Affinity-based methods for using transferrin receptor-binding proteins

Abstract

To provide methods for transporting agents across the blood brain barrier.SOLUTION: Provided is a method for transporting an agent that binds to a therapeutic target across the blood-brain barrier (BBB) of a mammal, comprising exposing the BBB to a protein that binds to a transferrin receptor (TfR) with an affinity of from about 400 nM to about 2 μM, the protein being linked to the agent and transporting the linked agent across the BBB. In some embodiments, the agents bind to therapeutic targets for the treatment of neurodegenerative diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to International Patent Application No. PCT / US2018 / 018371, filed February 15, 2018, U.S. Provisional Patent Application No. 62 / 583,314, filed November 8, 2017, and U.S. Provisional Patent Application No. 62 / 543,658, filed August 10, 2017, the disclosures of which are incorporated herein by reference in their entireties for all purposes. Summary of the Invention

[0002] [The present invention 1001] 1. A method for transporting an agent that binds to a therapeutic target across the blood-brain barrier (BBB) in a mammal, comprising: The method comprises exposing the BBB to a protein that binds to the transferrin receptor (TfR) with an affinity of about 400 nM to about 2 μM, wherein the protein is linked to the agent and transports the linked agent across the BBB. [The present invention 1002] 1001. The method of claim 1001, wherein the brain exposure time to said agent is extended. [The present invention 1003] 1001 or 1002, wherein the therapeutic target is involved in a neurodegenerative disease. [The present invention 1004] 1. A method for treating a neurodegenerative disease, comprising administering to a mammal a protein that binds to TfR with an affinity of about 400 nM to about 2 μM, wherein the protein is linked to an agent that binds to a therapeutic target involved in the neurodegenerative disease, thereby extending the exposure time of the mammal's brain to the agent. [The present invention 1005] The method of any of claims 1001 to 1004, wherein said protein increases brain exposure time to said agent compared to an agent linked to a reference protein that binds to said TfR with greater affinity. [The present invention 1006] The method of claim 1005, wherein brain exposure is determined by measuring the area under the curve (AUC) of a plot of brain concentration of said agent against time. [The present invention 1007] Any of the methods of claims 1001 to 1006, wherein the protein extends brain exposure time to the agent at therapeutically effective concentrations in the mammal compared to an agent linked to a reference protein that binds to the TfR with greater affinity. [The present invention 1008] 1008. The method of any of claims 1005 to 1007, wherein said reference protein binds to said TfR with an affinity of about 50 nM or stronger. [The present invention 1009] The method of any one of claims 1001 to 1008, wherein said TfR is a primate TfR. [The present invention 1010] 1009. The method of claim 10, wherein said primate TfR is a human TfR. [The present invention 1011] The method of any one of claims 1001 to 1010, wherein said protein binds to said TfR apical domain. [The present invention 1012] 1012. The method of any of claims 1001 to 1011, wherein said protein binds to said TfR with an affinity of about 420 nM to about 1.5 μM. [The present invention 1013] The method of any of claims 1001 to 1012, wherein said protein binds to said TfR with an affinity of about 600 nM to about 1.5 µM. [The present invention 1014] 14. The method of any of claims 1007 to 1013, wherein the therapeutically effective concentration of said agent is a concentration that treats one or more symptoms of a neurodegenerative disease in said mammal. [The present invention 1015] The method of any one of claims 1003 to 1014, wherein said neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), and combinations thereof. [The present invention 1016] The method of any one of claims 1001 to 1015, wherein said agent comprises an antibody variable region. [The present invention 1017] 1016. The method of claim 1016, wherein said agent comprises an antibody fragment. [The present invention 1018] 1017. The method of claim 1017, wherein said agent comprises a Fab or scFV. [The present invention 1019] The method of any of claims 1001 to 1018, wherein said protein is a modified Fc polypeptide comprising a non-native binding site capable of binding to TfR. [The present invention 1020] The method of any of claims 1001 to 1018, wherein the protein comprises an antibody variable region that specifically binds to TfR. [The present invention 1021] The method of claim 1020, wherein said protein comprises an antibody fragment. [The present invention 1022] 1021. The method of claim 1021, wherein said protein comprises a Fab or scFV. [The present invention 1023] Any of the methods of claims 1001 to 1022, wherein the therapeutic target is selected from the group consisting of beta-secretase 1 (BACE1) protein, tau protein, triggering receptor expressed on myeloid cells 2 (TREM2) protein, and alpha-synuclein protein. [The present invention 1024] Any of the methods of claims 1005 to 1023, wherein the therapeutic target is BACE1 and the agent, when linked to the protein, reduces the amount of amyloid beta protein (Aβ) present in the brain of the mammal for a longer period of time than when linked to the reference protein. [The present invention 1025] The method of any of claims 1004 to 1024, wherein said protein linked to said agent is administered as part of a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0003] [Figure 1] Pharmacokinetic (PK) analysis of CH3C polypeptides in wild-type mice shows that all polypeptide-Fab fusions exhibited comparable clearance to wild-type Fc-Fab fusions (i.e., anti-RSV antibody Ab122 and anti-BACE1 antibody Ab153), except for CH3C.3.2-5, which exhibited faster clearance. [Figure 2] Pharmacokinetic / pharmacodynamic (PK / PD) data in mouse brain tissue are shown. Chimeric huTfR heterozygous mice (n=4 / group) were intravenously administered 42 mg / kg of either Ab153 or monovalent CH3C.35.N163 (denoted as "CH3C.35.N163_mono"), and wild-type mice (n=3) were intravenously administered 50 mg / kg of control human IgG1 (denoted as "huIgG1"). Bars represent the mean ± SD. [Figure 3A] Shown are huIgG1 concentrations in the plasma of hTfR apical + / + knock-in (KI) mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). [Figure 3B] Shown are huIgG1 concentrations in brain lysates of hTfR apical + / + knock-in (KI) mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). [Figure 3C] Concentrations of endogenous mouse Aβ in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions are shown (mean ± SEM, n = 5 / group). [Figure 3D]Western blot quantification of brain TfR protein normalized to actin in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions is shown (mean ± SEM, n = 5 / group). [Figure 4A] Shown are huIgG1 concentrations in the plasma of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). [Figure 4B] Shown are huIgG1 concentrations in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). [Figure 4C] Concentrations of endogenous mouse Aβ in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusions are shown (mean ± SEM, n = 5 / group). [Figure 4D] Western blot quantification of brain TfR protein normalized to actin in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusion is shown (mean ± SEM, n = 4 / group). [Figure 5]Figure 1 shows the relationship between hTfR affinity of engineered TfR-binding polypeptides and brain exposure over time in hTfR apical + / + KI mice. Dots indicate the cumulative brain exposure over time (AUC) of different ATV affinity variants after a single dose of 50 mg / kg in hTfR apical + / + KI mice. Brain concentrations of the polypeptides (measured by huIgG1) were calculated at various days (ranging from 1 to 10 days) after administration. Data represent a summary of three independent studies with n = 4-5 mice per group in each experiment. [Figure 6] Figure 1 shows the relationship between hTfR affinity and maximum brain concentration of engineered TfR-binding polypeptides in hTfR apical + / + KI mice. Dots indicate maximum brain concentrations of different polypeptide affinity variants measured 1 day post-dose after a single 50 mg / kg dose. Data are a summary of three independent experiments with n = 4-5 mice per group for each study. [Figure 7] Figure 1 shows the relationship between hTfR affinity of engineered TfR-binding polypeptides in hTfR apical + / + KI mice and the ratio of brain concentration to plasma concentration of the polypeptide. Dots indicate the ratio of maximum brain concentration to plasma concentration of different polypeptide affinity variants measured 1 day after administration following a single 50 mg / kg dose. Data are a summary of three independent studies, with n = 4-5 mice per group in each experiment. DETAILED DESCRIPTION OF THE INVENTION

[0004] Detailed Description I. Introduction The present invention relates to the transport of therapeutic agents linked to TfR-binding polypeptides and proteins across the blood-brain barrier (BBB) for the treatment of disease. The invention is based in part on the discovery that the desired TfR binding affinity for transporting a therapeutic agent across the BBB depends on the target of the therapeutic agent and the mechanism of action that confers efficacy in treating disease. Specifically, polypeptides and proteins with relatively low TfR affinity exhibit C maxIt was found that the dose was lower but the clearance was slower, resulting in longer exposure times.

[0005] For some therapies, including inhibitors, including inhibitory antibodies such as anti-BACE1 and anti-tau agents (e.g., for treating Alzheimer's disease) and anti-α-synuclein agents (e.g., for treating Parkinson's disease), as well as others, achieving long-term or sustained brain exposure to the therapeutic over a dosing window is desirable to fully engage targets, including targets with short half-lives and / or fast turnover (e.g., tau, α-synuclein), and to sustain inhibition of BACE1 activity to reduce production of Aβ (also having a short half-life). Using polypeptides and proteins with TfR affinities in the range of 400-2,000 nM is particularly useful for achieving long-term or sustained brain exposure to the therapeutic.

[0006] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" may include two or more such molecules.

[0007] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that the numerical value and reasonable deviations from that value known to those of ordinary skill in the art, such as ±20%, ±10%, or ±5%, are within the expected meaning of the stated value.

[0008] "Transferrin receptor" or "TfR," as used in the context of the present invention, refers to transferrin receptor protein 1. The polypeptide sequence of human transferrin receptor 1 is set forth in SEQ ID NO: 6. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession numbers XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences, e.g., human sequences, encoded by genes at the chromosomal locus of transferrin receptor protein 1. The full-length transferrin receptor protein contains a short N-terminal intracellular region, a transmembrane region, 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 set forth in SEQ ID NO:4.

[0009] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a heavy chain polypeptide of a naturally occurring immunoglobulin, characterized by an Ig fold as a structural domain. An Fc polypeptide comprises a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may include at least a portion of a hinge region. Generally, an Fc polypeptide does not comprise a variable region.

[0010] "Modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion, or insertion, compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of a native Fc polypeptide.

[0011] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of IgG antibodies, a CH3 domain polypeptide refers to the segment of amino acids from about 341 to about 447 when numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about 231 to about 340 when numbered according to the EU numbering scheme. CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the CH2 domain is numbered 1-110 and the CH3 domain is numbered 1-107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. In the context of IgG antibodies, the Fc region refers to the segment of amino acids from about 231 to about 447 when numbered according to the EU numbering scheme. As used herein, the term "Fc region" may also include at least a portion of an antibody hinge region. An exemplary hinge region sequence is set forth in SEQ ID NO:5.

[0012] The term "variable region" refers to the domain within an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and confers to the antibody its specificity for binding to antigen. Antibody variable regions generally consist of four conserved "framework" regions sandwiched between three hypervariable "complementarity-determining regions."

[0013] The terms "wild-type," "native," and "naturally occurring" with respect to CH3 and CH2 domains are used herein to refer to domains having sequences that occur in nature.

[0014] In the context of the present invention, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." Variants with respect to a given wild-type (e.g., CH3 or CH2 domain) reference sequence can include naturally occurring allelic variants. A "non-naturally occurring" (e.g., CH3 or CH2) domain refers to a variant or mutant domain that does not naturally occur in cells and is generated by genetic modification of a polynucleotide or polypeptide of a native domain (e.g., CH3 domain or CH2 domain), for example, using genetic engineering techniques or mutagenesis. 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.

[0015] The term "binding affinity," as used herein, refers to the strength of a non-covalent interaction between two molecules, e.g., between one binding site of a polypeptide and a target to which the polypeptide binds, e.g., TfR. Thus, for example, the term may refer to a 1:1 interaction between a polypeptide and its target unless otherwise indicated or clear from the context. Binding affinity is measured by the dissociation rate constant (k d ,time -1 ) as the binding rate constant (k a ,time -1 M -1 ) is the equilibrium dissociation constant (K D ) can be quantified by measuring the K Dcan be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods such as the Biacore™ system (e.g., using the method described in Example 3 below); kinetic exclusion assays such as KinExA®; and biolayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, "binding affinity" includes not only formal binding affinity, such as that reflecting a 1:1 interaction between a polypeptide and its target, but also K values, which may reflect strong binding. D The apparent affinity calculated is also included.

[0016] As used herein, when referring to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody described herein, the term "specifically binds" or "selectively binds" to a target, e.g., TfR, refers to a binding reaction in which the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody binds to the target with greater affinity, greater avidity, and / or longer duration than it binds to a structurally different target. In typical embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody has 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 more, greater affinity for a particular target, e.g., TfR, compared to an unrelated target when assayed under the same affinity assay conditions. As used herein, the terms "specific binding," "specifically binds to," or "is specific for" a particular target (e.g., TfR) refer to, for example, the equilibrium dissociation constant K for the target to which it binds. D For example, 10 -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12M. In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody specifically binds to an epitope of the TfR that is conserved across species (e.g., structurally conserved across species), e.g., conserved between non-human primates and humans (e.g., structurally conserved between non-human primates and humans). In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can bind only to human TfR.

[0017] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0018] Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0019] 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 (Ile), 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. Naturally occurring stereoisomers of 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), and 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.

[0020] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0021] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. Such terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can be composed entirely of L-amino acids, entirely of D-amino acids, or a mixture of L and D amino acids.

[0022] As used herein, "protein" refers to a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single polypeptide chains. The single polypeptide chains of a protein can be linked by covalent bonds, such as disulfide bonds, or by non-covalent interactions.

[0023] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to a change that results in the replacement of an amino acid with another amino acid that can be classified as having similar properties. Examples of categories of conservative amino acid groups defined in this manner include "charged / polar groups" including 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); "aromatic groups" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "aliphatic groups" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Subgroups may also be identified within each group. For example, the group of charged or polar amino acids can be further divided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His, a "negatively charged subgroup" consisting of Glu and Asp, and a "polar subgroup" consisting of Asn and Gln. In another example, aromatic or cyclic groups can be further divided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp, and a "phenyl subgroup" consisting of Phe and Tyr. In yet another example, aliphatic groups can be further divided into subgroups such as an "aliphatic non-polar subgroup" consisting of Val, Leu, Gly, and Ala, and an "aliphatic weakly polar subgroup" consisting of Met, Ser, Thr, and Cys.Examples of conservative mutation categories include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to, Lys for Arg, or vice versa, to maintain a positive charge; Glu for Asp, or vice versa, to maintain a negative charge; Ser for Thr, or vice versa, to maintain a free -OH; Gln for Asn, or vice versa, to maintain a free -NH. In some embodiments, hydrophobicity is maintained, for example, by substituting a hydrophobic amino acid for a naturally occurring hydrophobic amino acid in the active site.

[0024] The terms "identical" or "percent identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that are the same over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as determined using a sequence comparison algorithm or by manual alignment and visual inspection, or that have a specified percentage (%) of amino acid residues that are identical, for example, 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 identical.

[0025] In polypeptide sequence comparison, one amino acid sequence generally serves as a reference sequence to which candidate sequences are compared. Alignment can be performed using various methods available to those skilled in the art to achieve maximum alignment, such as visual alignment or using publicly available software with known algorithms. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used in alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of this application, the standard protein BLAST of the BLASTP algorithm is used to align two protein sequences using default parameters.

[0026] The terms "corresponding to," "determined with respect to," or "numbered with respect to," when used in reference to identifying a given amino acid residue in a polypeptide sequence, refer to the position of the residue in a particular reference sequence when the given amino acid sequence is compared to the reference sequence for maximum alignment. Thus, for example, an amino acid residue in a modified Fc polypeptide "corresponds to" an amino acid in SEQ ID NO: 1 when that residue is aligned with that amino acid in SEQ ID NO: 1 when optimally aligned with SEQ ID NO: 1. A polypeptide that is aligned with a reference sequence need not be the same length as the reference sequence.

[0027] The terms "subject," "individual," and "patient," used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0028] The terms "treatment," "treating," and similar terms are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" can refer to any indication of success in treating or ameliorating an injury, disease, or condition, including any objective or subjective parameter, such as relief, remission, improved patient survival, increased survival time or rate, disappearance of symptoms or making the injury, disease, or condition more tolerable to the patient, slowing the rate of degeneration or decline, or improvement in the patient's physical or mental health. Treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or population of individuals not receiving treatment, or to the same patient before or at different time points during treatment.

[0029] "Pharmaceutically acceptable excipient" refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, a buffer, carrier, or preservative.

[0030] As used herein, a "therapeutic amount," "therapeutically effective amount," or "therapeutically effective concentration" of an agent is an amount or concentration of the agent that treats a sign or symptom of a disease in a subject.

[0031] The term "administering" refers to a method of delivering an agent, compound, or composition to a site where biological effect is desired. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal administration. In one embodiment, the compositions described herein are administered intravenously.

[0032] III. Treatment method A. Methods for the Treatment of Neurodegenerative Diseases In one aspect, the invention provides methods for transporting an agent (e.g., a therapeutic agent) that binds to (e.g., specifically binds to) a therapeutic target (e.g., a therapeutic target implicated in a neurodegenerative disease) across the blood-brain barrier (BBB) in a mammal. In some embodiments, the method includes exposing the BBB to a polypeptide or protein that binds to (e.g., specifically binds to) the transferrin receptor (TfR) with an affinity of about 400 nM to about 2 μM. In some embodiments, the polypeptide or protein is linked to the agent and transports the linked agent across the BBB. In some embodiments, the time of brain exposure to the agent is extended (e.g., compared to a reference agent).

[0033] In another aspect, the present invention provides methods for treating neurodegenerative diseases. In some embodiments, the methods include administering to a mammal a polypeptide or protein that binds (e.g., specifically binds) to TfR with an affinity of about 400 nM to about 2 μM. In some embodiments, the polypeptide or protein is linked to an agent (e.g., a therapeutic agent) that binds (e.g., specifically binds) to a therapeutic target involved in the neurodegenerative disease, thereby extending the exposure time of the mammal's brain to the agent. Non-limiting examples of suitable neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), and combinations thereof.

[0034] In some embodiments, the polypeptide or protein binds (e.g., specifically binds) to TfR with an affinity of about 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, or 2 μM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 420 nM to about 1.5 μM or 600 nM to 1.5 μM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 420 nM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 620 nM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 750 nM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 820 nM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 1,100 nM. In some embodiments, the polypeptide or protein binds to TfR with an affinity of about 1,440 nM.

[0035] In some embodiments, the polypeptide or peptide (e.g., linked to an agent) increases brain exposure time to a therapeutically effective concentration of the agent in a mammal (e.g., a concentration sufficient to treat one or more signs or symptoms of a neurodegenerative disease) compared to an agent linked to a reference polypeptide or protein that binds (e.g., specifically binds) TfR with greater affinity.

[0036] In some embodiments, brain exposure time (e.g., to an agent) is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.75-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 5-fold, or more, compared to the reference.

[0037] In some embodiments, brain exposure is quantified by calculating the area under the curve (AUC) of brain exposure (e.g., concentration of an agent in the brain) as a function of time. An increased AUC may indicate increased or prolonged brain exposure. In some embodiments, the duration of brain exposure to a therapeutically effective concentration of the agent is prolonged.

[0038] In some embodiments, the reference polypeptide or peptide binds (e.g., specifically binds) to TfR with an affinity of about 400 nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, or about 50 nM, or greater. In some embodiments, the reference polypeptide or protein binds to TfR with an affinity of about 50 nM or greater.

[0039] By way of non-limiting example, the therapeutic target can be the beta-secretase 1 (BACE1) protein, the tau protein, the triggering receptor expressed on myeloid cells 2 (TREM2) protein, or the alpha-synuclein protein. In some embodiments, the therapeutic target is BACE1, and the agent (e.g., therapeutic agent) when linked to this protein reduces the amount of amyloid beta protein (Aβ) present in the mammalian brain for a longer period of time compared to when linked to a reference protein.

[0040] In some embodiments, the mammal is a primate (e.g., a human). In some embodiments, the human is a patient in need of treatment for a neurological disease (e.g., a neurodegenerative disease). In some embodiments, the patient has one or more signs or symptoms of a neurological disease.

[0041] In some embodiments, the polypeptide or protein binds (e.g., specifically binds) to a primate TfR. In some embodiments, the primate TfR is a human TfR. In some embodiments, the polypeptide or protein binds to the TfR apical domain.

[0042] In some embodiments, an agent (e.g., a therapeutic agent) is linked to an engineered TfR-binding polypeptide. In some embodiments, the engineered TfR-binding polypeptide comprises a CH3 or CH2 domain with a modification that enables the polypeptide to specifically bind to the transferrin receptor. Non-limiting examples of suitable engineered TfR-binding polypeptides are described in Section IV, below. In some embodiments, the agent is linked to an engineered TfR-binding polypeptide described in Table 1 or Table 2. In some embodiments, the agent is linked to an engineered TfR-binding polypeptide selected from the group consisting of CH3C.35.23, CH3C.35.23.1.1, CH3C.35.23.3, and CH3C.35.23.4.

[0043] In some embodiments, an agent (e.g., a therapeutic agent) is linked to a TfR-binding peptide. In some embodiments, the TfR-binding polypeptide is a short peptide having a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Methods for generating, screening, and identifying suitable peptides (i.e., those that bind to TfR with an affinity within a desired range) are well known in the art. For example, suitable peptides can be identified using a phage display approach that uses alternating rounds of negative and positive selection. This approach is described, for example, in Lee et al., Eur. J. Biochem. (2001) 268:2004-2012, which is hereby incorporated by reference in its entirety for all purposes.

[0044] In some embodiments, the agent (e.g., a therapeutic agent) is linked to a TfR-binding antibody. A non-limiting example of a suitable TfR-binding polypeptide is the H67 antibody disclosed in Chinese Patent Application Publication No. CN101245107A, which has an affinity of about 480 nM.

[0045] In some embodiments, the protein comprises an antibody variable region that specifically binds to TfR. In some examples, the protein comprises an antibody fragment. In some examples, the protein comprises a Fab or scFv.

[0046] In some embodiments, the agent (e.g., a therapeutic agent) comprises an antibody variable region. In some embodiments, the agent comprises an antibody fragment. In some embodiments, the agent comprises a Fab or scFV.

[0047] In some embodiments, the agent (e.g., therapeutic agent) comprises a Fab and the polypeptide is in an Fc format (which may include a hinge or partial hinge region), thus generating a transferrin receptor-binding Fc-Fab fusion. In some embodiments, the Fc-Fab fusion (e.g., comprising a modified CH2 or CH3 domain polypeptide) is a subunit of a dimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer comprises one polypeptide that binds to the transferrin receptor (i.e., is monovalent for binding to the transferrin receptor). In some embodiments, the dimer comprises a second polypeptide that binds to the transferrin receptor. The second polypeptide may comprise the same modified CH3 domain polypeptide (or modified CH2 domain polypeptide) present in the Fc-Fab fusion, thereby providing a bivalent binding homodimer, or a second modified CH3 domain polypeptide (or modified CH2 domain polypeptide) may provide a second transferrin receptor-binding site. In some embodiments, the dimer comprises a first subunit comprising a modified CH3 domain polypeptide or a modified CH2 domain polypeptide and a second subunit comprising CH2 and CH3 domains, neither of which binds to the transferrin receptor.

[0048] In some embodiments, the agent (e.g., a Fab fragment) is linked to a polypeptide or protein and binds to a tau protein (e.g., a human tau protein) or a fragment thereof. In some embodiments, the agent can bind to phosphorylated tau protein, unphosphorylated tau protein, a splice isoform of tau protein, an N-terminally truncated tau protein, a C-terminally truncated tau protein, and / or fragments thereof.

[0049] In some embodiments, the agent (e.g., a Fab fragment) is linked to a polypeptide or protein and binds to a beta-secretase 1 (BACE1) protein (e.g., a human BACE1 protein) or a fragment thereof. In some embodiments, the agent can bind to one or more splice isoforms of the BACE1 protein or fragments thereof.

[0050] In some embodiments, the agent (e.g., a Fab fragment) is linked to a polypeptide or protein and binds to a triggering receptor expressed on myeloid cells 2 (TREM2) protein (e.g., a human TREM2 protein) or a fragment thereof.

[0051] In some embodiments, the agent (e.g., a Fab fragment) is linked to a polypeptide or protein and binds to an alpha-synuclein protein (e.g., a human alpha-synuclein protein) or a fragment thereof. In some embodiments, the agent can bind to monomeric alpha-synuclein, oligomeric alpha-synuclein, alpha-synuclein fibrils, soluble alpha-synuclein, and / or fragments thereof.

[0052] B. Further Embodiments and Linkers The polypeptide (e.g., a modified CH3 or CH2 domain polypeptide, described further below) can be linked to another domain of the Fc region. In some embodiments, the modified CH3 domain polypeptide can be linked to a CH2 domain, which can be a naturally occurring CH2 domain or a variant CH2 domain that is normally C-terminal to the CH2 domain. In some embodiments, the modified CH2 domain polypeptide can be linked to a CH3 domain, which can be a naturally occurring CH3 domain or a variant CH3 domain that is normally N-terminal to the CH3 domain. In some embodiments, a polypeptide comprising a modified CH2 domain linked to a CH3 domain or a polypeptide comprising a modified CH3 domain linked to a CH2 domain further comprises a partial or complete hinge region of an antibody, thereby forming the modified CH3 domain polypeptide or modified CH2 domain polypeptide as part of an Fc region with a partial or complete hinge region. The hinge region can be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, e.g., an IgG1 hinge region, e.g., a human IgG1 hinge amino acid sequence There is TIFF2025120209000001.tif5128.

[0053] In still other embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can be fused to a peptide or protein useful for protein purification, e.g., polyhistidine, an epitope tag such as FLAG, c-Myc, a hemagglutinin tag, glutathione S-transferase (GST), thioredoxin, protein A, protein G, or maltose-binding protein (MBP). Optionally, the peptide or protein fused to the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can include a protease cleavage site, such as a cleavage site for factor Xa or thrombin.

[0054] In the methods of the invention, an agent (e.g., a therapeutic agent) is linked to a polypeptide or protein (e.g., an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody). The linker can be any linker suitable for linking an agent to a polypeptide or protein. In some embodiments, the linkage is enzymatically cleavable. In certain embodiments, the linkage is cleavable by an enzyme present in the central nervous system.

[0055] In some embodiments, the linker is a peptide linker. The peptide linker can be configured to allow rotation of the agent (e.g., a therapeutic agent) and the polypeptide or protein relative to each other and / or to be resistant to digestion by proteases. In some embodiments, the linker can be a flexible linker comprising amino acids such as, for example, Gly, Asn, Ser, Thr, Ala, etc. Such linkers can be designed using well-known parameters. For example, the linker can have a repeating sequence such as a Gly-Ser repeat.

[0056] In various embodiments, linking of an agent (e.g., a therapeutic agent) to a polypeptide or protein (e.g., an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody) can be achieved using well-known chemical cross-linking reagents and protocols. For example, there are many chemical cross-linking agents known to those skilled in the art that are useful for cross-linking a polypeptide or protein to an agent of interest. For example, the cross-linking agent is a heterobifunctional cross-linker that can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers allow for the design of more specific linking methods for linking proteins, thereby reducing the occurrence of unwanted side reactions such as homoprotein polymers.

[0057] The agent (e.g., a therapeutic agent) can be linked to the N-terminal or C-terminal region of the polypeptide or protein, and can be attached to any region of the polypeptide or protein (e.g., an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody), so long as the agent does not interfere with binding of the polypeptide or protein to the transferrin receptor.

[0058] C. Measurement of Binding Affinity, Brain Concentration, and Brain Exposure In some embodiments, the affinity of a TfR-binding polypeptide can be measured in a monovalent format, while in other embodiments, the affinity can be measured in a bivalent format, e.g., as a dimer comprising a polypeptide-Fab fusion protein.

[0059] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are well known in the art. These 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), bio-layer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, kinetic exclusion assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, bio-layer interferometry is used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0060] A non-limiting example of a method for determining binding affinity (e.g., to TfR) is described in Example 3 below, where affinity is measured by surface plasmon resonance using a Bicaore™ instrument. In this method, an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody of interest is captured on a sensor chip, and serial dilutions of TfR are injected over the sensor chip at a specific flow rate (e.g., 30 μL / min) and temperature (e.g., room temperature). After analysis using specified association and dissociation times (e.g., 45 seconds and 180 seconds, respectively), the sensor chip is regenerated. The binding response can be corrected by subtracting the measured response from a control (e.g., an irrelevant IgG at a similar concentration), and steady-state affinity can be determined by fitting the equilibrium response to concentration using software.

[0061] The concentration of an agent (e.g., linked to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody) in the brain and / or plasma can be measured, for example, using a human transferrin receptor (hTfR) knock-in mouse model. Using such a model, for example, the maximum brain concentration (C max ) and / or brain exposure, e.g., C max It can be determined whether the concentration of TFR increases and / or the brain exposure time is extended. アピカル+ / +The generation of a mouse knock-in model is described in Example 2 below. To generate a suitable model, the CRISPR / Cas9 system can be used to generate mice expressing the human Tfrc apical domain within the mouse Tfrc gene (e.g., in vivo expression is under the control of the endogenous promoter). Specifically, Cas9, single guide RNA, and donor DNA (e.g., a coding sequence for the human apical domain that has been codon-optimized for expression in mice) can be introduced into mouse embryos (e.g., by pronuclear injection). The embryos can then be implanted into pseudopregnant females. Founder males from the offspring of the implanted females can be bred with wild-type females to generate F1 heterozygous mice. Homozygous mice can then be generated by breeding the F1 generation heterozygous mice.

[0062] For assessment of brain and / or plasma concentrations or exposure of an agent (e.g., linked to an engineered TfR-binding polypeptide, to a TfR-binding peptide, or to a TfR-binding antibody), the linked agent can be administered to a mouse model (e.g., hTfR). アピカル+ / + ) can be administered to the mouse. After a suitable time, a plasma sample can be obtained from the mouse, and the vasculature can be perfused with an appropriate solution. After perfusion, the brain (or a portion thereof) can be extracted, homogenized, and then lysed. The concentration of the agent in the plasma and / or brain lysate can be measured using routine methods known to those skilled in the art. A range of doses can be administered to the knock-in mouse model to generate a standard curve. By administering to the knock-in mouse model an agent linked to different engineered TfR-binding polypeptides, TfR-binding peptides, or TfR-binding antibodies (e.g., with different TfR affinities), or an agent linked to a reference polypeptide or protein (e.g., with a stronger affinity for TfR than the polypeptide or protein of interest), brain exposure to the agent and / or C of the agent in the brain can be measured. max Comparisons can be made regarding the effect of engineered TfR-binding polypeptides, TfR-binding peptides, or TfR-binding antibodies on the values.

[0063] D. Pharmaceutical Compositions Guidance for preparing formulations for use in the present invention can be found in any number of pharmaceutical and formulation handbooks well known to those skilled in the art.

[0064] In some embodiments, a polypeptide or protein linked to an agent (e.g., a therapeutic agent) is administered as part of a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the active agent. A variety of pharmaceutically acceptable excipients are well known.

[0065] In some embodiments, the carrier is suitable for intravenous, intrathecal, intracerebroventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can include one or more physiologically acceptable compounds, for example, to stabilize the composition or increase or decrease the absorption of the polypeptide. Physiologically acceptable compounds can include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, compositions that reduce the clearance or hydrolysis of the active agent, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0066] The pharmaceutical compositions described herein can be manufactured in a manner well known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The following methods and excipients are illustrative only and are not intended to be limiting in any way.

[0067] For oral administration, polypeptides or proteins linked to an active agent (e.g., a therapeutic agent) can be formulated by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, and the like, to be orally ingested by the patient to be treated. Oral pharmaceutical preparations can be obtained by mixing the polypeptide with a solid excipient, optionally milling the resulting mixture, adding appropriate excipients as needed, and then processing the granular mixture to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0068] Polypeptides or proteins linked to an active agent (e.g., a therapeutic agent) can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. For injection, the polypeptide can be formulated by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent, such as vegetable oil or similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, along with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed. In some embodiments, the polypeptide can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline. Formulations for injection can be provided in unit dosage form, e.g., in ampoules or multi-dose containers with added preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and can contain formulating agents, such as suspending agents, stabilizing agents, and / or dispersing agents.

[0069] Generally, pharmaceutical compositions to be used for in vivo administration are sterile. Sterilization can be accomplished by methods well known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0070] IV. Engineered Transferrin Receptor Binding Polypeptides This section describes non-limiting examples of engineered polypeptides that bind to the transferrin receptor and are capable of being transported across the blood-brain barrier (BBB).

[0071] In some embodiments, the engineered polypeptide comprises a CH3 or CH2 domain with modifications that enable the polypeptide to specifically bind to the transferrin receptor. Such modifications are introduced into a specific set of amino acids present on the surface of the CH3 or CH2 domain. In some embodiments, the polypeptide comprising the modified CH3 or CH2 domain specifically binds to an epitope within the apical domain of the transferrin receptor.

[0072] Those skilled in the art will appreciate that the CH2 and CH3 domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, and IgD, can be similarly modified by identifying amino acids within these domains that correspond to sets (i) through (vi) described above. Modifications can also be made to corresponding domains from immunoglobulins derived from other species, such as non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, and chickens.

[0073] CH3 Transferrin Receptor Binding Polypeptide In some embodiments, the domain to be modified is a human Ig CH3 domain, such as an IgG CH3 domain. The CH3 domain may be derived from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the segment of amino acids from about 341 to about 447 when numbered according to the EU numbering scheme. Positions within the CH3 domain for purposes of identifying the corresponding set of amino acid positions for transferrin receptor binding are determined relative to SEQ ID NO:3 or relative to amino acids 114-220 of SEQ ID NO:1, unless otherwise specified. Substitutions are also determined relative to SEQ ID NO:1, i.e., an amino acid is considered a substitution for the amino acid at the corresponding position in SEQ ID NO:1. SEQ ID NO:1 includes the partial hinge region sequence PCP as amino acids 1-3. The numbering of positions within the CH3 domain relative to SEQ ID NO:1 includes these first three amino acids.

[0074] As noted above, sets of residues in a CH3 domain that can be modified are numbered herein with reference to SEQ ID NO: 1. For example, any CH3 domain, such as the CH3 domain of IgG1, IgG2, IgG3, or IgG4, can have modifications, e.g., amino acid substitutions, at one or more sets of residues that correspond to the residues at the recited positions in SEQ ID NO: 1. The respective positions in the IgG2, IgG3, and IgG4 sequences that correspond to any particular position in SEQ ID NO: 1 can be readily determined.

[0075] In one embodiment, a modified CH3 domain polypeptide that specifically binds to the transferrin receptor binds to the apical domain of the transferrin receptor at an epitope that includes position 208 of the full-length human transferrin receptor sequence (SEQ ID NO: 6), which corresponds to position 11 of the human transferrin receptor apical domain sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 corresponds to amino acids 198-378 of the human transferrin receptor 1 uniprotein sequence P02786 (SEQ ID NO: 6). In some embodiments, a modified CH3 domain polypeptide binds to the apical domain of the transferrin receptor at an epitope that includes positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and / or 294 of the full-length human transferrin receptor sequence (SEQ ID NO: 6). The modified CH3 domain polypeptide may bind to the transferrin receptor without blocking or otherwise inhibiting the binding of transferrin to the receptor. In some embodiments, the binding of transferrin to the TfR is not substantially inhibited. In some embodiments, the binding of transferrin to the TfR is inhibited by 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, binding of transferrin to TfR is inhibited by 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%). Exemplary CH3 domain polypeptides that exhibit such binding specificity include polypeptides with amino acid substitutions at positions 153, 157, 159, 160, 161, 162, 163, 186, 188, 189, and 194, relative to amino acids 114-220 of SEQ ID NO:1.

[0076] CH3 Transferrin Receptor Binding Set (i): 153, 157, 159, 160, 161, 162, 163, 186, 188, 189, and 194 In some embodiments, the modified CH3 domain polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at one set of amino acid positions (Set i) including: 153, 157, 159, 160, 161, 162, 163, 186, 188, 189, and 194. Exemplary substitutions that can be introduced at these positions are shown in Tables 1 and 2.

[0077] In some embodiments, modified CH3 domain polypeptides that specifically bind to the transferrin receptor comprise at least one position having a substitution relative to SEQ ID NO:1, such as Glu, Leu, Ser, Val, Trp, Tyr, or Gln at position 153; Leu, Tyr, Phe, Trp, Met, Pro, or Val at position 157; Leu, Thr, His, Pro, Asn, Val, or Phe at position 159; Val, Pro, Ile, or an acidic amino acid at position 160; Trp at position 161; an aliphatic amino acid, Gly, Ser, Thr, or Asn at position 162; and Gly, His at position 163. , Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr, an acidic amino acid, Ala, Ser, Leu, Thr, Pro, Ile, or His, at position 186, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188, Thr, Arg, Asn, or an acidic amino acid at position 189, and / or an aromatic amino acid, His, or Lys, at position 194. In some embodiments, the modified CH3 domain polypeptide includes conservative substitutions of specific amino acids at one or more positions in the above sets, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), size, and / or polarity or nonpolarity. Thus, for example, Ile may be present at positions 157, 159, and / or 186. In some embodiments, the acidic amino acid at one, two, or each of positions 160, 186, and 189 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 160, 186, and 189 is Asp.

[0078] In some embodiments, the modified CH3 domain polypeptide further comprises one or two substitutions at positions including 164 and 165. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 164. In some embodiments, Gln, Phe, or His may be present at position 165.

[0079] In a further embodiment, the modified CH3 domain further comprises one, two, or three positions selected from the following: Lys, Arg, Gly, or Pro at position 187; Ser, Thr, Glu, or Lys at position 197; and Ser, Trp, or Gly at position 199.

[0080] CH3 Transferrin Receptor Binding Set (ii): 118, 119, 120, 122, 210, 211, 212, and 213 In some embodiments, the modified CH3 domain polypeptide comprises at least three or at least four, typically five, six, seven, or eight substitutions at one set of amino acid positions (set ii) consisting of: 118, 119, 120, 122, 210, 211, 212, and 213. In some embodiments, the modified CH3 domain polypeptide comprises a Gly at position 210, a Phe at position 211, and / or an Asp at position 213. In some embodiments, a Glu may be present at position 213. In particular embodiments, the modified CH3 domain polypeptide comprises at least one substitution at the following positions: That is, Phe or lie at position 118, Asp, Glu, Gly, Ala, or Lys at position 119, Tyr, Met, Leu, lie, or Asp at position 120, Thr or Ala at position 122, Gly at position 210, Phe at position 211, His, Tyr, Ser, or Phe at position 212, or Asp at position 213. In some embodiments, two, three, four, five, six, seven, or all eight of positions 118, 119, 120, 122, 210, 211, 212, and 213 have a substitution as specified in this paragraph. In some embodiments, the modified CH3 domain polypeptide may include conservative substitutions of particular amino acids at one or more of the positions in the above sets, e.g., amino acids in groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0081] In some embodiments, the modified CH3 domain polypeptide has 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 114-220 of SEQ ID NO: 1, provided that the percent identity excludes the set of positions 118, 119, 120, 122, 210, 211, 212, and 213.

[0082] CH2 Transferrin Receptor Binding Polypeptide In some embodiments, the domain to be modified is a human Ig CH2 domain, such as an IgG CH2 domain. The CH2 domain may be derived from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH2 domain refers to the segment of amino acids from about 231 to about 340 when numbered according to the EU numbering scheme. For purposes of identifying the corresponding set of amino acid positions for transferrin receptor binding, positions within the CH2 domain are determined relative to SEQ ID NO:2 or relative to amino acids 4-113 of SEQ ID NO:1. Substitutions are also determined relative to SEQ ID NO:1, i.e., an amino acid is considered a substitution for the amino acid at the corresponding position in SEQ ID NO:1. SEQ ID NO:1 includes the partial hinge region sequence PCP as amino acids 1-3. Although these three residues are not part of the Fc region, the numbering of positions within the CH2 domain relative to SEQ ID NO:1 includes these first three amino acids.

[0083] As noted above, sets of residues in a CH2 domain that can be modified are numbered herein with reference to SEQ ID NO: 1. For example, any CH2 domain, such as the CH2 domain of IgG1, IgG2, IgG3, or IgG4, can have modifications, e.g., amino acid substitutions, at one or more sets of residues that correspond to the residues at the recited positions in SEQ ID NO: 1. The respective positions in the IgG2, IgG3, and IgG4 sequences that correspond to any particular position in SEQ ID NO: 1 can be readily determined.

[0084] In one embodiment, the modified CH2 domain polypeptide that specifically binds to the transferrin receptor binds to an epitope within the apical domain of the transferrin receptor. The human transferrin receptor apical domain sequence is set forth in SEQ ID NO: 4, which corresponds to amino acids 198-378 of the uniprotein sequence P02786 of human transferrin receptor 1. The modified CH2 domain polypeptide can bind to the transferrin receptor without blocking or otherwise inhibiting transferrin binding to the receptor. In some embodiments, binding of transferrin to TfR is not substantially inhibited. In some embodiments, binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%). In some embodiments, binding of transferrin to TfR is inhibited by 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%).

[0085] CH2 Transferrin Receptor Binding Set (iii): 47, 49, 56, 58, 59, 60, 61, 62, and 63 In some embodiments, the modified CH2 domain polypeptide comprises at least three or at least four, typically five, six, seven, eight, or nine substitutions at a set of amino acid positions (set iii) including: 47, 49, 56, 58, 59, 60, 61, 62, and 63. In some embodiments, the modified CH2 domain polypeptide comprises a Glu at position 60 and / or a Trp at position 61. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp at position 47; Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49; Asp, Pro, Met, Leu, Ala, Asn, or Phe at position 56; Arg, Ser, Ala, or Gly at position 58; Tyr, Trp, Arg, or Val at position 59; Glu at position 60; Trp or Tyr at position 61; Gln, Tyr, His, Ile, Phe, Val, or Asp at position 62; or Leu, Trp, Arg, Asn, Tyr, or Val at position 63. In some embodiments, two, three, four, five, six, seven, eight, or all nine of positions 47, 49, 56, 58, 59, 60, 61, 62, and 63 have substitutions as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide may include conservative substitutions of particular amino acids at one or more of the positions in the above set, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0086] In some embodiments, the modified CH2 domain polypeptide has 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 4 to 113 of SEQ ID NO: 1, provided that the percent identity does not include the set of positions 47, 49, 56, 58, 59, 60, 61, 62, and 63.

[0087] CH2 Transferrin Receptor Binding Set (iv): 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72 In some embodiments, the modified CH2 domain polypeptide comprises at least three or at least four, typically five, six, seven, eight, nine, or ten substitutions at one set of amino acid positions (set iv) including: 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72. In some embodiments, the modified CH2 domain polypeptide comprises Pro at position 43, Glu at position 68, and / or Tyr at position 70. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Pro, Phe, Ala, Met, or Asp at position 39; Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr at position 40; Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu at position 41; Pro, Val, Ala, Thr, or Asp at position 42; Pro, Val, or Phe at position 43; Trp, Gln, Thr, or Glu at position 44; Glu, Val, Thr, Leu, or Trp at position 68; Tyr, His, Val, or Asp at position 70; Thr, His, Gln, Arg, Asn, or Val at position 71; and Tyr, Asn, Asp, Ser, or Pro at position 72. In some embodiments, two, three, four, five, six, seven, eight, nine, or all ten of positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72 have substitutions as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide may include conservative substitutions of particular amino acids at one or more of the positions in the above set, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0088] In some embodiments, the modified CH2 domain polypeptide has 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 4 to 113 of SEQ ID NO: 1, provided that the percent identity does not include the set of positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72.

[0089] CH2 Transferrin Receptor Binding Set (v): 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73 In some embodiments, the modified CH2 domain polypeptide comprises at least three or at least four, typically five, six, seven, eight, nine, or ten substitutions at one set of amino acid positions (set v) including positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Val or Asp at position 41; Pro, Met, or Asp at position 42; Pro or Trp at position 43; Arg, Trp, Glu, or Thr at position 44; Met, Tyr, or Trp at position 45; Leu or Trp at position 65; Thr, Val, Ile, or Lys at position 66; Ser, Lys, Ala, or Leu at position 67; His, Leu, or Pro at position 69; or Val or Trp at position 73. In some embodiments, two, three, four, five, six, seven, eight, nine, or all ten of positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73 have a substitution as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide may include conservative substitutions of particular amino acids at one or more of the positions in the above set, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0090] In some embodiments, the modified CH2 domain polypeptide has 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 4-113 of SEQ ID NO: 1, provided that the percent identity does not include the set of positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73.

[0091] CH2 Transferrin Receptor Binding Set (vi): 45, 47, 49, 95, 97, 99, 102, 103, and 104 In some embodiments, the modified CH2 domain polypeptide comprises at least three or at least four, typically five, six, seven, eight, or nine substitutions at one set of amino acid positions (set vi) including: 45, 47, 49, 95, 97, 99, 102, 103, and 104. In some embodiments, the modified CH2 domain comprises a Trp at position 103. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Trp, Val, Ile, or Ala at position 45; Trp or Gly at position 47; Tyr, Arg, or Glu at position 49; Ser, Arg, or Gln at position 95; Val, Ser, or Phe at position 97; Ile, Ser, or Trp at position 99; Trp, Thr, Ser, Arg, or Asp at position 102; Trp at position 103; and Ser, Lys, Arg, or Val at position 104. In some embodiments, two, three, four, five, six, seven, eight, or all nine of positions 45, 47, 49, 95, 97, 99, 102, 103, and 104 have a substitution as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide may include conservative substitutions of particular amino acids at one or more of the positions in the above sets, e.g., amino acids in groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0092] In some embodiments, the modified CH2 domain polypeptide has 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 4-113 of SEQ ID NO: 1, provided that the percent identity excludes the set of positions 45, 47, 49, 95, 97, 99, 102, 103, and 104.

[0093] V. Further mutations within the Fc region Polypeptides linked to agents used in the methods of the invention (e.g., modified to bind to the transferrin receptor and capable of initiating transport across the BBB) may also contain additional mutations, e.g., to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or enable knob-and-hole heterodimerization of the polypeptide.

[0094] In some embodiments, the polypeptide has at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to a corresponding wild-type Fc region (e.g., the Fc region of a human IgG1, IgG2, IgG3, or IgG4).

[0095] Polypeptides may have other mutations introduced outside the designated set of amino acids, for example, to affect glycosylation, increase serum half-life, or, in the case of CH3 domains, to enable knob-and-hole heterodimerization of polypeptides containing the modified CH3 domain. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding hole ("hole") at the interface of a second polypeptide, such that the protrusion can locate within the hole, promoting heterodimer formation and preventing homodimer formation. The protrusion is formed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary hole of the same or similar size as the protrusion is formed at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). Such additional mutations are introduced at positions within the polypeptide that do not negatively affect binding of the modified CH3 or CH2 domain to the transferrin receptor.

[0096] In one exemplary embodiment of a knobs-and-holes approach to dimerization, a first Fc polypeptide subunit to be dimerized has a tryptophan instead of the native threonine at a position corresponding to position 139 of SEQ ID NO: 1, and a second Fc polypeptide subunit of the dimer has a valine instead of the native tyrosine at a position corresponding to position 180 of SEQ ID NO: 1. The second subunit of the Fc polypeptide may further comprise a substitution of serine for the native threonine at a position corresponding to position 139 of SEQ ID NO: 1, and an alanine for the native leucine at a position corresponding to position 141 of SEQ ID NO: 1.

[0097] The polypeptides can also be engineered to include other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface, or hydrophobic patch modifications.

[0098] In some embodiments, modifications can be introduced to extend serum half-life. For example, in some embodiments, the Fc region comprises a CH2 domain comprising Tyr at position 25 of SEQ ID NO:1, Thr at position 27 of SEQ ID NO:1, and Glu at position 29 of SEQ ID NO:1.

[0099] In some embodiments, mutations, e.g., substitutions, are introduced at one or more of positions 17-30, 52-57, 80-90, 156-163, and 201-208 relative to SEQ ID NO: 1. In some embodiments, one or more mutations are introduced at positions 24, 25, 27, 28, 29, 80, 81, 82, 84, 85, 87, 158, 159, 160, 162, 201, 206, 207, or 209 relative to SEQ ID NO: 1. In some embodiments, mutations are introduced at one, two, or three of positions 25, 27, and 29 relative to SEQ ID NO: 1. In some embodiments, the mutations are M25Y, S27T, and T29E relative to SEQ ID NO: 1. In some embodiments, the polypeptides described herein further comprise the mutations M25Y, S27T, and T29E. In some embodiments, mutations are introduced at one or two of positions 201 and 207, as determined by reference to SEQ ID NO:1. In some embodiments, the mutations are M201L and N207S, as numbered by reference to SEQ ID NO:1. In some embodiments, the polypeptides described herein further comprise the mutation N207S, with or without M201L. In some embodiments, the polypeptides described herein comprise substitutions at one, two, or all three of positions T80, E153, and N207, as numbered by reference to SEQ ID NO:1. In some embodiments, the mutations are T80Q and N207A. In some embodiments, the polypeptides described herein comprise the mutations T80A, E153A, and N207A. In some embodiments, the polypeptides described herein comprise substitutions at positions T23 and M201, as numbered by reference to SEQ ID NO:1. In some embodiments, the polypeptides described herein comprise the mutations T23Q and M201L. In some embodiments, the polypeptides described herein comprise substitutions at positions M201 and N207, numbered relative to SEQ ID NO: 1. In some embodiments, the polypeptides described herein comprise substitutions M201L and N207S.In some embodiments, the polypeptides described herein comprise an N207S or N207A substitution.

[0100] Fc effector function In some embodiments, the Fc region (e.g., comprising a modified CH2 or CH3 domain) has effector function (i.e., the Fc region has the ability to induce a particular biological function upon binding to an Fc receptor expressed on an effector cell that mediates that function.) Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells.

[0101] Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions can vary depending on the antibody class. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity upon binding to the appropriate Fc receptor present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 antibodies can induce ADCP function upon binding to the appropriate Fc receptor present on immune cells.

[0102] In some embodiments, the polypeptides described herein can include additional modifications that reduce effector function, or in some embodiments, the polypeptides (e.g., including modified CH2 or CH3 domains) can include additional modifications that enhance effector function.

[0103] Exemplary Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions within the CH2 domain, e.g., substitutions at positions corresponding to positions 7 and 8 of SEQ ID NO: 1. In some embodiments, the substitutions within the modified CH2 domain comprise Ala at positions 7 and 8 of SEQ ID NO: 1. In some embodiments, the substitutions within the modified CH2 domain comprise Ala at positions 7 and 8 and Gly at position 102 of SEQ ID NO: 1.

[0104] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, one or more substitutions at positions 238, 265, 269, 270, 297, 327, and 329 (which, in the EU numbering scheme, correspond to positions 11, 38, 42, 43, 70, 100, and 102 when numbered relative to SEQ ID NO: 1). Exemplary substitutions (when numbered according to the EU numbering scheme) include the following: Position 329 can be mutated by substituting proline with glycine or arginine, or with an amino acid residue of sufficient size to disrupt the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may be present, for example, L234A and L235A in the Fc region of human IgG1, L234A, L235A, and P329G in the Fc region of human IgG1, S228P and L235E in the Fc region of human IgG4, L234A and G237A in the Fc region of human IgG1, L234A, L235A, and G237A in the Fc region of human IgG1, V234A and G237A in the Fc region of human IgG2, L235A, G237A, and E318A in the Fc region of human IgG4, and S228P and L236E in the Fc region of human IgG4. In some embodiments, the polypeptide may have one or more amino acid substitutions that modulate ADCC, for example, substitutions at positions 298, 333, and / or 334 in the Fc region according to the EU numbering scheme.

[0105] In some embodiments, the polypeptides described herein may have one or more amino acid substitutions that increase or decrease ADCC, or may have mutations that alter C1q binding and / or CDC.

[0106] Exemplary Polypeptides Containing Additional Mutations The polypeptide can include additional mutations, including knob mutations (e.g., T139W when numbered with reference to SEQ ID NO:1), hole mutations (e.g., T139S, L141A, and Y180V when numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered with reference to SEQ ID NO:1), and / or mutations that increase serum stability (e.g., (i) M25Y, S27T, and T29E when numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered with reference to SEQ ID NO:1). [Example]

[0107] VI. Working Examples The present invention will now be described in more detail by way of specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield substantially the same results. While efforts have been made to ensure accuracy with respect to values used (e.g., amounts, temperature, etc.), some experimental error and deviation can be expected. The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. Furthermore, it will be apparent to those skilled in the art that methods for manipulation applied to a particular library can also be applied to the other libraries described herein.

[0108] Example 1 Pharmacokinetic / Pharmacodynamic Characterization of CH3C Variants This example describes the pharmacokinetic / pharmacodynamic (PK / PD) characterization of CH3C variant polypeptides in mouse plasma and brain tissue. Specifically, anti-BACE1 agents exhibited lower C max This example demonstrates that when linked to a polypeptide with weaker affinity for TfR, it exhibited higher brain concentrations over time and greater inhibition of Aβ over time compared to a polypeptide with stronger affinity for TfR (see, e.g., Figures 3B, 3C, 4B, and 4C).

[0109] Pharmacokinetics of CH3C variants in wild-type mouse plasma Because the polypeptide-Fab fusions bind only to the human TfR but not to the mouse TfR, the pharmacokinetics (PK) of several CH3C variants was tested in wild-type mice to demonstrate in vivo stability in a model lacking TfR-mediated clearance. The experimental design is shown in Table 3 below. Six- to eight-week-old C57B16 mice were intravenously administered, and exsanguination was performed in situ by submandibular phlebotomy at the time points indicated in Table 3. Blood was collected into EDTA plasma tubes and centrifuged at 14,000 rpm for 5 minutes, after which plasma was isolated for subsequent analysis.

[0110] (Table 3) PK experiment design TIFF2025120209000002.tif77155

[0111] Ab122 was used as an anti-RSV control with normal PK in mice. Ab153 was used as an anti-BACE1 control with normal PK in mice. In this experiment, the Fab arm of Ab153 was fused to each polypeptide.

[0112] Polypeptide concentrations in mouse plasma were quantified using a generic human IgG assay (MSD® Human IgG Kit #K150JLD-4) according to the manufacturer's instructions. Briefly, pre-coated plates were blocked with MSD® Blocker A for 30 minutes. Plasma samples were diluted 1:2,500 using a Hamilton® NIMBUS liquid handling device and added to the blocked plates in duplicate. Dose solutions were also analyzed on the same plate to confirm the correct dose. A standard curve ranging from 0.78 to 200 ng / mL of IgG was fitted using a four-parameter logistic regression. Figure 1 and Table 4 show the results of the analysis of these data. All of the CH3C polypeptide variants had clearance and half-life values comparable to those of the standard Ab122, except for CH3C.3.2-5, which had a significantly faster clearance and shorter half-life. Interestingly, this variant was a point mutation of CH3C.3.2-19(N163D), the latter of which had a normal PK profile.

[0113] (Table 4) PK parameters of CH3C polypeptide-Fab fusions TIFF2025120209000003.tif43163

[0114] PK / PD evaluation of monovalent CH3C.35.N163 in wild-type mouse brain tissue We generated transgenic mice expressing the human Tfrc apical domain within the mouse Tfrc gene using CRISPR / Cas9 technology, and the resulting chimeric TfR was expressed in vivo under the control of the endogenous promoter.

[0115] Chimeric hTfR アピカル+ / +Heterozygous mice (n = 4 / group) were intravenously administered either 42 mg / kg of Ab153 or monovalent CH3C.35.N163, and wild-type mice (n = 3) were intravenously administered 50 mg / kg of control human IgG1. Ab153 was used as a control with normal PK in mice. All mice were perfused with PBS 24 hours after administration. Prior to perfusion, blood was collected by cardiac puncture into EDTA plasma tubes and centrifuged at 14,000 rpm for 5 minutes. Plasma was then isolated for subsequent PK and PD analysis. After perfusion, brains were removed, and the hemispheres were separated and homogenized in 10 times the tissue weight of PBS, 1% NP-40 (for PK) or 5 M GuHCl (for PD).

[0116] Figure 2 shows the results of the brain PK experiment. Uptake was greater in the monovalent CH3C.35.N163 group than in the Ab153 and control human IgG1 groups.

[0117] hTfR アピカル+ / + Brain and plasma PKPD of polypeptide-Fab fusions in mice: CH3C.35.21, CH3C.35.20, CH3C.35, CH3C.35.23, CH3C.35.23.3 To assess the effect of TfR binding affinity on PK and brain uptake, we generated anti-BACE1 Ab153 and engineered TfR-binding polypeptide fusions (CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions) with different binding affinities to the apical human TfR as measured by Biacore. The binding affinities of the CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions to the human TfR are 100 nM, 170 nM, and 620 nM, respectively. + / +Mouse knock-in mice were systemically administered 50 mg / kg of Ab153 or each polypeptide-Fab fusion, and plasma PK and brain PKPD were assessed 1, 3, and 7 days after administration. Brain and plasma PKPD analyses were performed as described in the previous sections. Due to TfR expression in peripheral tissues, the CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions showed more rapid plasma clearance compared to Ab153 alone, consistent with target-mediated clearance and indicative of TfR binding in vivo (Figure 3A). Strikingly, the brain concentrations of the CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions were significantly increased compared to Ab153, achieving a maximum brain concentration of >30 nM on day 1 post-injection compared to only approximately 3 nM for Ab153 at the same time point (Figure 3B). The increased brain exposure of the CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions resulted in approximately 55–60% lower endogenous mouse Aβ levels in the brain compared to Aβ levels in Ab153-treated mice (Figure 3C). Concentrations of CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions remained elevated in the brain, while lower brain Aβ levels were maintained, returning to levels similar to those observed in Ab153-treated mice when exposure was reduced by day 7. The reduction in brain exposure over time correlated with the reduced peripheral exposure of CH3C.35.21:Ab153, CH3C.35.20:Ab153, and CH3C.35:Ab153 fusions, demonstrating a clear PK / PD relationship in vivo (compare Figures 3A and 3C). Furthermore, total brain TfR levels were comparable between Ab153- and polypeptide-Fab fusion-treated mice after this single high-dose administration, indicating that increased brain exposure of the polypeptide-Fab fusions did not significantly affect TfR expression in the brain (Figure 3D).

[0118] To further evaluate the relationship between PK and brain uptake across a broader affinity range of engineered TfR-binding polypeptide-Fab fusions, additional fusions with a broader affinity range for binding to hTfR were generated. The binding affinities of the CH3C.35.23:Ab153 and CH3C.35.23.3:Ab153 fusions for human TfR are 420 nM and 1440 nM, respectively. hTfR apical + / + Knockin mice were dosed as described above. Plasma PK and brain PKPD were assessed 1, 4, 7, and 10 days after dosing. Peripheral PK of polypeptide-Fab fusions was dependent on hTfR affinity, with higher affinity CH3C.35.23:Ab153 fusions exhibiting more rapid clearance compared to the significantly lower affinity CH3C.35.23.3:Ab153 fusion (Figure 4A). Both CH3C.35.23:Ab153 and CH3C.35.23.3:Ab153 fusions had significantly higher brain exposure compared to Ab153 alone, with CH3C.35.23:Ab153 reaching approximately 36 nM in the brain at 1 day post-dose (Figure 4B). Despite similar plasma concentrations, this maximum brain uptake of the CH3C.35.23.3:Ab153 fusion was lower than that of the CH3.35.23:Ab153 fusion, likely due to the approximately 3.5-fold lower affinity of the latter fusion for hTfR. Interestingly, the lower affinity fusion provided more sustained peripheral exposure by day 10, and therefore its brain exposure was also higher than that of the higher affinity CH3C.35.23:Ab153 fusion. This suggests that the lower affinity engineered TfR-binding polypeptide-Fab fusions did not result in brain C maxThis demonstrates a trade-off: lower activity but more sustained PK over time. Compared to anti-BACE1 alone, significantly lower concentrations of Aβ40 were observed in the brains of mice treated with the anti-BACE1 polypeptide fusion (Figure 4C). The duration of this Aβ40 reduction coincided with the time-dependent level of huIgG1 exposure in the brain (Figure 4B). Strikingly, mice treated with the CH3C.35:Ab153 fusion showed sustained brain Aβ40 reduction for 7–10 days after a single dose. Overall brain TfR levels were comparable between mice treated with the CH3C.35:Ab153 fusion and Ab153 at day 1 post-dose (Figure 4D). Together, these data demonstrate that engineered TfR-binding polypeptide fusions can significantly reduce brain Aβ40 after a single dose by increasing brain exposure of anti-BACE1.

[0119] Example 2 Selection of TfR-binding polypeptide affinities This example describes the relationship between the affinity of a TfR-binding polypeptide for the transferrin receptor (TfR) and the resulting brain exposure to a therapeutic agent linked to the TfR-binding polypeptide.

[0120] 5 shows that brain exposure to therapeutic agents (as assessed by the area under the curve (AUC) of brain concentration versus time) was greatest when the therapeutic agents were linked to polypeptides with relatively low affinity for the TfR. Specifically, brain exposure was significantly increased when the therapeutic agents were linked to polypeptides with an affinity for the TfR weaker than about 250 nM.

[0121] As shown in Figure 6, the lower peak blood concentration (C max ) was observed when the therapeutic agent was linked to a polypeptide with a relatively weak affinity for TfR. max The values were significantly lower when the TfR-binding polypeptide had an affinity lower than about 250 nM.

[0122] Figure 7 shows the relationship between plasma concentration and brain C of therapeutic agents when linked to a range of polypeptides with affinity for TfR. max The ratio of

[0123] method hTfR アピカル+ / + Preparation of KI Methods for generating knock-in / knock-out mice have been published in the literature and are well known to those skilled in the art. In summary, hTfR expressing the human Tfrc apical domain within the mouse Tfrc gene using CRISPR / Cas9 technology has been developed. アピカル+ / + KI mice were generated, and the resulting chimeric TfR was expressed in vivo under the control of the endogenous promoter. A knock-in human apical TfR mouse line was generated using C57B16 mice by pronuclear microinjection into single-cell embryos followed by embryo transfer into pseudopregnant females, as described in International Application No. PCT / US2018 / 018302, incorporated herein by reference in its entirety. Specifically, Cas9, single-guide RNA, and donor DNA were introduced into the embryos. The donor DNA contained a human apical domain coding sequence codon-optimized for mouse expression. The apical domain coding sequence was flanked by left and right homology arms. The donor sequence was designed so that the apical domain was inserted after the fourth mouse exon and immediately adjacent to the ninth exon at the 3′ end. Founder males from the offspring of the females into which the embryos were transferred were mated with wild-type females to generate F1 heterozygous mice. Next, homozygous mice were produced by mating the heterozygous mice of the F1 generation.

[0124] Mouse PKPD For PK / PD evaluation, hTfR アピカル+ / +KI mice received a single systemic dose of 50 mg / kg via tail vein injection. Prior to perfusion, blood was collected by cardiac puncture into EDTA plasma tubes and centrifuged at 14,000 rpm for 5 minutes. Plasma was then isolated for subsequent PK / PD analysis. After perfusion, brains were removed, and the hemispheres were separated and homogenized in 10x the tissue weight of PBS containing 1% NP-40 (for PK) or 5M GuHCl (for PD).

[0125] Antibody concentrations in mouse plasma and brain lysates were quantified using a generic human IgG assay (MSD Human IgG Kit #K150JLD) according to the manufacturer's instructions. Briefly, precoated plates were blocked with MSD Blocker A for 30 minutes. Plasma samples were diluted 1:10,000 using a Hamilton Nimbus liquid handling device and added to the blocked plates in duplicate. Brain samples were homogenized in 1% NP-40 lysis buffer, and the lysates were diluted 1:10 for PK analysis. Dose solutions were also analyzed on the same plate to confirm the appropriate dose. A standard curve ranging from 0.78 to 200 ng / mL of IgG was fitted using four-parameter logistic regression.

[0126] Example 3. Characterization of Binding of CH3C Variants Using Biacore™ The affinity of clonal variants of the recombinant TfR apical domain was measured by surface plasmon resonance using a Biacore™ T200 instrument. A Biacore™ Series S CM5 sensor chip was immobilized with anti-human Fab (human Fab capture kit, available from GE Healthcare). 5 μg / mL of polypeptide-Fab fusion was captured on each flow cell for 1 min, and serial 3-fold dilutions of human or cynomolgus monkey apical domain were injected at a flow rate of 30 μL / min at room temperature. Each sample was analyzed with 45 s of association and 3 min of dissociation. After each injection, the chip was regenerated with 10 mM glycine-HCl (pH 2.1). Binding responses were corrected by subtracting the RU from a flow cell in which an irrelevant IgG was captured at a similar density. Steady-state affinities were obtained by fitting the equilibrium responses to concentration using Biacore™ T200 Evaluation Software v3.1.

[0127] To determine the affinity of clonal variants of recombinant TfR extracellular domain (ECD), Biacore™ Series S CM5 sensor chips were immobilized with streptavidin. Biotinylated human or cynomolgus TfR ECD was captured on each flow cell for 1 min, and serial 3-fold dilutions of each clonal variant were injected at a flow rate of 30 μL / min at room temperature. Each sample was analyzed with 45 s of binding and 3 min of dissociation. Binding responses were corrected by subtracting the RU from a flow cell without TfR ECD at a similar density. Steady-state affinities were obtained by fitting the equilibrium responses to concentration using Biacore™ T200 Evaluation Software v3.1.

[0128] The binding affinities are summarized in Table 5. The affinities were obtained by steady-state fitting.

[0129] (Table 5) Binding affinities of additional CH3C variants TIFF2025120209000004.tif103138

[0130] Additional CH3C variants CH3C.35.20.1.1, CH3C.35.23.2.1, CH3C.35.23.1.1, CH3C.35.S413, CH3C.35.23.3.1, CH3C.35.N390.1, and CH3C.35.23.6.1 were generated and their binding affinities to human TfR were measured following the same protocol described above. The binding affinities of CH3C.35.20.1.1, CH3C.35.23.2.1, CH3C.35.23.1.1, CH3C.35.S413, CH3C.35.23.3.1, CH3C.35.N390.1, and CH3C.35.23.6.1 are 620 nM, 690 nM, 750 nM, 1700 nM, 1900 nM, 2000 nM, and 2100 nM, respectively.

[0131] It will be understood that the examples and embodiments set forth herein are for illustrative purposes only, and that various modifications or variations will be suggested to those skilled in the art in light of the same, and that such modifications and variations are intended to be included within the spirit and scope of this application and the appended claims. Sequences with sequence accession numbers cited herein are hereby incorporated by reference.

[0132] (Table 1) CH3C register location and mutations TIFF2025120209000005.tif158170

[0133] (Table 2) Search for permissible diversity and hotspot locations within the register for CH3C.35.21 TIFF2025120209000006.tif219170

[0134] Unofficial sequence listing TIFF2025120209000007.tif156169

[0135] Sequence information SEQUENCE LISTING <110> DENALI THERAPEUTICS INC. <120> AFFINITY-BASED METHODS FOR USING TRANSFERRIN RECEPTOR-BINDING PROTEINS <150> PCT / US2018 / 018371 <151> 2018-02-15 <150> US 62 / 583,314 <151> 2017-11-08 <150> US 62 / 543,658 <151> 2017-08-10 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 220 <212> PRT <213> Homo sapiens <400> 1 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 115 120 125 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 2 <211> 113 <212> PRT <213> Homo sapiens <400> 2 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys <210> 3 <211> 107 <212> PRT <213> Homo sapiens <400> 3 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 4 <211> 181 <212> PRT <213> Homo sapiens <400> 4 Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val 1 5 10 15 Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr 20 25 30 Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp 35 40 45 Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys 50 55 60 Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile 65 70 75 80 Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala 85 90 95 Glu Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr 100 105 110 Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg 115 120 125 Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala 130 135 140 Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp 145 150 155 160 Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val 165 170 175 Lys Leu Thr Val Ser 180 <210> 5 <211> 15 <212> PRT <213> Homo sapiens <400> 5 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 6 <211> 760 <212> PRT <213> Homo sapiens <400> 6 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60 Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly 65 70 75 80 Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95 Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110 Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120 125 Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile 130 135 140 Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln 145 150 155 160 Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe 165 170 175 Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190 Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205 Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220 Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys 225 230 235 240 Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile 245 250 255 Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu 260 265 270 Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285 Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300 Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln 305 310 315 320 Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335 Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350 Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360 365 Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile 370 375 380 Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp 385 390 395 400 His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415 Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430 Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445 Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460 Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr 465 470 475 480 Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val 485 490 495 Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn 500 505 510 Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp 515 520 525 Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540 Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp 545 550 555 560 Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575 Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590 Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600 605 Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp 610 615 620 Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln 625 630 635 640 Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655 Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys 660 665 670 Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro 675 680 685 Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700 Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys 705 710 715 720 Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala 725 730 735 Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp 740 745 750 Val Trp Asp Ile Asp Asn Glu Phe 755 760

Claims

1. 1. A method for transporting an agent that binds to a therapeutic target across the blood-brain barrier (BBB) in a mammal, comprising: The method includes exposing the BBB to a protein that binds to the transferrin receptor (TfR) with an affinity of about 400 nM to about 2 μM, wherein the protein is linked to the agent and transports the linked agent across the BBB.

2. The method of claim 1 , wherein the brain exposure time to the agent is extended.

3. The method of claim 1 or 2, wherein the therapeutic target is involved in a neurodegenerative disease.

4. 1. A method for treating a neurodegenerative disease, comprising administering to a mammal a protein that binds to TfR with an affinity of about 400 nM to about 2 μM, wherein said protein is linked to an agent that binds to a therapeutic target involved in said neurodegenerative disease, thereby extending the exposure time of the mammal's brain to said agent.

5. 5. The method of any one of claims 1 to 4, wherein the protein extends brain exposure time to the agent compared to an agent linked to a reference protein that binds to the TfR with greater affinity.

6. 6. The method of claim 5, wherein brain exposure is determined by measuring the area under the curve (AUC) of a plot of brain concentration of the agent against time.

7. 7. The method of any one of claims 1-6, wherein the protein extends brain exposure time to the agent at therapeutically effective concentrations in the mammal compared to an agent linked to a reference protein that binds to the TfR with greater affinity.

8. The method of any one of claims 5 to 7, wherein the reference protein binds to the TfR with an affinity of about 50 nM or stronger.

9. The method of any one of claims 1 to 8, wherein the TfR is a primate TfR.

10. 10. The method of claim 9, wherein the primate TfR is a human TfR.

11. The method of any one of claims 1 to 10, wherein the protein binds to the TfR apical domain.

12. The method of any one of claims 1 to 11, wherein the protein binds to the TfR with an affinity of about 420 nM to about 1.5 μM.

13. 13. The method of any one of claims 1 to 12, wherein the protein binds to the TfR with an affinity of about 600 nM to about 1.5 μM.

14. 14. The method of any one of claims 7 to 13, wherein the therapeutically effective concentration of the agent is a concentration that treats one or more symptoms of a neurodegenerative disease in the mammal.

15. 15. The method of any one of claims 3 to 14, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), and combinations thereof.

16. The method of any one of claims 1 to 15, wherein the agent comprises an antibody variable region.

17. The method of claim 16 , wherein the agent comprises an antibody fragment.

18. 18. The method of claim 17, wherein the agent comprises a Fab or scFv.

19. The method of any one of claims 1 to 18, wherein the protein is a modified Fc polypeptide comprising a non-native binding site capable of binding to TfR.

20. The method of any one of claims 1 to 18, wherein the protein comprises an antibody variable region that specifically binds to TfR.

21. 21. The method of claim 20, wherein the protein comprises an antibody fragment.

22. 22. The method of claim 21, wherein the protein comprises a Fab or scFv.

23. 23. The method of any one of claims 1 to 22, wherein the therapeutic target is selected from the group consisting of beta-secretase 1 (BACE1) protein, tau protein, triggering receptor expressed on myeloid cells 2 (TREM2) protein, and alpha-synuclein protein.

24. 24. The method of any one of claims 5 to 23, wherein the therapeutic target is BACE1 and the agent, when linked to the protein, reduces the amount of amyloid beta protein (Aβ) present in the brain of the mammal for a longer period of time compared to when linked to the reference protein.

25. The method of any one of claims 4 to 24, wherein the protein linked to the agent is administered as part of a pharmaceutically acceptable carrier.

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