, and donkey anti-human IgG, Jackson, 709-606-149). Next, the slides were washed and incubated with secondary antibodies (donkey anti-rat, Invitrogen, SA5-10027; donkey anti-rabbit, Invitrogen, A21206) and DAPI (5 μg / mL, Invitrogen, D1306). Afterward, they were washed and mounted on coverslips using Prolong Glass (Invitrogen, P36984). Slides were imaged at 20x magnification using a Zeiss Axioscan.Z1 slide scanner and processed using custom macros in Zeiss ZEN software to generate binary masks using dynamic thresholds for each target channel (amyloid beta, human IgG, CD68, and tissue regions using DAPI). Data analysis was performed using Microsoft Excel and GraphPad Prism 9.

[0274] 5XFAD;TfR mu / hu The plasma PK profile in KI mice is described in ATV35.23.4 cisLALA and ATV35.23.4 cisLALAPG The results were the same between the two groups, and they showed the expected TfR-mediated clearance against control IgG (Figure 10A). Furthermore, the brain concentrations of ATV were similar (Figure 10B), indicating that different cisLALAPG mutations did not have an overall effect on plasma clearance or brain uptake compared to cisLALA mutations.

[0275] The relatively low dose was used to avoid reaching a ceiling effect. This allowed for the detection of small differences in microglia recruitment (showing an Fc-dependent response) between Fc mutations on ATV. Given the short experimental time, this endpoint focused on the smallest plaque, as it was anticipated that there would not be enough time for a substantial response to occur around larger plaques. Immunostaining of all plaques with both ATVs was comparable (Figure 10C), which corresponds to the overall human IgG concentration in the brain. Interestingly, ATV35.23.4 cisLALAPG The ability to mobilize microglia into small patches is described in ATV35.23.4. cisLALA This demonstrated that it was equivalent to (Figure 10D). This indicates that the added effector function silencing mutation did not affect microglia recruitment. This suggests that the molecule can retain virtually complete Fab-mediated effector function even with asymmetric modifications.

[0276] Example 11: ARIA safety test in 5XFAD;TfRmu / huKI mice 5xFAD:TfRmu / huKI mice (male and female, 10-12 months old at the start of the study) were assigned to the following five treatment groups (n=10): anti-amyloid beta (10 mg / kg, n=6M, 4F) treatment group, anti-amyloid beta with LALA mutation (10 mg / kg, n=6M, 4F) treatment group, ATV35.23.3:amyloid beta (3 mg / kg, n=6M, 3F) treatment group, ATV35.23.3cisLALA:amyloid beta (3 mg / kg, n=4M, 4F) treatment group, and untreated group (n=3M, 2F).

[0277] All treatments were administered intraperitoneally weekly for 10 weeks. MRI images were acquired first at baseline, before the start of treatment, and then weekly within 1-3 days after administration. Blood was collected from all animals before the first dose. Untreated animals were imaged only at baseline and after the 10th dose. Anti-CD4 antibody was administered intraperitoneally every two weeks, starting one day before the first dose of the compound (0.5 mg / animal). Blood was collected at the end of the study, before the 10th and final doses, and at slaughter 24 hours after the final dose.

[0278] Analysis of weekly MRI scans revealed the development of MRI lesions corresponding to ARIA-E and ARIA-H in 8 out of 10 animals in the anti-amyloid beta group and 3 out of 10 animals in the LALA mutation amyloid beta group. ARIA could not be detected in either of the two ATV treatment groups (Figure 11).

[0279] MRI lesions detected by T2-weighted sequencing were found in the meninges surrounding the brain (high signal intensity on convex areas) or in the deep cortex (diffuse or localized high signal intensity on the cortex). MRI lesions detected by T2* sequencing appeared as localized low signal intensity in the meninges or cortex. The underlying lesions were characterized by meningovasculitis and microvascular lesions (visualized using H&E staining), leakage of proteinaceous fluids (visualized by immunohistochemistry of albumin and / or mouse IgG), and the presence of microhemorrhages (visualized by Perls staining).

[0280] MRI scans and histological section analyses were performed independently by different observers who were blinded to their treatment assignments.

[0281] Another experiment was conducted to evaluate the pathways by which ATV-amyloid beta enters the brain. As shown in Figure 12, mice administered a single dose of ATV35.23.3cisLALA:amyloid beta showed less vascular connectivity compared to mice administered a single dose of an anti-amyloid beta drug. These results indicate different pathways of entry into the brain, which may lead to a reduced incidence of ARIA events.

Claims

1. (a) Amyloid-beta (amyloid-beta) binding region, (b) A TfR binding domain that specifically binds to the transferrin receptor (TfR) with an affinity of approximately 900 nM to approximately 10,000 nM, and (c) A protein that targets amyloid beta and contains an Fcγ receptor (FcγR) binding domain.

2. The amyloid-beta-targeting protein according to claim 1, wherein the TfR-binding domain specifically binds to TfR with an affinity of approximately 900 nM to approximately 2500 nM.

3. The amyloid-beta-targeting protein according to claim 1, wherein the TfR-binding domain specifically binds to TfR with an affinity of approximately 900 nM to approximately 1300 nM.

4. The amyloid-beta-targeting protein according to claim 3, wherein the TfR binding domain specifically binds to TfR with an affinity of approximately 1100 nM.

5. The amyloid-beta-targeting protein according to any one of claims 1 to 4, wherein the TfR-binding region binds to the apical domain of the TfR.

6. A protein targeting amyloid beta according to any one of claims 1 to 5, wherein the TfR binding domain binds to the TfR without inhibiting the binding of transferrin to the TfR.

7. A protein that targets amyloid beta according to any one of claims 1 to 6, which can be actively transported across the blood-brain barrier.

8. The TfR binding region is (a) TfR-binding polypeptide, (b) TfR-binding polypeptide linked to Fc polypeptide, (c) A first Fc polypeptide containing a CH3 domain modified to bind to TfR, (d) an anti-TfR antibody or its antigen-binding fragment, (e) an amyloid-beta targeting protein according to any one of claims 1 to 7, comprising an anti-TfR antibody or its antigen-binding fragment linked to an Fc polypeptide.

9. The amyloid-beta targeting protein according to any one of claims 1 to 8, wherein the FcγR binding region comprises a second Fc polypeptide, and optionally the second Fc polypeptide does not contain any modifications that reduce FcγR binding.

10. The amyloid-beta-targeting protein according to claim 9, wherein the amyloid-beta-targeting protein comprises an Fc dimer.

11. The Fc dimer is (a) the first Fc polypeptide, and (b) The amyloid-beta targeting protein according to claim 10, comprising the second Fc polypeptide.

12. A protein that targets amyloid beta according to any one of claims 8 to 11, wherein the first Fc polypeptide, the second Fc polypeptide, or the first Fc polypeptide and the second Fc polypeptide are derived from human IgG1, IgG2, IgG3, or IgG4.

13. The amyloid-beta-targeting protein according to any one of claims 8 to 12, wherein the modified CH3 domain comprises amino acid substitutions 384Y, 386T, 387E, 388W, 389V, 413T, 415E, 416E, and 421F according to the EU numbering system.

14. An amyloid-beta-targeting protein according to any one of claims 8-10 and 12-13, wherein the anti-TfR antibody or its antigen-binding fragment comprises an antibody, an F(ab)2 fragment, a Fab fragment, or a single-chain variable fragment (scFv).

15. The amyloid beta-targeting protein according to any one of claims 1 to 14, wherein binding to TfR is reduced when binding to FcγR.

16. The amyloid-beta-targeting protein according to any one of claims 9 to 15, wherein the Fc dimer comprises the first Fc polypeptide and the second Fc polypeptide, the first Fc polypeptide comprises one or more amino acid substitutions that reduce binding to FcγR, and the second Fc polypeptide does not contain any modifications that reduce FcγR binding.

17. The amyloid-beta-targeting protein according to claim 16, wherein the one or more amino acid substitutions that reduce binding to FcγR include substitutions of 234A and 235A according to the EU numbering system.

18. The amyloid-beta-targeting protein according to claim 16 or 17, wherein the one or more amino acid substitutions that reduce binding to FcγR include a 329G substitution according to the EU numbering system.

19. The amyloid-beta-targeting protein according to claim 16, wherein the one or more amino acid substitutions that reduce binding to FcγR include substitutions 234A, 235A, and 329G according to the EU numbering system.

20. The amyloid-beta-targeting protein according to any one of claims 10 to 19, wherein the Fc dimer contains one or more heterodimerizing mutations.

21. The amyloid-beta targeting protein according to claim 20, wherein the first Fc polypeptide contains a knob mutation and the second Fc polypeptide contains a hole mutation, or the first Fc polypeptide contains a hole mutation and the second Fc polypeptide contains a knob mutation.

22. The amyloid-beta-targeting protein according to claim 21, wherein the knob mutation comprises a T366W substitution according to the EU numbering system, and the hole mutation comprises T366S, T368A, and Y407V substitutions according to the EU numbering system.

23. The amyloid-beta-targeting protein according to claim 22, wherein the first Fc polypeptide comprises the substitutions T366S, T368A, and Y407V according to the EU numbering system, and the second Fc polypeptide comprises the substitution T366W according to the EU numbering system.

24. The amyloid-beta-targeting protein according to claim 22, wherein the first Fc polypeptide comprises the substitution of T366W according to the EU numbering system, and the second Fc polypeptide comprises the substitutions of T366S, T368A, and Y407V according to the EU numbering system.

25. The amyloid-beta targeting protein according to claim 24, wherein the modified CH3 domain of the first Fc polypeptide has at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 348 to 454 of any of SEQ ID NOs. 8, 10, and 17 to 31.

26. The amyloid-beta targeting protein according to claim 25, wherein the modified CH3 domain comprises an amino acid sequence of amino acids 348-453 or 348-454 of any of SEQ ID NOs. 8, 10, and 17-31.

27. The protein according to any one of claims 24 to 26, wherein the first Fc polypeptide has at least 85% identity, at least 90% identity, or at least 95% identity with respect to 228 to 454 amino acids of any of SEQ ID NOs. 8, 10, and 17 to 31.

28. The amyloid-beta-targeting protein according to claim 27, wherein the first Fc polypeptide comprises an amino acid sequence of amino acids 228-453 or amino acids 228-454 of any of SEQ ID NOs. 8, 10, and 17-31.

29. The amyloid-beta targeting protein according to any one of claims 24 to 27, wherein the second Fc polypeptide has at least 85% identity, at least 90% identity, or at least 95% identity with respect to amino acids 228 to 454 of SEQ ID NO:

9.

30. The amyloid-beta-targeting protein according to claim 29, wherein the second Fc polypeptide comprises the amino acid sequence of amino acids 228-453 or amino acids 228-454 of SEQ ID NO:

9.

31. The amyloid-beta targeting protein according to any one of claims 1 to 30, wherein the amyloid-beta binding region comprises an anti-amyloid-beta antibody or its antigen-binding fragment.

32. The amyloid-beta targeting protein according to claim 31, wherein the anti-amyloid-beta antibody or its antigen-binding fragment comprises at least one Fab region, at least two Fab regions, an F(ab)2 region, or at least one scFv.

33. The anti-amyloid-beta antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), (a) The VH includes a first heavy chain complementarity determination region (VHCDR1) having the amino acid sequence of SEQ ID NO: 1, a second heavy chain complementarity determination region (VHCDR2) having the amino acid sequence of SEQ ID NO: 2, and a third heavy chain complementarity determination region (VHCDR3) having the amino acid sequence of SEQ ID NO:

3. (b) The amyloid-beta targeting protein according to claim 31 or 32, wherein the VL comprises a first light chain complementarity determining region (VLCDR1) having the amino acid sequence of SEQ ID NO: 4, a second light chain complementarity determining region (VLCDR2) having the amino acid sequence of SEQ ID NO: 5, and a third light chain complementarity determining region (VLCDR3) having the amino acid sequence of SEQ ID NO:

6.

34. The amyloid-beta targeting protein according to claim 33, wherein VH comprises an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 120 of SEQ ID NO: 8 or 10, and VL comprises an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 109 of SEQ ID NO:

7.

35. The amyloid-beta targeting protein according to claim 34, wherein VH comprises the amino acid sequence of amino acids 1 to 120 of SEQ ID NO: 8 or 10, and VL comprises the amino acid sequence of amino acids 1 to 109 of SEQ ID NO:

7.

36. The amyloid-beta targeting protein according to claim 33, wherein the anti-amyloid-beta antibody or its antigen-binding fragment comprises two heavy chain Fab regions and two light chain Fab regions, each heavy chain Fab region comprising an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 227 of SEQ ID NO: 8 or 10, and each light chain Fab region comprising an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with SEQ ID NO:

7.

37. The amyloid-beta targeting protein according to claim 36, wherein each heavy chain Fab region contains the amino acid sequence of amino acids 1 to 227 of SEQ ID NO: 8 or 10, and each light chain Fab region contains the amino acid sequence of SEQ ID NO:

7.

38. The aforementioned amyloid-beta targeting protein, (a) A first light chain comprising an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with respect to Sequence ID No. 7, (b) A first heavy chain comprising an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with respect to Sequence ID No. 8 or 10, (c) A second light chain comprising an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with respect to Sequence ID No. 7, and (d) an amyloid-beta targeting protein according to any one of claims 1 to 37, comprising a second heavy chain having an amino acid sequence having at least 85% identity, at least 90% identity, or at least 95% identity with respect to SEQ ID NO:

9.

39. (a) The first light chain comprises the amino acid sequence of SEQ ID NO: 7, (b) The first heavy chain comprises the amino acid sequence of SEQ ID NO: 8 or 10, and optionally has the C-terminal lysine removed. (c) The second light chain comprises the amino acid sequence of SEQ ID NO: 7, (d) The amyloid-beta targeting protein according to claim 38, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 9, and optionally the lysine at its C-terminus is removed.

40. The amyloid-beta-targeting protein according to any one of claims 1 to 39, wherein the amyloid-beta-targeting protein binds to amyloid plaques, vascular amyloid-beta, or diffuse amyloid-beta deposits.

41. The amyloid-beta-targeting protein according to any one of claims 1 to 40, wherein the amyloid-beta-targeting protein does not deplete reticulocytes in vivo.

42. A composition comprising a protein that targets amyloid beta according to any one of claims 1 to 41.

43. A pharmaceutical composition comprising an amyloid-beta targeting protein according to any one of claims 1 to 41 or the composition according to claim 42, and a pharmaceutically acceptable additive.

44. The pharmaceutical composition according to claim 43, further comprising an additional agent useful for treating Alzheimer's disease.

45. The pharmaceutical composition according to claim 43, wherein the pharmaceutical composition is formulated for use in combination with additional agents useful for the treatment of Alzheimer's disease.

46. A method for reducing amyloid plaques in the brain of a subject, comprising administering to the subject a protein that targets amyloid beta according to any one of claims 1 to 41, or a composition according to claim 42, or a pharmaceutical composition according to any one of claims 43 to 45.

47. The method according to claim 46, wherein the subject has cognitive impairment, memory loss, dementia, or loss of neural connections in the brain.

48. The method according to claim 46 or 47, wherein the subject has a disease associated with the accumulation of amyloid plaques, or is at risk of developing a disease associated with the accumulation of amyloid plaques.

49. The method according to any one of claims 46 to 48, wherein the subject has Alzheimer's disease, has been diagnosed with Alzheimer's disease, or is at high risk of developing Alzheimer's disease.

50. A method for treating neurodegenerative diseases in a subject, comprising administering to the subject an amyloid-beta-targeting protein according to any one of claims 1 to 41, or a composition according to claim 42, or a pharmaceutical composition according to any one of claims 43 to 45.

51. The method according to claim 50, wherein the neurodegenerative disease is Alzheimer's disease.

52. The method according to any one of claims 46 to 51, further comprising administering to the subject at least one additional agent useful for treating Alzheimer's disease.

53. A pharmaceutical composition according to any one of claims 43 to 45 for use in the treatment of Alzheimer's disease.

54. A pharmaceutical composition according to any one of claims 43 to 45, for use in increasing the phagocytosis of amyloid beta in a subject, and / or for use in increasing the recruitment of microglia to amyloid beta-positive plaques in a subject.

55. A nucleic acid encoding a protein that targets amyloid beta, as described in any one of claims 1 to 41.

56. A nucleic acid encoding a polypeptide having at least 85% identity, at least 90% identity, or at least 95% identity with any of sequence numbers 8, 10, and 17-31, wherein the encoded polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 as shown in Table 1, in accordance with the EU numbering system.

57. The coded polypeptide, (a) Contains alanine at position 234 and alanine at position 235, (b) Does it contain glycine at position 329? (c) Contains alanine at position 234, alanine at position 235, and glycine at position 329, (d) Does it contain tryptophan at position 366? (e) Contains serine at position 366, alanine at position 368, and valine at position 407, (f) Contains alanine at position 234, alanine at position 235, and tryptophan at position 366, (g) Contains glycine at position 329 and tryptophan at position 366, (h) Contains alanine at position 234, alanine at position 235, glycine at position 329, and tryptophan at position 366, (i) Contains alanine at 234th position, alanine at 235th position, serine at 366th position, alanine at 368th position, and valine at 407th position, (j) containing glycine at position 329, serine at position 366, alanine at position 368, and valine at position 407, or (k) Contains alanine at position 234, alanine at position 235, glycine at position 329, serine at position 366, alanine at position 368, and valine at position 407. Herein, each position is the nucleic acid according to claim 56, in accordance with the EU numbering system.

58. The nucleic acid according to claim 57, wherein the encoded polypeptide has the amino acid sequence of any of SEQ ID NOs: 8, 10, and 17-31, and optionally the polypeptide has glycine at position 329 and / or tryptophan at position 366, in accordance with the EU numbering system.

59. A combination of nucleic acids comprising a first nucleic acid encoding a first polypeptide having the amino acid sequence of SEQ ID NOs: 8, 10, and 17-31, and a second nucleic acid encoding a second polypeptide having the amino acid sequence of SEQ ID NO:

9.

60. The combination according to claim 59, wherein the combination further comprises a third nucleic acid encoding a third polypeptide having the amino acid sequence of SEQ ID NO:

7.

61. (a) Culturing recombinant host cells comprising one or more nucleic acids encoding a protein that targets amyloid beta as described in any one of claims 1 to 41, (b) A method for producing an amyloid-beta-targeting protein, comprising isolating the amyloid-beta-targeting protein from a culture.

62. A cell expressing an amyloid-beta-targeting protein according to any one of claims 1 to 41.

63. The aforementioned cells, (a) A first nucleic acid encoding a first polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with any of sequence numbers 8, 10, and 17-31, wherein the encoded polypeptide has amino acids at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 as shown in Table 1, according to the EU numbering system, (b) A second nucleic acid encoding a second polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to Sequence ID No. 9, (c) The cell according to claim 62, comprising a third nucleic acid encoding a third polypeptide having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity with respect to Sequence ID No.

7.

64. The first polypeptide is (a) Contains alanine at position 234 and alanine at position 235, (b) Does it contain glycine at position 329? (c) Contains alanine at position 234, alanine at position 235, and glycine at position 329, (d) Does it contain tryptophan at position 366? (e) Contains alanine at position 234, alanine at position 235, and tryptophan at position 366, (f) Contains glycine at position 329 and tryptophan at position 366, (g) Contains alanine at position 234, alanine at position 235, glycine at position 329, and tryptophan at position 366. The cell according to claim 63, wherein each position here conforms to the EU numbering system.

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