Dual transporter and instructions for use

Abstract

A dual transporter and methods for producing and using the dual transporter are described. The dual transporter binds to a blood-brain barrier transporter and a second protein which is expressed on the luminal surface of the blood-brain barrier or is a brain-retained protein. The dual transporter improves delivery to the central nervous system. A dual transporter is described comprising a first binding domain that specifically binds to the BBB transporter and a second binding domain that specifically binds to the second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transporter.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 509,148, filed on June 20, 2023, which is incorporated herein by reference.

[0002] Sequence List The sequence listings contained in the DNL-027-05_SeqListing.xml file are 131 kilobytes in size, were created on June 14, 2024, and are incorporated herein by reference. [Background technology]

[0003] The blood-brain barrier (BBB) ​​blocks the passage of most macromolecules from the periphery to the brain, thus limiting the use of macromolecular therapeutics that require brain exposure. Therefore, the BBB poses a significant obstacle to delivering therapeutic molecules to the brain or CNS to treat neurological disorders or conditions of the central nervous system (CNS). In addition to the need to improve therapeutic molecule delivery across the BBB, improvements in peripheral delivery to specific tissues are also desirable.

[0004] To deliver cargo across the blood-brain barrier (BBB), moieties that bind to individual receptors such as transferrin receptors (TfRs) or CD98hc are used. Nevertheless, improved compositions and methods that enhance BBB transport or target specific peripheral tissues beyond what these individual binding moieties offer alone are therefore desired. [Overview of the project]

[0005] A dual transporter is described comprising a first binding region that specifically binds to a BBB transporter protein and a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is distinct from the BBB transporter protein. In some embodiments, the first binding region specifically binds to the BBB transporter protein to increase the transport of the dual transporter (or a molecule linked to the dual transporter) across the BBB, and the second binding protein specifically binds to the second protein to increase the retention (i.e., exposure time) of the dual transporter (or molecular linker to the dual transporter) in the CNS. In some embodiments, the BBB transporter protein is transferrin receptor 1 (TfR) or CD98hc (also known as the 4F2 cell surface antigen heavy chain). In some embodiments, the BBB transporter protein is alkaline phosphatase (ALPL). The second protein may be a BBB surface protein (a protein expressed on the luminal surface of brain endothelial cells contained in the BBB) or a brain-retained protein. In some embodiments, the BBB surface protein is a second BBB transport protein.In some embodiments, the second protein is TfR, CD98hc, large neutral amino acid transporter small subunit 1 (CD98 light chain or CD98lc), glucose transporter 1 (GLUT1), major promoter superfamily domain-containing protein 2A (MFSD2A), carbonic anhydrase IV (CA-IV), alkaline phosphatase (ALPL), low-density lipoprotein receptor, insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane The group is selected from the following: protein 50A, basidine, leptin receptor, claudin-5, P-selectin, lactoferrin receptor, folate receptor, sodium-dependent lysophosphatidylcholine cotransporter 1, solute transporter organic anion transporter family member 1C1, sodium-coupled neutral amino acid transporter 5, LDL receptor-related protein 8, high affinity cationic amino acid transporter 1, sodium chloride-dependent taurine transporter, insulin-like growth factor-binding protein 7, solute transporter family 40 member 1, zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and myelin oligodendrocyte glycoprotein (MOG). By binding to the BBB transporter and the second protein, the dual transporter, and optionally the therapeutic agent linked to the dual transporter, exhibit significantly increased uptake into the brain, significantly increased retention in the brain (i.e., brain exposure time), or both, compared to molecules that bind to the BBB transporter but not to the second protein.In some embodiments, by binding to a BBB transport protein and a second protein, the uptake of a dual transporter, and optionally a therapeutic agent linked to the dual transporter, into cells or tissues expressing the BBB transport protein and the second protein is significantly increased, or the retention of the dual transporter, and optionally a therapeutic agent linked to the dual transporter, in cells or tissues expressing the BBB transport protein and the second protein is significantly increased, or both, as compared to a molecule that binds to the BBB transport protein but not to the second protein.

[0006] In some embodiments, the BBB transport protein is TfR and the second protein is a brain retention protein. In some embodiments, the brain retention protein is CD98hc or MOG, or a protein selected from the group consisting of GLUT1, MFSD, IGF1R, and CA-IV. In some embodiments, the BBB transport protein is TfR and the second protein is CD98hc. In some embodiments, the dual transporter comprises a TfR binding region and a CD98hc binding region. In some embodiments, the BBB transport protein is TfR and the second protein is MOG. In some embodiments, the dual transporter comprises a TfR binding region and a MOG binding region. In some embodiments, the BBB transport protein is CD98hc and the second protein is a brain retention protein. In some embodiments, the dual transporter comprises a CD98hc binding region and a brain retention protein binding region.

[0007] Using the described dual transporter, the delivery of therapeutic agents to the CNS can be improved or increased. In some embodiments, the dual transporter is conjugated to the therapeutic agent. By conjugating the dual transporter to the therapeutic agent, the delivery of the therapeutic agent to the CNS is increased compared to the level of delivery of the therapeutic agent to the CNS in the absence of conjugation to the dual transporter. In some embodiments, by conjugating the dual transporter to the therapeutic agent, the delivery of the therapeutic agent to the CNS is increased compared to the level of delivery of the therapeutic agent conjugated to a molecule that binds to the BBB transport protein but not to the second protein. In some embodiments, a therapeutic agent such as an antibody is modified to contain a first binding region that specifically binds to the BBB transport protein and a second binding region that specifically binds to the second protein, and the second protein is expressed on the luminal surface of the BBB or is a brain retention protein, and the second protein is different from the BBB transport protein (e.g., a therapeutic agent such as an antibody can be modified to contain a dual transporter function). By modifying a therapeutic agent such as an antibody to contain a dual transporter function, the delivery of the therapeutic agent to the CNS is increased compared to the level of delivery of the therapeutic agent to the CNS in the absence of a drug modification that contains a dual transporter function.

[0008] A method for improving the delivery of therapeutic agents and other drugs to the CNS is described, which includes linking a dual transporter to the drug, or modifying the drug to include a first binding region that specifically binds to a BBB transporter protein and a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the modification (for example, modifying the drug to include dual transporter function) is different from the BBB transporter protein. Improving drug delivery to the CNS includes, but is not limited to, (a) increasing the concentration of the drug delivered to the CNS compared to the concentration of the drug delivered to the CNS in the absence of the dual transporter, (b) decreasing the dose (amount) of the drug that needs to be administered to the subject to achieve a therapeutic effect or concentration of the drug delivered to the CNS compared to the dose that needs to be administered to the subject to achieve a therapeutic effect or concentration of the drug delivered to the CNS in the absence of the dual transporter, (c) enhancing the potency of the drug compared to the potency of the drug in the absence of the dual transporter, (d) improving the safety of the drug compared to the safety of the drug in the absence of the dual transporter (e.g., reducing toxicity), (e) improving the biodistribution and / or pharmacokinetics of the drug compared to the biodistribution and / or pharmacokinetics of the drug in the absence of the dual transporter, (f) increasing the brain uptake Cmax of the drug compared to the brain uptake Cmax of the drug in the absence of the dual transporter, and (g) increasing the specificity of drug delivery to the brain compared to the specificity of drug delivery to the brain in the absence of the dual transporter. By linking a dual transporter to a drug, or by modifying a drug to have dual transporter function, drug delivery to the CNS is also improved compared to linking a drug to a binding domain that specifically binds to a BBB transporter protein, where there is no second binding domain that specifically binds to a second protein.

[0009] In some embodiments, by binding to a BBB transporter protein and a second protein, the dose of the therapeutic agent required to be administered to a subject to achieve a desired or therapeutic level of the agent in the CNS (e.g., the brain) is reduced compared to the dose required for the therapeutic agent alone or for the therapeutic agent linked to a molecule that binds to the BBB transporter protein but not to the second protein. Reducing the dosage can provide an improved safety margin for the patient.

[0010] The dual transporter described can be used to improve the delivery of therapeutic agents to peripheral cells or tissues (cells that are not part of the CNS) expressing the BBB transporter protein and the second protein. Peripheral cells or tissues expressing the BBB transporter protein and the second protein include, but are not limited to, proliferating cells, cancer cells, and tumors. In some embodiments, the dual transporter is ligated to the therapeutic agent. Ligation of the therapeutic agent to the dual transporter improves the delivery of the therapeutic agent to peripheral cells or tissues compared to the level of delivery of the therapeutic agent to peripheral cells or tissues in the absence of ligation to the dual transporter. In some embodiments, ligation of the therapeutic agent to the dual transporter improves the delivery of the therapeutic agent to peripheral cells or tissues compared to the level of delivery of the therapeutic agent linked to a molecule that binds to the BBB transporter protein but not to the second protein. In some embodiments, therapeutic agents such as antibodies are modified to include a first binding region that specifically binds to a BBB transporter protein and a second binding region that specifically binds to a second protein, the second protein being expressed on the luminal surface of the BBB or a protein retained in the brain, and the second protein being different from the BBB transporter protein (for example, therapeutic agents such as antibodies may be modified to contain dual transporter function). By modifying therapeutic agents such as antibodies to contain dual transporter function, the delivery of the therapeutic agent to peripheral cells or tissues is improved compared to the level of delivery of the therapeutic agent to peripheral cells or tissues when there is no drug modification containing dual transporter function.

[0011] A method for improving the delivery of therapeutic agents or other drugs to peripheral cells or tissues (cells not part of the CNS) expressing a BBB transporter protein and a second protein is described, comprising: linking a dual transporter to the drug; or modifying the drug to include a first binding region that specifically binds to the BBB transporter protein and a second binding region that specifically binds to the second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the modification (e.g., modifying the drug to include dual transporter function) is different from the BBB transporter protein. Peripheral cells or tissues expressing the BBB transporter protein and the second protein include, but are not limited to, proliferating cells, cancer cells, and tumors. Improving the delivery of drugs to peripheral cells or tissues includes, but is not limited to, (a) increasing the concentration of drugs delivered to peripheral cells or tissues compared to the concentration of drugs delivered to peripheral cells or tissues in the absence of dual transporters, (b) decreasing the dose (amount) of drugs that need to be administered to a subject to achieve a therapeutic effect or concentration of drugs delivered to peripheral cells or tissues compared to the dose that needs to be administered to a subject to achieve a therapeutic effect or concentration of drugs delivered to peripheral cells or tissues in the absence of dual transporters, (c) enhancing the potency of drugs compared to the potency of drugs in the absence of dual transporters, (d) increasing the uptake of drugs into peripheral cells or tissues compared to the level of drug uptake into peripheral cells or tissues in the absence of dual transporters, (e) improving the safety of drugs compared to the safety of drugs in the absence of dual transporters, and / or (f) improving the biodistribution and / or pharmacokinetics of drugs compared to the biodistribution and / or pharmacokinetics of drugs in the absence of dual transporters.By linking a dual transporter to a drug, or by modifying a drug to have dual transporter function, drug delivery to peripheral cells or tissues is also improved compared to linking a drug to a binding domain that specifically binds to a BBB transporter protein, where there is no second binding domain that specifically binds to a second protein.

[0012] In some embodiments, by binding to the BBB transport protein and a second protein, the dose of the therapeutic agent required to be administered to a subject to achieve a desired or therapeutic level of the agent in peripheral cells or tissues is reduced compared to the dose required for the therapeutic agent alone, or the dose required for the therapeutic agent linked to a molecule that binds to the BBB transport protein but not to the second protein. Reducing the dosage can provide an improved safety margin for the patient. [Brief explanation of the drawing]

[0013] [Figure 1] A is a schematic diagram of an exemplary dual transporter having a heterodimeric Fc polypeptide modified to specifically bind to a BBB transporter protein (first binding domain) and a second protein (second binding domain). The heterodimeric Fc polypeptide is fused to Fab. Fab can bind to one or two therapeutic targets. B is a schematic diagram of an exemplary dual transporter having a heterodimeric Fc polypeptide modified to specifically bind to TfR (anti-TfR) and CD98hc (anti-CD98hc). The heterodimeric Fc polypeptide is fused to Fab. Fab can bind to one or two therapeutic targets.

[0014] [Figure 2A] This is a schematic diagram of an exemplary dual transporter having a VHH-Fc / Fab-Fc format. It shows a dual transporter having a Fab domain with a first binding region and a VHH domain with a second binding region. [Figure 2B]This is a schematic diagram of an exemplary dual transporter having a VHH-Fc / Fab-Fc format. It shows a dual transporter having a Fab domain that specifically binds to TfR (anti-TfR) and a VHH domain that specifically binds to CD98hc (anti-CD98hc).

[0015] [Figure 2C] This is a schematic diagram of an exemplary dual transporter having an scFv domain and a VHH domain ligated to a full-length antibody. The scFv domain is ligated to the C-terminus of one heavy chain of the antibody, and the VHH domain is ligated to the C-terminus of the other heavy chain of the antibody. The diagram shows a dual transporter having an scFv domain with a first binding region and a VHH domain with a second binding region. [Figure 2D] This is a schematic diagram of an exemplary dual transporter having an scFv domain and a VHH domain ligated to a full-length antibody. The scFv domain is ligated to the C-terminus of one heavy chain of the antibody, and the VHH domain is ligated to the C-terminus of the other heavy chain. The diagram shows a dual transporter having scFv that specifically binds to TfR (anti-TfR) and VHH that specifically binds to CD98hc (anti-CD98hc).

[0016] [Figure 3A] This is a schematic diagram of an additional exemplary dual transporter. It shows a dual transporter containing a full-length antibody, having first and second binding regions (e.g., scFv), each ligated to the C-terminus of the antibody's light chain. The antibody Fab can bind to one or two therapeutic targets. [Figure 3B] This is a schematic diagram of an additional exemplary dual transporter. It shows a dual transporter containing a full-length antibody, having a first binding region (e.g., scFab) linked to the antibody's heavy chain, and an antibody Fab arm that specifically binds to a second protein. The second Fab arm of the antibody can bind to a therapeutic target. [Figure 3C]This is a schematic diagram of an additional exemplary dual transporter. It shows a dual transporter molecule having a DVD-Ig format. DVD-Ig contains a full-length antibody with additional Fv fragments ligated to each Fab arm. Example C shows an additional Fv fragment having a first binding region ligated to a Fab arm having a second binding region. Other additional Fv fragments and other Fab arms can bind to one or two therapeutic targets.

[0017] [Figure 3D] This is a schematic diagram of an additional exemplary dual transporter. It shows a dual transporter containing a full-length antibody, having an anti-TfR scFv ligated to the C-terminus of one light chain of the antibody and an anti-Cd98hc scFv ligated to the C-terminus of the other light chain of the antibody. [Figure 3E] This is a schematic diagram of an additional exemplary dual transporter. It shows a dual transporter containing a full-length antibody, having one Fab arm bound to CD98hc, and an anti-TfR scFab ligated to the C-terminus of one of the antibody's heavy chains. The other Fab arm of the antibody can bind to a therapeutic target. [Figure 3F] This is a schematic diagram of an additional exemplary dual transporter. It shows a DVD-Ig dual transporter molecule having an anti-TfR Fv fragment ligated to a Fab arm that specifically binds to CD98hc. The other Fv fragment of DVD-Ig and the Fab arm can bind to one or two therapeutic targets.

[0018] [Figure 4] This graph shows the binding of exemplary antibody transport vehicles (ATVs) that bind only to TfR, ATVs that bind only to CD98hc, dual transporters that bind to both TfR and CD98hc, and negative control antibodies that do not bind to either TfR or CD98hc, to HEK293T cells expressing both TfR and CD98hc.

[0019] [Figure 5] This graph shows the binding of specific ATVs to HEK293T cells expressing both TfR and CD98hc: ATVs that bind only to TfR, ATVs that bind only to CD98hc, dual ATVs that bind to both TfR and CD98hc, and negative control antibodies that do not bind to either TfR or CD98hc.

[0020] [Figure 6] This is a graph from one experiment showing the plasma and brain concentrations of a specific dual transporter and its corresponding single-binding control in mice after a single intravenous administration of 50 mg / kg.

[0021] [Figure 7] Example 8 shows graphs illustrating the plasma and brain concentrations of a specific dual transporter in mice after a single intravenous administration, using molecular format matching standards.

[0022] [Figure 8-1] This graph shows the plasma and brain concentrations of a specific dual transporter and its corresponding single-binding control after a single intravenous administration of 25 mg / kg or 10 mg / kg in mice. [Figure 8-2] This graph shows the plasma and brain concentrations of a specific dual transporter and its corresponding single-binding control after a single intravenous administration of 25 mg / kg or 10 mg / kg in mice.

[0023] [Figure 9] This graph shows the plasma pharmacokinetics of a specific dual transporter with different binding affinities for TfR and CD98hc. These data indicate that, in the case of a double-bound molecule, the affinity for TfR can be a stronger plasma clearance driver than that for CD98hc.

[0024] [Figure 10]This graph shows the brain pharmacokinetics of a specific dual transporter with different binding affinities to TfR and CD98hc. These data indicate that, when tested in a double-binding format, stronger TfR affinity induces rapid and high uptake, while stronger CD98hc affinity induces increased brain retention (i.e., brain exposure time).

[0025] [Figure 11] This graph shows the pharmacokinetics in the kidney of a specific dual transporter with different binding affinities for TfR and CD98hc. These data indicate that the in vivo distribution of the double-bound molecule in the kidney is strongly correlated with the affinity for CD98hc.

[0026] [Figure 12] This graph shows the pharmacokinetics in the bone marrow of a specific dual transporter with different binding affinities to TfR and CD98hc. These data indicate that the biodistribution of the double-binding molecule in the bone marrow is strongly correlated with its affinity to TfR.

[0027] [Figure 13-1] This study quantifies the local biodistribution in the brain of a specific dual transporter with different binding affinities for TfR and CD98hc. These data demonstrate that binding to both TfR and CD98hc results in a relative increase in biodistribution to the medulla oblongata and pons, which is not observed with molecules that bind to only CD98hc or only TfR. [Figure 13-2] This study quantifies the local biodistribution in the brain of a specific dual transporter with different binding affinities for TfR and CD98hc. These data demonstrate that binding to both TfR and CD98hc results in a relative increase in biodistribution to the medulla oblongata and pons, which is not observed with molecules that bind to only CD98hc or only TfR.

[0028] [Figure 14-1]This study quantifies the in vivo distribution of a specific dual transporter with different binding affinities to TfR and CD98hc in CNS cells. These data suggest that for double-binding molecules, the TfR-mediated in vivo distribution to neurons can be reduced in a CD98hc affinity-dependent manner. [Figure 14-2] This study quantifies the in vivo distribution of a specific dual transporter with different binding affinities to TfR and CD98hc in CNS cells. These data suggest that for double-binding molecules, the TfR-mediated in vivo distribution to neurons can be reduced in a CD98hc affinity-dependent manner.

[0029] [Figure 15] This graph shows the pharmacokinetics in the peripheral and brain of dual transporters with different formats (Example 7) and transporter affinity.

[0030] [Figure 16] This graph shows the peripheral and cerebral pharmacokinetics of the ATVTfR:MOG1 molecule, indicating that MOG1 binding induces retention and reduces TfR-mediated efflux from the brain. [Modes for carrying out the invention]

[0031] I. Definition As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to "a polypeptide" may include two or more such molecules.

[0032] As used herein, the terms “about” and “approximately,” when used as modifiers for a quantity specified by a number or range, indicate the number and a reasonable deviation from that number, such deviation being known to those skilled in the art. In some embodiments, the term “about” means within a typical acceptable range in the art. In some embodiments, the term “about” means within one standard deviation or two standard deviations from the mean. In some embodiments, the term “about” means ±10%. In some embodiments, the term “about” refers to ±5%. When the term “about” precedes a series of numbers or a range, it is understood that “about” may modify each of the numbers in that series or range.

[0033] As used in connection with this invention, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1 (encoded by the TfR1(CD71) gene). The human transferrin receptor 1 polypeptide sequence is described in Sequence ID No. 15. Sequences of transferrin receptor protein 1 from other species are also known (e.g., chimpanzee, accession number XP003310238.1; rhesus macaque, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP035768.1; rat, P_073203.1; and chicken, P_990587.1). The term “transferrin receptor” also includes allele variants of exemplary reference sequences, e.g., human sequences, encoded by the gene at the chromosomal locus of transferrin receptor protein 1. The full-length transferrin receptor protein contains a short N-terminal intracellular domain, a transmembrane domain, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is described in SEQ ID NO: 1.

[0034] "CD98hc" or "CD98 heavy chain" refers to the 4F2 cell surface antigen heavy chain, encoded by the SLC3A2 gene. CD98hc is also known as the 4F2 heavy chain. The human CD98hc sequence is described in SEQ ID NO: 5 and UNIPROT accession number P08195. CD98hc sequences from other species are also known (e.g., mouse, UNIPROT accession number P10852 and cynomolgus monkey, UNIPROT accession number G8F3Z0).

[0035] "MOG" refers to myelin oligodendrocyte glycoprotein, encoded by the MOG gene (also known as BTNL11, BTN6, MOG alpha-G, MOG AluA, MOG AluB, MOGIG2, and NRCLP7). Human MOG sequences are described in NCBI Gene:4340 and UniProtKB / Swiss-Prot:Q16653.

[0036] The term "BBB transport protein" includes any membrane-bound receptor that is expressed at the blood-brain barrier (BBB) ​​and capable of transporting receptor-binding molecules across the BBB. Examples of such receptors include TfR and CD98hc, as well as others described herein. In some embodiments, the BBB transport protein is TfR. In some embodiments, the BBB transport protein is CD98hc.

[0037] "BBB surface proteins" are proteins that are highly expressed in endothelial cells of the blood-brain barrier (BBB) ​​compared to peripheral endothelial cells. Although not required, BBB surface proteins may have limited or even absent expression in the periphery of animals (tissues or cells outside the BBB or CNS). BBB surface proteins can be either transport proteins, such as proteins that use receptor-mediated transcytosis to transport other molecules, or proteins that are not transporters.

[0038] "Brain-retained proteins" are proteins that are (1) expressed in the brain, (2) present on the cell surface or extracellularly, and (3) not therapeutic targets in themselves in terms of binding by dual transporters. Examples of brain-retained proteins include, but are not limited to, extracellular structural proteins such as MOG and receptors such as CD98hc. Although not required, brain-retained proteins may have limited or no expression in the peripheral regions of animals. Brain-retained proteins may be, but are not limited to, CD98hc, MOG, GLUT1, MFSD2A, IGF1R, or CA-IV. In some embodiments, brain-retained proteins are selected from the group consisting of CD98hc, MOG, GLUT1, MFSD2A, IGF1R, and CA-IV.

[0039] "Fc polypeptide" refers to the C-terminal region of naturally occurring immunoglobulin heavy chain polypeptides characterized by an Ig fold as a structural domain. Fc polypeptides contain a constant region sequence that includes at least a CH2 peptide and / or a CH3 peptide, and may contain at least a portion of the hinge region. Generally, Fc polypeptides do not contain a variable region.

[0040] A "modified Fc polypeptide" is an Fc polypeptide that has at least one mutation, e.g., substitution, deletion, or insertion, compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0041] An "Fc polypeptide dimer" refers to a dimer of two Fc polypeptides. In some embodiments, an Fc polypeptide dimer can bind to an Fc receptor (e.g., FcγR). In an Fc polypeptide dimer, the two Fc polypeptides dimerize through an interaction between two CH3 antibody constant domains. In some embodiments, the two Fc polypeptides may also dimerize via one or more disulfide bonds formed between the hinge domains of the two dimerizing Fc domain monomers. An Fc polypeptide dimer can be a heterodimer or a homodimer. An Fc polypeptide dimer may contain two wild-type Fc polypeptides, a wild-type Fc polypeptide and a modified Fc polypeptide, or two modified Fc polypeptides. In the case of an Fc polypeptide dimer containing two modified Fc polypeptides, the two modified Fc polypeptides may be the same or different.

[0042] "CH3 peptide" and "CH2 peptide" refer to polypeptides of the constant region domain of immunoglobulins. In relation to IgG antibodies, the CH3 peptide refers to the amino acid segment from approximately position 341 to 447, numbered according to the EU numbering scheme, and the CH2 peptide polypeptide refers to the amino acid segment from approximately position 231 to 340, numbered according to the EU numbering scheme. CH2 and CH3 peptide polypeptides may also be numbered according to the EVIGT (ImMunoGeneTics) numbering scheme, in which case, according to the IMGT Scientific chart numbering (IMGT website), the numbering of CH2 peptides is 1 to 110 and the numbering of CH3 peptides is 1 to 107. CH2 and CH3 peptides are part of the Fc region of immunoglobulins. In relation to IgG antibodies, the Fc region refers to the amino acid segment from approximately position 231 to 447, numbered according to the EU numbering scheme. As used herein, the term “Fc region” may also include at least a portion of the hinge region of the antibody.

[0043] The term "antibody" refers to a protein having an immunoglobulin fold that specifically binds to an antigen via the variable region (antibody-antigen binding domain) of the immunoglobulin fold. This term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term "antibody" includes full-length antibodies (e.g., variable heavy chain (V)). H ), two heavy chains composed of CH1 domain, CH2 domain, and CH3 domain, and a variable light chain (V L The antibody may contain both an IgG1 antibody (having two light chains composed of a CL domain) and an antibody fragment that retains antigen-binding specificity. Examples of antibody fragments include, but are not limited to, Fab, F(ab')2, scFab, Fv, scFv, bivalent scFv, VHH, vNAR, and nanobodies. The antibody may contain a light chain classified as either kappa or lambda. The antibody may contain a heavy chain classified as gamma, mu, alpha, delta, or epsilon, which then define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0044] With respect to CH3 peptides or CH2 peptides, the terms “wild-type,” “natural,” and “naturally occurring” are used herein to refer to domains having sequences that exist in nature.

[0045] The term "Fcγ receptor" or "FcγR" refers to a type of Fc receptor, which is classified based on the type of antibody it recognizes. FcγR includes several members: FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), and FcγRIIIB(CD16b), which differ in their antibody affinity due to differences in molecular structure. FcγRs are important for binding to the Fc portion of the IgG class of antibodies and inducing phagocytosis of opsonized microorganisms. FcγRs are present on the cell surface of immune system cells. FcγRs are involved in inducing effector function in the immune system and are activated when the Fc portion of an antibody binds to the receptor. FcγR mediates immune function by binding to antibodies attached to infected cells or invading pathogens, for example, and stimulating phagocytic or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis or ADCC (antibody-dependent cell-mediated cytotoxicity).

[0046] The term "variable region" refers to a domain in the heavy or light chain of an antibody that originates from germline variable (V) genes, diversity (D) (heavy chain only) genes, and linking (J) genes (not from constant (Cμ and Cδ) gene segments) and confers specificity to the antibody for binding to an antigen. Antibody variable regions typically consist of four conserved "framework" regions with three hypervariable "complementarity-determining regions (CDRs)" incorporated between them.

[0047] A variant with respect to a given sequence is a change in the nucleic acid or amino acid sequence relative to a reference sequence (e.g., wild-type or most common). Variants can be naturally occurring sequences (e.g., allelic variants) or sequences that do not naturally occur. A naturally occurring variant domain refers to a variant or mutant domain that is not present in natural cells and is produced by genetic modification of the original domain (e.g., using genetic engineering or mutagenesis). Changes in the nucleic acid or amino acid sequence (e.g., mutations) can include one or more substitutions, one or more insertions, one or more deletions, or combinations thereof.

[0048] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to mutations in which one amino acid is substituted with another amino acid that can be classified as having similar characteristics. Examples of classifications of such conservative amino acid groups include the following: The "charged / polar group" includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); the "aromatic group" includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Tip (tryptophan or W), and (histidine or H); and the "aliphatic group" includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), He (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Subgroups can also be identified within each group. For example, the group of charged amino acids or polar amino acids can be subdivided into subgroups such as the "positively charged subgroup" containing Lys, Arg, and His, the "negatively charged subgroup" containing Glu and Asp, and the "polar subgroup" containing Asn and Gln. In another example, the aromatic group or cyclic group can be subdivided into subgroups such as the "nitrogen ring subgroup" containing Pro, His, and Trp, and the "phenyl subgroup" containing Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups such as the "aliphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala, and the "aliphatic weakly polar subgroup" containing Met, Ser, Thr, and Cys.Examples of the classification of conservative mutations include amino acid substitutions of amino acids within the above subgroups, for example, without limitation, substitutions of Arg by Lys to maintain a positive charge or vice versa; substitutions of Asp by Glu to maintain a negative charge or vice versa; substitutions of Thr by Ser to maintain free -OH or vice versa; and substitutions of Asn by Gln to maintain free -NH2 or vice versa. In some embodiments, hydrophobic amino acids are substituted with naturally occurring hydrophobic amino acids, for example, at the active site, thereby preserving hydrophobicity.

[0049] "Binding affinity" refers to the strength of the non-covalent interaction between two molecules, for example, between a protein and a single-binding region or site on a target. Binding affinity can be quantified by measuring the equilibrium dissociation constant (KD). The equilibrium dissociation constant (KD) is the same as the dissociation rate constant (k d ,time -1 ) and the association rate constant (k a ,time -1 M -1 KD refers to the result obtained by dividing by ). KD can be determined by measuring the dynamics of complex formation and dissociation using methods such as surface plasmon resonance (SPR), e.g., the Biacore® system; binding equilibrium exclusion methods such as KinExA®; and biolayer interferometry (e.g., using the ForteBio® Octet® platform). The term "binding affinity" includes not only conventional binding affinity, such as that which reflects a 1:1 interaction between a polypeptide and its target, but also apparent affinity from which KD is calculated, which may reflect strong binding (binding strength).

[0050] When referring to a binding region (e.g., a BBB binding region), the terms "specifically binds" or "selectively binds" to a target (e.g., a BBB protein, e.g., TfR or CD98hc) refer to a binding reaction in which the binding region binds to that target with a higher affinity, higher binding strength, and / or longer duration than when binding to structurally different targets. The binding region may have 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 affinity for a particular target compared to an unrelated target when assayed under the same affinity assay conditions. The terms "specific binding", "specifically binds", or "specific" with respect to a particular target, for example, the equilibrium dissociation constant KD for the binding target is, for example, 10 -4 M or less (e.g., 10 -5 M, 10 -6 M (1000 nM), 10 -7 M (100 nM), 10 -8 M (10 nM), 10 -9 M (1 nM), 10 -10 M, 10 -11 M, or 10 -12 M) and can be shown by a molecule. In some embodiments, the binding region specifically binds to a target (e.g., a protein) that is conserved across species (e.g., structurally conserved across species).

[0051] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.

[0052] Naturally occurring amino acids include those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and carbon bonded to the R group) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimes" refer to chemical substances that have a different structure from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0053] Naturally occurring α-amino acids include, without limitation, alanine (Ala, A), cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), phenylalanine (Phe, F), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), arginine (Arg, R), lysine (Lys, K), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), proline (Pro, P), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), valine (Val, V), tryptophan (Trp, W), tyrosine (Tyr, Y), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. In this specification, amino acids may be represented by either the commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0054] As used herein, the term “variant” is used interchangeably with “variant” with respect to a mutant polypeptide or mutant polynucleotide. A variant with respect to a given wild-type CH3 peptide or CH2 peptide reference sequence may include naturally occurring allelic variants. A “non-naturally occurring” CH3 peptide or CH2 peptide refers to a variant domain or mutant domain that is not present in naturally occurring cells and is produced by genetic modification (e.g., using genetic engineering or mutagenesis techniques) of a polynucleotide or polypeptide of a naturally occurring CH3 peptide or CH2 peptide. A “variant” includes any domain that contains at least one amino acid mutation with respect to the wild type. Mutations can include substitutions, insertions, and deletions. A single amino acid substitution can be indicated by a single amino acid symbol indicating the amino acid before substitution, followed by a number indicating the amino acid position, and then by a single amino acid symbol indicating the subsequent amino acid substitution (for example, T366W indicates that the threonine at position 366 has been replaced with tryptophan). Furthermore, single amino acid substitutions can also be indicated by a number indicating the position of the substituted amino acid and a single-letter amino acid symbol indicating the subsequent amino acid substitution (for example, 366W indicates tryptophan at that position).

[0055] A polypeptide is a single-chain polymer of two or more amino acid residues. This term applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. Amino acid polymers may be entirely L-amino acids, entirely D-amino acids, or mixtures of L-amino acids and D-amino acids.

[0056] As used herein, the term “protein” refers to a polypeptide, a polypeptide dimer, or a polypeptide polymer. A polypeptide dimer may be a heterodimer or a homodimer. A polypeptide polymer may be a homopolymer or a heteropolymer. A heteropolymer can contain two or more copies of any given single-chain polypeptide. For example, immunoglobulins are heteropolymers containing two heavy chains and two light chains. The two heavy chains may be the same or different, and the two light chains may be the same or different. A single polypeptide chain in a dimer or polymer may be linked by one or more covalent bonds (e.g., disulfide bonds), by non-covalent interactions, or by a combination thereof.

[0057] In relation to two or more nucleic acid sequences or polypeptide sequences, the term “identical” or “identity” percentage means that two or more sequences or subsequences are the same, or that a certain percentage (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more) of nucleic acid or amino acid residues are identical across a particular region when compared and aligned for maximum agreement across a comparison frame or designated region, as measured by sequence comparison algorithms or manual alignment and visual inspection. For sequence comparison, typically one sequence is used as a reference sequence and compared with the candidate sequence. Alignment can be performed using various methods available to those skilled in the art to achieve maximum alignment (e.g., visual alignment or alignment 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 for alignment to obtain the maximum alignment can be determined by those skilled in the art. For the purpose of this application, the sequence comparison of polypeptide sequences uses the standard protein BLAST of the BLASTP algorithm to align two protein sequences using default parameters.

[0058] The expressions "corresponding to," "determined by reference to," or "numbered by reference to," used in relation to identifying a given nucleotide or amino acid residue in a nucleic acid or polypeptide sequence, refer to the position of a residue in a particular reference sequence when the given amino acid sequence is maximally aligned and compared to that reference sequence. The sequence aligned to the reference sequence does not need to be the same length as the reference sequence.

[0059] The "EU numbering scheme" is generally used in the art to refer to residues within the constant region of an antibody heavy chain. The EU numbering scheme for Sequence ID No. 2 (clone CH3C.35.23.2 with a knob mutation) is shown below: [ka]

[0060] The terms “subject,” “individual,” and “patient,” as used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammalian species. In some embodiments, the subject is human.

[0061] The terms “to treat” and “treatment” refer to methods or steps taken to reduce, improve or alleviate the number, severity, side effects, and / or frequency of at least one symptom or pathological consequence of a disease, disorder, or illness in a subject. Treatment can be preventive in that it prevents or partially prevents the disease, or the symptoms or conditions of the disease. Prevention includes preventing the onset or recurrence of the disease in subjects who are susceptible to the disease but have not yet been diagnosed with the disease. Prevention also includes preventing the onset or recurrence of symptoms or pathological consequences of the disease in subjects who are susceptible to the symptoms or pathological consequences of the disease but have not yet been diagnosed with the symptoms or pathological consequences of the disease. Treatment can also be preventive in that it delays the onset of the disease or delays the symptoms or conditions of the disease. Delaying the onset of the disease or delaying the symptoms or pathological consequences of the disease means delaying, hindering, slowing, postponing, stabilizing, suppressing, and / or postponing the onset of the disease or the onset of symptoms or pathological consequences of the disease. This delay may vary in duration depending on the history of the disease being treated and / or the individual being treated. Treatment may include suppressing a disease, disorder, or illness, e.g., preventing its progression, and may also include alleviating a disease, disorder, or illness, e.g., causing the disease, disorder, and / or illness to regress. Treatment may also mean extending survival time compared to the predicted survival time without treatment. Treatment may aim to cure in the sense of partial or complete cure of a disease, disorder, symptom, or side effects caused by a disease, disorder, or illness. The term “treatment” may encompass (a) to (c) the following: (a) preventing the onset of a disease in an object susceptible to the disease but not yet diagnosed with it; (b) inhibiting the disease, i.e., preventing its onset; and (c) reducing the disease, i.e., reducing or improving the disease and / or its symptoms or condition. Treatment may refer to treatment aimed at curing only, treatment aimed at prevention only, or treatment aimed at both curing and prevention.Those who require treatment (or are subject to treatment) may include individuals who have been previously diagnosed with a disease, disorder, or illness, or who have been identified as being at risk of developing a disease, disorder, or illness. Treating a disease, disorder, or illness may include improving at least one symptom of a particular disease, disorder, or illness, even if it does not affect the underlying pathophysiology.

[0062] The term "pharmaceutically acceptable excipient" refers to non-active pharmaceutical ingredients, such as buffers, carriers, or preservatives, that are biologically or pharmacologically compatible for use in humans or animals.

[0063] In relation to administration, the “effective dose” of a drug (e.g., a dual transporter or a pharmaceutical formulation containing a dual transporter) refers to the amount that is effective in achieving the desired outcome, such as a therapeutic or prophylactic result, in the required dosage / amount and duration.

[0064] The “therapeutic dose” of a drug, such as a dual transporter or a pharmaceutical formulation containing a dual transporter, refers to the amount effective in achieving the desired therapeutic outcome, such as the treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of that treatment, in the required dosage and duration. The therapeutic dose may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the population of cells being administered.

[0065] "Dosage," "unit dose," or "administration" refers to a physical individual unit suitable for use on a subject, each unit containing a predetermined amount of active pharmaceutical ingredient and / or pharmaceutical composition.

[0066] The term “administer” refers to a method of delivering a drug, compound, or composition to a desired biological site of action. Such methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the composition described herein is administered intravenously. A “peripherally administered” molecule means that the molecule is not administered directly to the CNS (e.g., not intrathecal delivery or directly to the brain (e.g., intraventricular delivery)).

[0067] II. Dual Transporter This document describes a dual transporter, a composition containing a dual transporter, a nucleic acid encoding a dual transporter, and a method for producing a dual transporter.

[0068] Methods for treating diseases or conditions of the brain or CNS by using dual transporters to deliver therapeutic agents to the brain or CNS are also described.

[0069] The dual transporters and compositions described can cross the blood-brain barrier (BBB). The dual transporters may be directly or indirectly linked to a therapeutic agent. The therapeutic agent may be, but is not limited to, a drug, a cytotoxic agent, a DNA or RNA molecule, or a chemical moiety.

[0070] A dual transporter comprises a first binding region (means for binding to the BBB transporter) that specifically binds to a BBB transporter protein (e.g., TfR or CD98hc), and a second binding region (means for binding to the second protein) that specifically binds to a second protein, the second protein being expressed on the luminal surface of the BBB or a protein retained in the brain, and distinct from the BBB transporter. By combining binding to two different proteins, it is possible to increase the transport of molecules across the BBB, decrease the efflux or degradation of the dual transporter from the brain, or a combination thereof. While we do not wish to be bound by any particular theory, the use of a dual transporter that binds to two targets expressed on the luminal surface of the BBB may increase the local concentration of molecules on the BBB surface, thereby promoting association with the BBB transporter protein (e.g., TfR or CD98hc). Furthermore, by binding to CNS targets (brain-retaining proteins) that promote brain retention, which can reduce the excretion or degradation of molecules, it is possible to enhance brain concentration and increase the persistence (retention) of molecules in the brain.

[0071] The dual transporters described may have a first binding region which has a weaker affinity for the BBB transporter compared to a molecule that binds to the BBB transporter but not to the second protein, while maintaining equivalent or increased brain exposure (e.g., intracranial concentration) compared to a molecule that binds to the BBB transporter but not to the second protein. The first binding region of the dual transporter can bind to the BBB transporter with an affinity about 1.1 to about 10 times weaker than a molecule that binds to the BBB transporter but not to the second protein, while maintaining equivalent or increased brain exposure (e.g., intracranial concentration) compared to a molecule that binds to the BBB transporter but not to the second protein. In some embodiments, the first binding region of the dual transporter can bind to the BBB transporter with an affinity about 1.1 to about 5 times weaker than a molecule that binds to the BBB transporter but not to the second protein. In some embodiments, the first binding region of a dual transporter can bind to the BBB transport protein with an affinity approximately 2 to 10 times weaker than a molecule that binds to the BBB transport protein but not to the second protein. In some embodiments, the first binding region of a dual transporter can bind to the BBB transport protein with an affinity approximately 2 to 5 times weaker than a molecule that binds to the BBB transport protein but not to the second protein. This weaker affinity for the BBB transport protein allows for lower therapeutic doses and / or improved safety margins for dual transporters compared to therapeutics delivered solely by the BBB transport protein in the absence of binding to the second protein.

[0072] In some embodiments, the first binding region of the dual transporter specifically binds to the BBB transporter with an affinity equal to or weaker than the specific binding of the second binding region to the second protein. In some embodiments, the first binding region of the dual transporter specifically binds to the BBB transporter with a weaker affinity than the specific binding of the second binding region to the second protein. In some embodiments, the first binding region of the dual transporter specifically binds to TfR with a weaker affinity than the specific binding of the second binding protein to CD98hc.

[0073] A dual transporter is described comprising a first binding region that specifically binds to a BBB transporter protein (e.g., TfR or CD98hc) and a second binding region that specifically binds to a different second BBB protein, which is either a BBB surface protein or a brain-retaining protein. The dual transporter may be monovalent or polyvalent with respect to binding to the BBB transporter protein and / or the second protein independently. In some embodiments, the dual transporter is monovalent with respect to binding to both the BBB transporter protein and the second protein. While we do not wish to be bound to any particular theory, the binding of a dual transporter to a second BBB transporter protein may improve brain delivery by increasing the transport efficiency or transport rate across the BBB, increasing the local concentration of the transporter on the BBB surface and thereby enhancing transport mediated by the binding of the dual transporter to the BBB transporter protein, and / or increasing the retention of the dual transporter in the brain (i.e., brain exposure time).

[0074] BBB surface proteins are proteins present on the luminal or anti-luminal surface of BBB endothelial cells. In some embodiments, a second protein is abundant in BBB cells. Abundance means that the protein is present in BBB cells (e.g., BBB endothelial cells) at higher concentrations than it is present in most other cells of the body. BBB is a term used to describe the microvascular system of the CNS. The BBB includes a network of blood vessels and related tissues, including a neurovascular network composed of densely spaced cells that plays a role in preventing harmful substances from reaching the brain. The blood vessels of the CNS are continuous, windowless vessels, and their properties allow for tight regulation of the movement of molecules, ions, and cells between the blood and the CNS. The blood vessels of the BBB are composed of two main types of cells: endothelial cells that form the walls of the vessels, and parietal cells present on the anti-luminal surface of the endothelial cell layer. In some embodiments, BBB proteins include proteins present on the surface of CNS endothelial cells.

[0075] In some embodiments, the BBB transport protein and / or the second protein are proteins that undergo receptor-mediated transcytosis. In some embodiments, the BBB transport protein undergoes receptor-mediated transcytosis. In some embodiments, the second protein undergoes receptor-mediated transcytosis. In some embodiments, the BBB transport protein and the second protein undergo receptor-mediated transcytosis.

[0076] In some embodiments, the BBB transport protein is TfR, the second protein is CD98hc, large neutral amino acid transporter small subunit 1 (CD98 light chain), glucose transporter 1 (GLUT1), major promoter superfamily domain-containing protein 2A (MFSD2A), carbonic anhydrase IV (CA-IV), low-density lipoprotein receptor, insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 5 The group is selected from the group including 0A, basidine, leptin receptor, claudin-5, P-selectin, lactoferrin receptor, folate receptor, sodium-dependent lysophosphatidylcholine cotransporter 1, solute transporter organic anion transporter family member 1C1, sodium-conjugated neutral amino acid transporter 5, LDL receptor-related protein 8, high affinity cationic amino acid transporter 1, sodium chloride-dependent taurine transporter, insulin-like growth factor-binding protein 7, solute transporter family 40 member 1, zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and myelin oligodendrocyte glycoprotein (MOG). In some embodiments, the BBB transporter protein is TfR, and the second protein is CD98hc.

[0077] In some embodiments, the BBB transport protein is TfR or CD98hc, and the second protein is a brain-retained protein.

[0078] In some embodiments, the BBB transport protein is ALPL, and the second protein is a brain-retaining protein. For an exemplary description of the ALPL binding region, see Moyer TC et al., BioRxiv. 2024 Mar 14;doi:10.1101 / 2024.03.12.584703.

[0079] In some embodiments, the BBB transport protein is TfR or CD98hc, and the second protein is MOG. For an illustrative description of the MOG binding region, see Nakano R. et al., PLoS One. 2019 Apr 12;14(4):e0214404.

[0080] In some embodiments, the BBB transport protein is TfR or CD98hc, and the second protein is GLUT1 (also known as solute transporter family 2 member 1, or SLC2A1).

[0081] In some embodiments, the BBB transport protein is TfR or CD98hc, and the second protein is IGF1R.

[0082] In some embodiments, the BBB transport protein is TfR or CD98hc, and the second protein is MFSD2A (Major Promoter Superfamily Domain-containing Protein 2A).

[0083] In some embodiments, the BBB transport protein is TfR or CD98hc, and the second protein is CA-IV. See Shay T. et al., Sci.Adv.9, eadg6618 (2023) 19 April 2023).

[0084] In some embodiments, the BBB transport protein is TfR, and the second protein is a brain-retained protein.

[0085] In some embodiments, the BBB transport protein is TfR, and the second protein is CD98hc.

[0086] In some embodiments, the BBB transport protein is TfR, and the second protein is MOG.

[0087] In some embodiments, the BBB transport protein is TfR, and the second protein is GLUT1.

[0088] In some embodiments, the BBB transport protein is TfR, and the second protein is IGF1R.

[0089] In some embodiments, the BBB transport protein is TfR, and the second protein is MFSD2A.

[0090] In some embodiments, the BBB transport protein is TfR, and the second protein is CA-IV.

[0091] In some embodiments, the BBB transport protein is CD98hc, the second protein is TfR, large neutral amino acid transporter small subunit 1 (CD98 light chain), low-density lipoprotein receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 50A, basidine, leptin receptor, claudin-5, P-selectin, lactoferrin The group is selected from the following: BBB receptor, folate receptor, sodium-dependent lysophosphatidylcholine cotransporter 1, solute transporter organic anion transporter family member 1C1, sodium-coupled neutral amino acid transporter 5, LDL receptor-related protein 8, high affinity cationic amino acid transporter 1, sodium chloride-dependent taurine transporter, insulin-like growth factor-binding protein 7, solute transporter family 40 member 1, zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and myelin oligodendrocyte glycoprotein (MOG). In some embodiments, the BBB transporter protein is CD98hc, and the second protein is TfR.

[0092] In some embodiments, the BBB transport protein is CD98hc, and the second protein is MOG.

[0093] In some embodiments, the BBB transport protein is CD98hc, and the second protein is GLUT1.

[0094] In some embodiments, the BBB transport protein is CD98hc, and the second protein is IGF1R.

[0095] In some embodiments, the BBB transport protein is CD98hc, and the second protein is MFSD2A.

[0096] In some embodiments, the BBB transport protein is CD98hc, and the second protein is CA-IV.

[0097] Additional BBB transport proteins include those expressed by brain endothelial cell genes associated with either receptor-mediated transcytosis or small molecule transport (see Zhang et al. Fluids and Barriers or the CNS (2020) 17:47 and Yang AC et al. Nature (2022) 603:885-892).

[0098] The first binding region is, (a) A first peptide that specifically binds to the BBB transport protein, (b) A first antibody-antigen binding domain that specifically binds to the BBB transport protein, (c) First Fc polypeptide modified to specifically bind to BBB transport protein, (d) A first CH3 peptide modified to specifically bind to the BBB transport protein, (e) A first fibronectin type III domain peptide modified to specifically bind to a BBB transport protein, or (f) A first bicyclic peptide that specifically binds to a BBB transport protein, but is not limited to these. The antibody-antigen-binding domain may, but is not limited to, a Fab, a single-strand Fab (scFab), a heavy-chain-only antibody-variable domain (nanobody, e.g., VHH or vNAR), an Fv fragment, or a single-strand-variable fragment (scFv).

[0099] In some embodiments, the first bonding region is (a) A first peptide that specifically binds to TfR, (b) A first antibody-antigen binding domain that specifically binds to TfR, (c) A first Fc polypeptide modified to specifically bind to TfR, (d) A first CH3 peptide modified to specifically bind to TfR, (e) A first fibronectin type III domain peptide modified to specifically bind to TfR, or (f) This may be, but is not limited to, a first bicyclic peptide that specifically binds to TfR. The antibody-antigen-binding domain may, but is not limited to, a Fab, a single-strand Fab (scFab), a heavy-chain-only antibody-variable domain (nanobody, e.g., VHH or vNAR), an Fv fragment, or a single-strand-variable fragment (scFv).

[0100] In some embodiments, the first bonding region is (a) A first peptide that specifically binds to CD98hc, (b) A first antibody-antigen binding domain that specifically binds to CD98hc, (c) A first Fc polypeptide modified to specifically bind to CD98hc, (d) A first CH3 peptide modified to specifically bind to CD98hc, (e) A first fibronectin type III domain peptide modified to specifically bind to CD98hc, or (f) A first bicyclic peptide that specifically binds to CD98hc, but is not limited to these. The antibody-antigen-binding domain may, but is not limited to, a Fab, a single-strand Fab (scFab), a heavy-chain-only antibody-variable domain (nanobody, e.g., VHH or vNAR), an Fv fragment, or a single-strand-variable fragment (scFv).

[0101] The second binding region is, (a) A second peptide that specifically binds to the second protein, (b) A second antibody-antigen binding domain that specifically binds to the second protein, (c) A second Fc polypeptide modified to specifically bind to the second protein, (d) A second CH3 peptide modified to specifically bind to the second protein, (e) A second fibronectin type III domain peptide modified to specifically bind to the second protein, or (f) A second bicyclic peptide that specifically binds to the second protein, but is not limited to these. The antibody-antigen-binding domain may be, but is not limited to, Fab, scFab, heavy chain-only antibody variable domains (nanobodies, e.g., VHH or vNAR), Fv fragments, or scFv.

[0102] In some embodiments, the second bonding region is (a) A second peptide that specifically binds to CD98hc, (b) A second antibody-antigen binding domain that specifically binds to CD98hc, (c) A second Fc polypeptide modified to specifically bind to CD98hc, (d) A second CH3 peptide modified to specifically bind to CD98hc, (e) A second fibronectin type III domain peptide modified to specifically bind to CD98hc, or (f) A second bicyclic peptide that specifically binds to CD98hc, but is not limited to these. The antibody-antigen-binding domain may be, but is not limited to, Fab, scFab, heavy chain-only antibody variable domains (nanobodies, e.g., VHH or vNAR), Fv fragments, or scFv.

[0103] In some embodiments, the second bonding region is (a) A second peptide that specifically binds to MOG, (b) A second antibody-antigen binding domain that specifically binds to MOG, (c) A second Fc polypeptide modified to specifically bind to MOG, (d) A second CH3 peptide modified to specifically bind to MOG, (e) A second fibronectin type III domain peptide modified to specifically bind to MOG, or (f) A second bicyclic peptide that specifically binds to the second protein, but is not limited to these. The antibody-antigen-binding domain may be, but is not limited to, Fab, scFab, heavy chain-only antibody variable domains (nanobodies, e.g., VHH or vNAR), Fv fragments, or scFv.

[0104] The antibody-antigen-binding domain includes the antigen-binding domain of an immunoglobulin or a peptide having a structure similar to that of the antigen-binding domain of an immunoglobulin. The immunoglobulin may be, but is not limited to, IgG, IgM, IgE, IgA, IgD, or a heavy-chain antibody. The antibody-antigen-binding domain may be, but is not limited to, a Fab, scFab, Fv fragment, scFv, or a heavy-chain-only antibody variable domain (nanobody, e.g., VHH or vNAR).

[0105] The term "Fab" refers to the light chain variable region (V L ) and the constant region of the light chain (collectively known as the antibody light chain), and the variable region of the heavy chain (V H This refers to an antigen-binding fragment consisting of the heavy chain CH1 constant region (collectively known as the antibody Fd fragment).

[0106] The term "single-stranded Fab" or "scFab" refers to an antigen-binding fragment consisting of a Fab, where the Fd fragment and light chain are linked together by a peptide linker. The linker can connect the N-terminus of the Fd fragment to the C-terminus of the light chain, or vice versa.

[0107] The term "Fv fragment" refers to the V fragments that together form a binding site for the antigen. H and V L This refers to antigen-binding fragments consisting of [the specified components].

[0108] The term "single-chain variable fragment" or "scFv" refers to an antigen-binding fragment consisting of a heavy-chain variable region and a light-chain variable region linked via a peptide linker. The linker is V H The N-terminus of V L The C-terminus of, or V L The N-terminus of V H It can be connected to the C-terminus of . scFv lacks a constant region. Modified scFv and methods for modifying scFv to bind to target proteins are described in WO2022 / 258841 (incorporated herein by reference). Exemplary scFv that bind to TfR are also described herein, for example, in Table 15.

[0109] The term “nanobody” refers to an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies derived from camelid heavy chain antibodies may be referred to as “VHH” fragments. Nanobodies derived from cartilaginous fish heavy chain antibodies may be referred to as “vNARs.” Modified VHH fragments, and methods for modifying VHH fragments to bind to target proteins such as TfR and CD98hc, are described in WO2019 / 246288, WO2021 / 205361, WO2020 / 056327, WO2022 / 103769, and WO2023 / 023166 (each of which is incorporated herein by reference). Exemplary nanobodies that bind to TfR are also described herein, for example, in Table 15.

[0110] In some embodiments, the modified CH3 peptide includes a CH3 peptide modified to bind to a target protein, such as a BBB transport protein (e.g., TfR or CD98hc), a BBB cell surface protein, or a brain-retaining protein. Modified CH3 peptides and methods for modifying CH3 peptides to bind to target proteins are described in US20180237496, US20200223935, and PCT / US2022 / 053220 (each of which is incorporated herein by reference). Exemplary modified CH3 peptides that bind to TfR and CD98 are also described herein, for example, in Tables 2, 8, 17, and 18.

[0111] In some embodiments, the peptide that specifically binds to a BBB transport protein, a BBB cell surface protein, or a brain-retained protein comprises a heavy chain complementarity-determining region 3 (CDRH3) peptide that specifically binds to a BBB transport protein, a BBB cell surface protein, or a brain-retained protein. CDRH3 may be inserted into the constant domain of an antibody, for example, within a CH3 peptide, CH2 peptide, CH1 peptide, or CL peptide, as described in WO2023 / 087017 (incorporated herein by reference).

[0112] The Fc polypeptides or CH3 peptides used herein may be derived from IgG, for example, human IgG1, IgG2, IgG3, or IgG4.

[0113] Modified fibronectin type III (FN3) domains include FN3 domains modified to bind to target proteins, such as BBB transport proteins (e.g., TfR or CD98hc), BBB cell surface proteins, or brain-retaining proteins. The FN3 domain is a consensus sequence of multiple FN3 domains from human tenascin-C. Modified FN3 domains and methods for modifying FN3 domains to bind to target proteins are described in US20100216708 (incorporated herein by reference). Specific examples of FN3 domains binding to TfR (CD71) are described in WO2021 / 076546 and WO2022 / 221505, both incorporated herein by reference.

[0114] Bicyclic peptides include synthetic short-chain peptides constrained by a chemical linker to form two loops. The chemical linker can form bonds to three different amino acids within the peptide. Bicyclic peptides can be prepared to specifically bind to target proteins, such as BBB transport proteins (e.g., TfR or CD98hc), BBB cell surface proteins, or brain-retaining proteins. Bicyclic peptides that bind to target proteins and methods for preparing bicyclic peptides are described in U.S. Patents 8680022, 8685890, and 8778844 (each incorporated herein by reference). A specific bicyclic peptide that binds to TfR is described in WO2022 / 101633, which is incorporated herein by reference.

[0115] In some embodiments, the dual transporter includes a multispecific antibody or a multispecific antibody-like molecule.

[0116] In some embodiments, the dual transporter includes a bispecific antibody or a bispecific antibody-like molecule.

[0117] In some embodiments, the dual transporter comprises a bispecific antibody comprising a first antibody-antigen binding domain that specifically binds to a BBB transport protein (e.g., TfR or CD98hc) and a second antibody-antigen binding domain that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retained protein. Each antibody-antigen binding domain may, but are not limited to, a Fab, scFab, Fv fragment, scFv, or nanobody. A bispecific antibody dual transporter may have two antigen-binding domains of the same type (e.g., two Fabs, two scFabs, two Fv fragments, two scFvs, or two nanobodies), or it may have two antigen-binding domains of two different types (e.g., Fab and scFab, Fv fragment, scFv, or nanobodies; scFab and Fab, Fv fragment, scFv, or nanobodies; Fv fragment and Fab, scFab, scFv, or nanobodies; scFv and Fab, scFab, Fv fragment, or nanobodies; or nanobodies and Fab, scFab, Fv fragment, or scFv). In certain embodiments, the bispecific antibody contains Fab and scFv.

[0118] In some embodiments, the dual transporter includes a bispecific antibody comprising: a first antibody heavy chain and a first antibody light chain, wherein the first antibody heavy chain and the first antibody light chain form an antibody variable domain that specifically binds to a BBB transport protein (e.g., TfR or CD98hc); and a second antibody heavy chain and a second antibody light chain, wherein the second antibody heavy chain and the second antibody light chain form an antibody variable domain that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retained protein.

[0119] In some embodiments, the dual transporter includes a bispecific heavy chain antibody in which the first heavy chain of the heavy chain antibody specifically binds to a BBB transport protein (e.g., TfR or CD98hc), and the second heavy chain of the heavy chain antibody specifically binds to a different second protein, which is either a BBB surface protein or a brain-retained protein.

[0120] In some embodiments, the dual transporter comprises a bispecificity F(ab')2 comprising a first Fab that specifically binds to a BBB transport protein (e.g., TfR or CD98hc) and a second Fab that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retaining protein. The dual transporter bispecificity F(ab')2 may or may not be ligated to one or more Fc polypeptides, one or more CH2 peptides, one or more CH3 peptides, an Fc dimer, or a CH3 peptide dimer. In some embodiments, the first Fab is ligated to a first Fc polypeptide, a first CH2 peptide, or a first CH3 peptide, and the second Fab is ligated to a second Fc polypeptide, a second CH2 peptide, or a second CH3 peptide.

[0121] In some embodiments, the dual transporter comprises a bispecific sc(Fv)2 including a first scFv that specifically binds to a BBB transport protein (e.g., TfR or CD98hc) and a second scFv that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retaining protein. The dual transporter bispecific sc(Fv)2 may or may not be ligated to one or more Fc polypeptides, one or more CH2 peptides, one or more CH3 peptides, an Fc dimer, or a CH3 peptide dimer. In some embodiments, the first scFv is ligated to a first Fc polypeptide, a first CH2 peptide, or a first CH3 peptide, and the second scFv is ligated to a second Fc polypeptide, a second CH2 peptide, or a second CH3 peptide. In some embodiments, the dual transporter bispecific sc(Fv)2 includes a diabody. In some embodiments, the dual transporter comprises a bispecific minibody comprising a first scFv linked to a first CH3 peptide and a second scFv linked to a second CH3 peptide, wherein the first scFv specifically binds to a BBB transport protein (e.g., TfR or CD98hc) and the second scFv specifically binds to a different second protein, which is either a BBB surface protein or a brain-retaining protein, and the first CH3 peptide and the second CH3 peptide form a dimer.

[0122] In some embodiments, the dual transporter comprises a bispecific nanobody including a first nanobody that specifically binds to a BBB transport protein (e.g., TfR or CD98hc) and a second nanobody that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retained protein. The dual transporter bispecific nanobody may or may not be ligated to one or more Fc polypeptides, one or more CH2 peptides, one or more CH3 peptides, an Fc dimer, or a CH3 peptide dimer. In some embodiments, the first nanobody is ligated to a first Fc polypeptide, a first CH2 peptide, or a first CH3 peptide, and the second nanobody is ligated to a second Fc polypeptide, a second CH2 peptide, or a second CH3 peptide. The first and second nanobodies may independently be vNARs or VHHs. In some embodiments, the dual transporter bispecific nanobody comprises a (vNAR)2 peptide, a (VHH)2 peptide, a vNAR-VHH peptide, or a VHH-vNAR peptide.

[0123] In some embodiments, the dual transporter includes a first binding region that specifically binds to a BBB transporter protein (e.g., TfR or CD98hc), which is linked to a bispecific antibody that binds to a second protein and a therapeutic target. In some embodiments, the dual transporter includes a second binding region that specifically binds to a BBB surface protein or a brain-retained protein, which is linked to a bispecific antibody that binds to a BBB transporter protein and a therapeutic target.

[0124] In some embodiments, the dual transporter comprises a triplicate antibody or a triplicate antibody-like molecule (dual transporter triplicate antibody). In some embodiments, the dual transporter triplicate antibody comprises a first antibody-antigen binding domain that specifically binds to a BBB transporter protein (e.g., TfR or CD98hc), a second antibody-antigen binding domain that specifically binds to a different second protein that is either a BBB surface protein or a brain-retained protein, and a third antibody-antigen binding domain that specifically binds to a target protein.

[0125] Dual-transporter triplicate antibodies are not limited to any particular configuration of the antigen-binding domain. Triplicate antibody-like molecules may be, but are not limited to, trimer Fab ((Fab)3), trimer scFv (triabody or (scFv)3), or combinations thereof. In some embodiments, dual-transporter triplicate antibodies include a bispecific immunoglobulin (e.g., IgG) linked to Fab, scFab, Fv fragment, scFv, or nanobody.

[0126] In some embodiments, the dual transporter comprises a quadruplespecific antibody or a quadruplespecific antibody-like molecule (dual transporter quadruplespecific antibody). In some embodiments, the dual transporter quadruplespecific antibody comprises a first antibody-antigen binding domain that specifically binds to a BBB transporter protein (e.g., TfR or CD98hc), a second antibody-antigen binding domain that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retained protein, a third antibody-antigen binding domain that specifically binds to a first target protein, and a fourth antibody-antigen binding domain that specifically binds to a second target protein. The first and second target proteins may be the same or different.

[0127] Dual-transporter quadruspecific antibodies are not limited to any particular stereochemistry of the antigen-binding domain. Quadruple-specific antibody-like molecules may be, but are not limited to, tetrameric Fab ((Fab)3), tetrameric scFv (tetrabody or (scFv)4), tetrameric nanobody, or a combination thereof. In some embodiments, the dual-transporter quadruspecific antibody comprises an immunoglobulin (e.g., IgG) ligated to two antigen-binding domains independently selected from the group consisting of Fab, scFab, Fv fragments, scFv, or nanobody. In some embodiments, the dual transporter comprises a bivariate-domain immunoglobulin (DVD-Ig). DVD-Ig comprises a tetravalent immunoglobulin having two variable domains in each of the Fab arms.

[0128] In some embodiments, a dual transporter quadruspecific antibody comprises a therapeutic antibody ligated to a first Fab, scFab, scFv, or nanobody that specifically binds to a BBB transport protein, and a second Fab, scFab, scFv, or nanobody that specifically binds to a second protein. The first Fab, scFab, scFv, or nanobody and the second Fab, scFab, scFv, or nanobody are independently ligated to the heavy chain, light chain, or Fc of the therapeutic antibody. In some embodiments, the first Fab, scFab, scFv, or nanobody and / or the second Fab, scFab, scFv, or nanobody are ligated to the C-terminus of the heavy chain of the therapeutic antibody. In some embodiments, the BBB transport protein is TfR. In some embodiments, the BBB transport protein is CD98hc. In some embodiments, the BBB transport protein is TfR and the second protein is CD98hc.

[0129] In some embodiments, the dual transporter includes a first binding region that specifically binds to a BBB transport protein (e.g., TfR or CD98hc), and a second binding region that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retaining protein, with the first and second binding regions linked to each other.

[0130] In some embodiments, the dual transporter includes a first binding region that specifically binds to a BBB transport protein (e.g., TfR or CD98hc), and a second binding region that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retaining protein, with the first and second binding regions linked to a scaffold.

[0131] The scaffold may be, but is not limited to, a polypeptide, protein, antibody, or antibody fragment. The antibody scaffold may be, but is not limited to, IgG, IgA, IgD, IgE, IgM, or heavy chain antibodies. The antibody scaffold may be monospecific or multispecific (e.g., bispecific) antibodies. The antibody fragment scaffold may be, but is not limited to, an Fc polypeptide, Fc dimer, CH2 peptide, CH2 dimer, CH3 peptide, CH3 dimer, Fab fragment, multimer Fab, F(ab')2 fragment, multimer F(ab')2 fragment, scFab, multimer scFab, scFv, multimer scFv, nanobody, or multimer nanobody. The antibody fragment scaffold may be monospecific or multispecific (e.g., bispecific). The antibody scaffold or antibody fragment scaffold may include a therapeutic antibody or therapeutic antibody fragment. The antibody scaffold or antibody fragment scaffold may be derived from a therapeutic antibody or therapeutic antibody fragment. The antibody scaffold or antibody fragment scaffold may contain antibody-antigen-binding domains from therapeutic antibodies.

[0132] In some embodiments, the dual transporter includes a first binding region that specifically binds to a BBB transport protein (e.g., TfR or CD98hc), and a second binding region that specifically binds to a different second protein, which is either a BBB surface protein or a brain-retaining protein, with the first and second binding regions ligated to an antibody scaffold. The first and second binding regions may independently be ligated to a variable domain region, a light chain, the C-terminus of the light chain, the N-terminus of the light chain, a light chain variable region, a light chain constant region, a heavy chain, the C-terminus of the heavy chain, the N-terminus of the heavy chain, a heavy chain variable region, a heavy chain CH1 region, a heavy chain CH2 region, or a heavy chain CH3 region.

[0133] In some embodiments, the first binding region is ligated to the C-terminus of one light chain of the antibody scaffold, and the second binding region is ligated to the C-terminus of the other light chain of the antibody scaffold.

[0134] In some embodiments, the first binding region is linked to the C-terminus of one heavy chain of the antibody scaffold, and the second binding region is linked to the C-terminus of the other heavy chain of the antibody scaffold.

[0135] In some embodiments, the first binding region is ligated to the C-terminus of the light chain of the antibody scaffold, and the second binding region is ligated to the C-terminus of the heavy chain of the antibody scaffold.

[0136] In some embodiments, the first binding region is ligated to the N-terminus of one light chain of the antibody scaffold, and the second binding region is ligated to the N-terminus of the other light chain of the antibody scaffold.

[0137] In some embodiments, the first binding region is ligated to the N-terminus of one heavy chain of the antibody scaffold, and the second binding region is ligated to the N-terminus of the other heavy chain of the antibody scaffold.

[0138] In some embodiments, the first binding region is ligated to the N-terminus of the light chain of the antibody scaffold, and the second binding region is ligated to the N-terminus of the heavy chain of the antibody scaffold.

[0139] In some embodiments, the first binding region is ligated to the C-terminus of the light chain of the antibody scaffold, and the second binding region is ligated to the N-terminus of either the light or heavy chain of the antibody scaffold.

[0140] In some embodiments, the first binding region is ligated to the C-terminus of the heavy chain of the antibody scaffold, and the second binding region is ligated to the N-terminus of the light or heavy chain of the antibody scaffold.

[0141] In some embodiments, the first binding region is ligated to the N-terminus of the light chain of the antibody scaffold, and the second binding region is ligated to the C-terminus of either the light or heavy chain of the antibody scaffold.

[0142] In some embodiments, the first binding region is ligated to the N-terminus of the heavy chain of the antibody scaffold, and the second binding region is ligated to the C-terminus of the light or heavy chain of the antibody scaffold.

[0143] The first binding region may be, but is not limited to, a peptide, a modified CH3 peptide, a modified tenascin C peptide, a bicyclic peptide, a Fab, scFab, scFv, or a nanobody. The second binding region may be, but is not limited to, a peptide, a modified CH2 peptide, a modified CH3 peptide, a modified tenascin C peptide, a bicyclic peptide, a Fab, scFab, scFv, or a nanobody.

[0144] In some embodiments, the first binding region includes an scFv ligated to the C-terminus of one light chain of the antibody scaffold, and the second binding region includes an scFv ligated to the C-terminus of the other light chain of the antibody scaffold. In some embodiments, the first binding region specifically binds to TfR. In some embodiments, the first binding region specifically binds to CD98hc. In some embodiments, the first binding region specifically binds to TfR, and the second binding region specifically binds to CD98hc.

[0145] In some embodiments, the first binding region and / or the second binding region comprises an scFv, the N-terminus of which is ligated to the C-terminus of one light chain of the antibody scaffold, and the C-terminus of which is ligated to the N-terminus of the other light chain of the antibody scaffold.

[0146] In some embodiments, the dual transporter comprises DVD-Ig, where each Fab arm of DVD-Ig contains two Fv fragments (antibody variable regions). Thus, DVD-Ig contains four Fv fragments, namely a first Fv fragment, a second Fv fragment, a third Fv fragment, and a fourth Fv fragment. At least one of the Fv fragments specifically binds to the BBB transport protein, and at least one of the Fv fragments specifically binds to the second protein. In some embodiments, one of the Fv fragments binds to the BBB transport protein, one of the Fv fragments binds to the second protein, and the other two Fv fragments specifically bind to one or two target proteins (e.g., therapeutic targets). The DVD-Ig dual transporter is not limited to any particular arrangement of the Fv fragments. The Fv fragment that specifically binds to the BBB transport protein may be the first, second, third, or fourth Fv fragment. Similarly, the Fv fragment that specifically binds to the second protein may be the first, second, third, or fourth Fv fragment, provided that the Fv fragment that binds to the second protein is different from the Fv fragment that specifically binds to the BBB transport protein. In some embodiments, the first Fab arm of DVD-Ig includes a first Fv fragment that specifically binds to the target protein and a second Fv fragment that specifically binds to the BBB transport protein, and the second Fab arm of DVD-Ig includes a third Fv fragment that specifically binds to the target protein and a fourth Fv fragment that specifically binds to the second protein. In some embodiments, the first Fab arm of DVD-Ig includes a first Fv fragment that specifically binds to the BBB transport protein and a second Fv fragment that specifically binds to the second protein, and the second Fab arm of DVD-Ig includes third and fourth Fv fragments that specifically bind to one or two target proteins.

[0147] In some embodiments, the dual transporter includes a first binding region comprising a first Fc polypeptide or CH3 modified to specifically bind to a BBB transport protein (e.g., TfR or CD98hc). The second binding region may be provided by a second peptide that specifically binds to a second protein, a second antibody-antigen binding domain that specifically binds to a second protein, a second Fc polypeptide modified to specifically bind to a second protein, a second CH3 peptide modified to specifically bind to a second protein, a second fibronectin type III domain peptide modified to specifically bind to a second protein, or a second bicyclic peptide that specifically binds to a second protein. In some embodiments, the BBB transport protein is TfR. In some embodiments, the BBB transport protein is CD98hc. In some embodiments, the BBB transport protein is TfR and the second protein is CD98hc.

[0148] In some embodiments, the dual transporter includes a second binding region comprising a second Fc polypeptide or CH3 modified to specifically bind to a different second protein, which is either a BBB surface protein or a brain-retained protein. The first binding region may be provided by a first peptide that specifically binds to a BBB transport protein (e.g., TfR or CD98hc), a first antibody-antigen binding domain that specifically binds to a BBB transport protein, a first Fc polypeptide modified to specifically bind to a BBB transport protein, a first CH3 peptide modified to specifically bind to a BBB transport protein, a first fibronectin type III domain peptide modified to specifically bind to a BBB transport protein, or a first bicyclic peptide that specifically binds to a BBB transport protein. In some embodiments, the second protein is CD98hc. In some embodiments, the BBB transport protein is TfR and the second protein is CD98hc.

[0149] In some embodiments, the dual transporter comprises an Fc dimer, the Fc dimer comprising a first Fc polypeptide containing a CH3 peptide modified to specifically bind to TfR, and a second Fc polypeptide containing a CH3 peptide modified to specifically bind to CD98hc.

[0150] The Fc dimers or Fc polypeptides described herein may further comprise a partial or complete hinge region. The hinge region may be from any subclass or isotype of immunoglobulin. An exemplary immunoglobulin hinge is the IgG hinge region, e.g., the IgG1 hinge region, e.g., the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 3). Fc polypeptides, which may comprise a hinge or partial hinge region, may further be fused to a therapeutic molecule (e.g., a therapeutic peptide; e.g., a therapeutic antibody or a fragment thereof).

[0151] In some embodiments, the dual transporter containing the Fc dimer is linked to one or more Fabs, one or more F(ab')2s, one or more scFabs, one or more scFvs, or one or more nanobodies. In some embodiments, the dual transporter containing the Fc dimer is linked to one or more therapeutic molecules. The therapeutic molecules may be, but are not limited to, therapeutic Fabs, therapeutic F(ab')2s, therapeutic scFabs, scFvs, or therapeutic nanobodies.

[0152] In some embodiments, the dual transporter comprises a therapeutic antibody having a first Fc polypeptide or CH3 peptide modified to specifically bind to a BBB transport protein (e.g., TfR or CD98hc), and a second Fc polypeptide or CH3 peptide modified to specifically bind to a different second protein, which is either a BBB surface protein or a brain-retained protein. The therapeutic antibody may be monospecific or bispecific.

[0153] In some embodiments, the dual transporter comprises a therapeutic antibody having a first Fc polypeptide or CH3 peptide modified to specifically bind to TfR, and a second Fc polypeptide or CH3 peptide modified to specifically bind to a different second protein, which is either a BBB surface protein or a brain-retained protein. The therapeutic antibody may be monospecific or bispecific.

[0154] In some embodiments, the dual transporter comprises a therapeutic antibody having a first Fc polypeptide or CH3 peptide modified to specifically bind to CD98hc, and a second Fc polypeptide or CH3 peptide modified to specifically bind to a different second protein, which is a BBB surface protein or a brain-retained protein. The therapeutic antibody may be monospecific or bispecific.

[0155] In some embodiments, the dual transporter comprises a therapeutic antibody having a first Fc polypeptide or CH3 peptide modified to specifically bind to TfR and a second Fc polypeptide or CH3 peptide modified to specifically bind to CD98hc. The therapeutic antibody may be monospecific or bispecific.

[0156] Any of the dual transporters described may further contain one or more therapeutic molecules. The therapeutic molecules may be, but are not limited to, small molecule drugs or therapeutic peptides. The therapeutic peptides may be, but are not limited to, antibodies, Fab, F(ab')2, scFab, Fv fragments, scFv, or nanobodies.

[0157] In some embodiments, the dual transport comprises a TfR binding domain and a second binding domain that specifically binds to a second protein, the second protein being expressed on the luminal surface of the BBB or a brain-retained protein, and the second protein being different from the BBB transport protein. In some embodiments, the dual transporter comprises a TfR binding domain and a second binding domain that binds to CD98hc, MOG, GLUT1, MFSD2A, IGF1R, or CA-IV. It has been demonstrated herein that dual transporters having both TfR and CD98hc binding unexpectedly provide both (i) higher brain Cmax (maximum concentration in the brain) and (ii) longer brain exposure time (i.e., AUC (area under the curve)) compared to BBB transporters having only TfR or CD98hc binding (see examples). TfR binding alone results in lower brain Cmax concentrations and shorter brain exposure times compared to the dual transporters provided herein. CD98hc binding alone resulted in lower brain Cmax concentrations compared to the dual transporter provided herein. With the dual transporter combining the TfR-binding and MOG-binding regions, improvements in brain Cmax and brain activity were also observed.

[0158] By combining two different binding regions, the dual transporter provides increased brain Cmax and increased brain exposure time (i.e., retention). Therefore, the described dual transporter can be used to develop therapeutic agents that cross the blood-brain barrier (BBB) ​​or to improve brain delivery of therapeutic compounds ligated to the dual transporter. Ligating therapeutic compounds to the described dual transporter can also be used to improve the efficacy or therapeutic index of therapeutic compounds whose targets are in the brain or CNS.

[0159] Cmax and brain exposure time can be further optimized by adjusting the affinity of the first (e.g., TfR) binding region and the second (e.g., CD98hc, MOG, GLUT1, MFSD2A, or CA-IV) binding region. For example, it has been demonstrated that a relatively strong TfR affinity combined with a relatively strong CD98hc affinity results in higher brain Cmax concentrations and moderate brain exposure time compared to a strong TfR affinity combined with a weaker CD98hc affinity (see examples). A moderate to weak TfR affinity combined with a strong CD98hc affinity results in moderate brain Cmax and longer brain exposure time compared to a relatively strong TfR affinity combined with a relatively strong CD98hc affinity. A moderate to weak TfR affinity combined with a weak CD98hc affinity results in lower brain concentrations and moderate brain exposure time. The longest brain exposure time was observed with a moderate to weak TfR affinity combined with a strong CD98hc affinity. Therefore, in some embodiments, the dual transporter includes a first binding region that specifically binds to a blood-brain barrier (BBB) ​​transport protein (e.g., TfR) with moderate to weak affinity, and a second binding region that specifically binds to a second protein (e.g., CD98hc, MOG, GLUT1, MFSD2A, or CA-IV) with strong affinity, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein (and is different from the BBB transport protein).

[0160] It has also been demonstrated herein that as the ratio of TfR binding affinity to CD98hc binding affinity increases (i.e., more than 10-fold), brain Cmax increases and brain exposure time decreases. Conversely, as the ratio of CD98hc binding affinity to TfR binding affinity increases (i.e., more than 10-fold), brain Cmax decreases and brain exposure time increases. Therefore, by modulating the affinity of the first and second binding regions to their respective target proteins, dual transporters with desired brain Cmax concentrations and brain exposure times can be provided. In some embodiments, brain Cmax and brain retention can be modulated by altering the affinity of the first and second binding regions to their respective target proteins. Using such modulation, brain Cmax and brain retention can be altered for various therapeutic, discovery, or research purposes.

[0161] In some embodiments, the first and second binding regions of the dual transporter may independently have weak, moderate, or strong affinity for their respective targets.

[0162] In some embodiments, the first binding region has a weak affinity for the target protein, and the second binding region has a weak affinity for the target protein. In some embodiments, the first binding region has a weak affinity for the target protein, and the second binding region has a moderate affinity for the target protein. In some embodiments, the first binding region has a weak affinity for the target protein, and the second binding region has a strong affinity for the target protein. In some embodiments, the first binding region has a weak affinity for TfR, and the second binding region has a weak affinity for CD98hc. In some embodiments, the first binding region has a weak affinity for TfR, and the second binding region has a moderate affinity for CD98hc. In some embodiments, the first binding region has a weak affinity for TfR, and the second binding region has a strong affinity for CD98hc. In some embodiments, the first binding region has a weak affinity for TfR, and the second binding region has a weak affinity for MOG. In some embodiments, the first binding region has a weak affinity for TfR, and the second binding region has a moderate affinity for MOG. In some embodiments, the first binding region has a weak affinity for TfR, and the second binding region has a strong affinity for MOG.

[0163] In some embodiments, the first binding region has moderate affinity for the target protein, and the second binding region has weak affinity for the target protein. In some embodiments, the first binding region has moderate affinity for the target protein, and the second binding region has moderate affinity for the target protein. In some embodiments, the first binding region has moderate affinity for the target protein, and the second binding region has strong affinity for the target protein. In some embodiments, the first binding region has moderate affinity for TfR, and the second binding region has weak affinity for CD98hc. In some embodiments, the first binding region has moderate affinity for TfR, and the second binding region has moderate affinity for CD98hc. In some embodiments, the first binding region has moderate affinity for TfR, and the second binding region has strong affinity for CD98hc. In some embodiments, the first binding region has moderate affinity for TfR, and the second binding region has weak affinity for MOG. In some embodiments, the first binding region has moderate affinity for TfR, and the second binding region has moderate affinity for MOG. In some embodiments, the first binding region has moderate affinity for TfR, and the second binding region has strong affinity for MOG.

[0164] In some embodiments, the first binding region has a strong affinity for its target protein, and the second binding region has a weak affinity for its target protein. In some embodiments, the first binding region has a strong affinity for its target protein, and the second binding region has a moderate affinity for its target protein. In some embodiments, the first binding region has a strong affinity for its target protein, and the second binding region has a strong affinity for its target protein. In some embodiments, the first binding region has a strong affinity for TfR, and the second binding region has a weak affinity for CD98hc. In some embodiments, the first binding region has a strong affinity for TfR, and the second binding region has a moderate affinity for CD98hc. In some embodiments, the first binding region has a strong affinity for TfR, and the second binding region has a strong affinity for CD98hc. In some embodiments, the first binding region has a strong affinity for TfR, and the second binding region has a weak affinity for MOG. In some embodiments, the first binding region has a strong affinity for TfR, and the second binding region has a moderate affinity for MOG.

[0165] Strong affinity indicates that the affinity of the first or second binding region to its respective target protein is less than 250 nM. In some embodiments, the first or second binding region with strong affinity has an affinity of about 50 nM to about 250 nM to its target protein. In some embodiments, the first or second binding region with strong affinity has an affinity of 150 nM ± 100 nM, 150 nM ± 75 nM, or 150 nM ± 50 nM to its target protein. In some embodiments, the first or second binding region with strong affinity has an affinity of about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, or about 250 nM to its target protein. Moderate affinity indicates that the affinity of the first or second binding region to its respective target protein is approximately 300 nM to 900 nM, 400 nM to 800 nM, 500 to 700 nM, or 600 nM. In some embodiments, the first or second binding region with moderate affinity has an affinity of 600 nM ± 300 nM, 600 nM ± 200 nM, or 600 nM ± 100 nM to its target protein. In some embodiments, the first or second binding region with moderate affinity has an affinity of approximately 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, or 800 nM to its target protein. Weak affinity is when the affinity of the first or second binding region to its respective target protein is approximately 900 nM to 6000 nM, 1000 nM to 6000 nM, 900 nM to 5000 nm, 1000 nM to 5000 nm, 900 to 4000 nM, 1000 to 4000 nM, 900 to 3000 nM, 1000 to 3000 nM, and 900 This indicates that the value is approximately 2500 nM, 1000 to 2500 nM, 1500 to 6000 nM, 1500 to 5000 nM, 1500 to 4000 nM, 1500 to 3000 nM, 1500 to 2500 nM, 2000 to 6000 nM, 2000 to 5000 nM, 2000 to 4000 nM, or 2000 to 3000 nM.In some embodiments, affinity is measured using surface plasmon resonance (SPR) (e.g., using the Biacore® system), binding equilibrium exclusion (e.g., using the KinExA® system), and / or biolayer interferometry (e.g., using the ForteBio® Octet® platform). Those skilled in the art will recognize that affinity measurements are subject to some degree of variability. Therefore, the above affinity values ​​may vary within a reasonable range depending on the assay or protein.

[0166] The affinity of a binding region for its target protein can vary depending on the context of the binding region. For example, the affinity of a single TfR-binding polypeptide for TfR may differ when measured for a single TfR-binding polypeptide compared to its affinity for TfR when combined with a second binding region to form a dual transporter. Therefore, in some embodiments, the affinity values ​​and ranges provided above correspond to the affinity of the binding region for its target protein when the binding region is part of a dual transporter. In other words, the affinity of the first and second binding regions for their respective target proteins corresponds to the affinity of the dual transporter for the target proteins of the first and second binding regions.

[0167] In some embodiments, the first binding region that binds to TfR with strong affinity binds to TfR with an affinity of less than 250 nM. In some embodiments, the first binding region that binds to TfR with strong affinity binds to TfR with an affinity of 50 nM to about 250 nM. In some embodiments, the first binding region that binds to TfR with strong affinity binds to TfR with an affinity of 150 nM ± 100 nM, 150 nM ± 75 nM, or 150 nM ± 50 nM. In some embodiments, the first binding region that binds to TfR with strong affinity binds to TfR with an affinity of about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, or about 250 nM. In some embodiments, the first binding region that binds to TfR with moderate affinity binds to TfR with affinity of approximately 300 nM to approximately 900 nM, approximately 400 nM to approximately 800 nM, approximately 500 to approximately 700 nM, or approximately 600 nM. In some embodiments, the first binding region that binds to TfR with moderate affinity binds to TfR with affinity of 600 nM ± 300 nM, 600 nM ± 200 nM, or 600 nM ± 100 nM. In some embodiments, the first binding region that binds to TfR with moderate affinity binds to TfR with affinity of approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, or approximately 800 nM. In some embodiments, the first binding region that binds to TfR with weak affinity is such that TfR is bound to TfR in the following ranges: approximately 900 nM to approximately 6000 nM, approximately 1000 nM to approximately 6000 nM, approximately 900 nM to approximately 5000 nm, approximately 1000 nM to approximately 5000 nm, approximately 900 to approximately 4000 nM, approximately 1000 to approximately 4000 nM, approximately 900 to approximately 3000 nM, approximately 1000 to approximately 3000 nM, and approximately 900 It binds with affinity levels of approximately ~2500 nM, approximately 1000~2500 nM, approximately 1500~6000 nM, approximately 1500~5000 nM, approximately 1500~4000 nM, approximately 1500~3000 nM, approximately 1500~2500 nM, approximately 2000~6000 nM, approximately 2000~5000 nM, approximately 2000~4000 nM, or approximately 2000~3000 nM.

[0168] In some embodiments, the first binding region that binds to CD98hc with strong affinity binds to CD98hc with an affinity of less than 250 nM. In some embodiments, the first binding region that binds to CD98hc with strong affinity binds to CD98hc with an affinity of 50 nM to about 250 nM. In some embodiments, the first binding region that binds to CD98hc with strong affinity binds to CD98hc with an affinity of 150 nM ± 100 nM, 150 nM ± 75 nM, or 150 nM ± 50 nM. In some embodiments, a first binding region that binds to CD98hc with strong affinity binds to CD98hc with affinity of about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, or about 250 nM. In some embodiments, a first binding region that binds to CD98hc with moderate affinity binds to CD98hc with affinity of about 300 nM to about 900 nM, about 400 nM to about 800 nM, about 500 to about 700 nM, or about 600 nM. In some embodiments, the first binding region that binds to CD98hc with moderate affinity binds to CD98hc with affinity of 600 nM ± 300 nM, 600 nM ± 200 nM, or 600 nM ± 100 nM. In some embodiments, the first binding region that binds to CD98hc with moderate affinity binds to CD98hc with affinity of approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, or approximately 800 nM. In some embodiments, the first binding region that binds to CD98hc with weak affinity is such that CD98hc has a range of approximately 900 nM to 6000 nM, approximately 1000 nM to 6000 nM, approximately 900 nM to 5000 nM, approximately 1000 nM to 5000 nM, approximately 900 nM to 5000 nM, approximately 1000 to 4000 nM, approximately 900 nM to 3000 nM, and approximately 1000 to 3000 nM. It binds with affinity levels of approximately 900 nM to 2500 nM, 1000 to 2500 nM, 1500 to 6000 nM, 1500 to 5000 nM, 1500 to 4000 nM, 1500 to 3000 nM, 1500 to 2500 nM, 2000 to 6000 nM, 2000 to 5000 nM, 2000 to 4000 nM, or 2000 to 3000 nM.

[0169] In some embodiments, the first binding region that binds to MOG with strong affinity binds to MOG with an affinity of less than 250 nM. In some embodiments, the first binding region that binds to MOG with strong affinity binds to MOG with an affinity of 50 nM to about 250 nM. In some embodiments, the first binding region that binds to MOG with strong affinity binds to MOG with an affinity of 150 nM ± 100 nM, 150 nM ± 75 nM, or 150 nM ± 50 nM. In some embodiments, the first binding region that binds to MOG with strong affinity binds to MOG with an affinity of about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, or about 250 nM. In some embodiments, the first binding region that binds to MOG with moderate affinity binds to MOG with affinity of approximately 300 nM to approximately 900 nM, approximately 400 nM to approximately 800 nM, approximately 500 to approximately 700 nM, or approximately 600 nM. In some embodiments, the first binding region that binds to MOG with moderate affinity binds to MOG with affinity of 600 nM ± 300 nM, 600 nM ± 200 nM, or 600 nM ± 100 nM. In some embodiments, the first binding region that binds to MOG with moderate affinity binds to MOG with affinity of approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, or approximately 800 nM. In some embodiments, the first binding region that binds to MOG with weak affinity is such that the MOG is approximately 900 nM to 6000 nM, approximately 1000 nM to 6000 nM, approximately 900 nM to 5000 nM, approximately 1000 nM to 5000 nM, approximately 900 nM to 4000 nM, approximately 1000 to 4000 nM, approximately 900 nM to 3000 nM, approximately 1000 to 3000 nM, and approximately 90 It binds with affinity levels of 0 nM to approximately 2500 nM, approximately 1000 to approximately 2500 nM, approximately 1500 to approximately 6000 nM, approximately 1500 to approximately 5000 nM, approximately 1500 to approximately 4000 nM, approximately 1500 to approximately 3000 nM, approximately 1500 to approximately 2500 nM, approximately 2000 to approximately 6000 nM, approximately 2000 to approximately 5000 nM, approximately 2000 to approximately 4000 nM, or approximately 2000 to approximately 3000 nM.

[0170] In some embodiments, the dual transporter is (a) A first binding region that binds to TfR with an affinity of approximately 900 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 900 nM to approximately 6000 nM, (b) A first binding region that binds to TfR with an affinity of approximately 900 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 300 nM to approximately 900 nM, or (c) Includes a first binding region that binds to TfR with an affinity of approximately 900 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of less than approximately 250 nM.

[0171] In some embodiments, the dual transporter is (a) A first binding region that binds to TfR with an affinity of approximately 1000 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 1000 nM to approximately 6000 nM, (b) A first binding region that binds to TfR with an affinity of approximately 1000 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 300 nM to approximately 900 nM, or (c) Includes a first binding region that binds to TfR with an affinity of approximately 1000 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of less than approximately 250 nM.

[0172] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 900 nM to about 6000 nM, and a second binding region that binds to CD98hc with an affinity of about 50 nM to about 250 nM.

[0173] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 900 nM to about 6000 nM, and a second binding region that binds to CD98hc with an affinity of about 300 nM to about 900 nM.

[0174] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 900 nM to about 6000 nM, and a second binding region that binds to CD98hc with an affinity of about 50 nM to about 900 nM.

[0175] In some embodiments, the dual transporter is (a) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 900 nM, and a second binding region that binds to CD98hc with an affinity of approximately 900 nM to approximately 6000 nM. (b) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 900 nM, and a second binding region that binds to CD98hc with an affinity of approximately 1000 nM to approximately 6000 nM. (c) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 900 nM, and a second binding region that binds to CD98hc with an affinity of approximately 300 nM to approximately 900 nM, or (d) comprising a first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 900 nM, and a second binding region that binds to CD98hc with an affinity of less than approximately 250 nM, or

[0176] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 300 nM to about 900 nM, and a second binding region that binds to CD98hc with an affinity of about 50 nM to about 250 nM.

[0177] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 300 nM to about 900 nM, and a second binding region that binds to CD98hc with an affinity of about 300 nM to about 900 nM.

[0178] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 300 nM to about 900 nM, and a second binding region that binds to CD98hc with an affinity of about 50 nM to about 900 nM.

[0179] In some embodiments, the dual transporter is (a) A first binding region that binds to TfR with an affinity of less than approximately 250 nM, and a second binding region that binds to CD98hc with an affinity of approximately 1000 nM to approximately 6000 nM. (b) A first binding region that binds to TfR with an affinity of less than approximately 250 nM, and a second binding region that binds to CD98hc with an affinity of approximately 300 nM to approximately 900 nM, or (c) Includes a first binding region that binds to TfR with an affinity of less than approximately 250 nM, and a second binding region that binds to CD98hc with an affinity of less than approximately 250 nM.

[0180] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 50 nM to about 250 nM, and a second binding region that binds to CD98hc with an affinity of about 300 nM to about 900 nM.

[0181] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 50 nM to about 250 nM, and a second binding region that binds to CD98hc with an affinity of about 50 nM to about 250 nM.

[0182] In some embodiments, the dual transporter includes a first binding region that binds to TfR with an affinity of about 50 nM to about 250 nM, and a second binding region that binds to CD98hc with an affinity of about 50 nM to about 900 nM.

[0183] In some embodiments, the dual transporter is (a) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 900 nM to approximately 6000 nM, (c) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 300 nM to approximately 900 nM. (d) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of less than approximately 250 nM. (e) A first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 50 nM to 250 nM, or (f) It includes a first binding region that binds to TfR with an affinity of approximately 300 nM to approximately 6000 nM, and a second binding region that binds to CD98hc with an affinity of approximately 50 nM to 900 nM.

[0184] In some embodiments, to increase both the uptake and retention of the dual transporter in the brain, the affinity of the dual transporter to the BBB transporter protein (e.g., TfR) is about 50 nM to about 1000 nM, and the affinity to the second protein is about 1 nM to about 500 nM. In some embodiments, the affinity of the dual transporter to the BBB transporter protein (e.g., TfR) is about 100 to about 600 nM, and the affinity to the second protein is about 50 nM to about 300 nM. In some embodiments, the affinity of the dual transporter to the BBB transporter protein (e.g., TfR) is about 100 nM to about 400 nM, and the affinity to the second protein is about 50 nM to about 200 nM.

[0185] In some embodiments, to maximize brain retention of the dual transporter while increasing brain uptake, the affinity of the dual transporter to the BBB transporter protein (e.g., TfR) is about 250 to about 6000 nM, and the affinity to the second protein is about 1 to about 250 nM. In some embodiments, the affinity of the dual transporter to the BBB transporter protein (e.g., TfR) is about 250 to about 1000 nM, and the affinity to the second protein is about 1 to about 100 nM. In some embodiments, the affinity of the dual transporter to the BBB transporter protein (e.g., TfR) is about 50 to about 250 nM, and the affinity to the second protein is about 1 to about 100 nM.

[0186] In some embodiments, when low affinity to the BBB transport protein is desired, the affinity of the dual transporter to the BBB transport protein (e.g., TfR) is about 1000 to about 6000 nM, and the affinity to the second protein is about 1 to about 500 nM. In some embodiments, the affinity of the dual transporter to the BBB transport protein (e.g., TfR) is about 1000 to about 6000 nM, and the affinity to the second protein is about 250 to about 1000 nM. In some embodiments, the affinity of the dual transporter to the BBB transport protein (e.g., TfR) is about 1000 to about 6000 nM, and the affinity to the second protein is about 500 to about 6000 nM.

[0187] In some embodiments, to increase both the uptake and retention of the dual transporter in the brain, the affinity of the dual transporter to TfR is about 50 to about 1000 nM, and the affinity to CD98hc is about 1 to about 500 nM. In some embodiments, the affinity of the dual transporter to TfR is about 100 to about 600 nM, and the affinity to CD98hc is about 50 to about 300 nM. In some embodiments, the affinity of the dual transporter to TfR is about 100 to about 400 nM, and the affinity to CD98hc is about 50 to about 200 nM.

[0188] In some embodiments, to maximize brain retention of the dual transporter while increasing brain uptake, the affinity of the dual transporter to TfR is approximately 250 to 6000 nM, and the affinity to CD98hc is approximately 1 to 250 nM. In some embodiments, the affinity of the dual transporter to TfR is approximately 250 to 1000 nM, and the affinity to CD98hc is approximately 1 to 100 nM. In some embodiments, the affinity of the dual transporter to TfR is approximately 50 to 250 nM, and the affinity to CD98hc is approximately 1 to 100 nM.

[0189] In some embodiments, when a low TfR affinity is desired, the affinity of the dual transporter to TfR is about 1000 to about 6000 nM, and the affinity to CD98hc is about 1 to about 500 nM. In some embodiments, the affinity of the dual transporter to TfR is about 1000 to about 6000 nM, and the affinity to CD98hc is about 250 to about 1000 nM. In some embodiments, the affinity of the dual transporter to TfR is about 1000 to about 6000 nM, and the affinity to CD98hc is about 500 to about 6000 nM.

[0190] In some embodiments, to increase the transport of the dual transporter across the blood-brain barrier (BBB), the affinity of the dual transporter to the BBB transporter protein is about 5 to 10 times higher than its affinity to a second brain-retained protein. In some embodiments, the BBB transporter protein is TfR, and the affinity of the dual transporter to TfR is about 50 to 1000 nM, while the affinity of the dual transporter to the second protein is about 5 to 10 times lower than its affinity to TfR.

[0191] In some embodiments, to enhance retention in the CNS, the affinity of the dual transporter to the second protein is 5 to 10 times higher than its affinity to the BBB transporter protein. In some embodiments, the BBB transporter protein is TfR, and the affinity of the dual transporter to TfR is about 50 to 6000 nM, and the affinity of the dual transporter to the second brain-retained protein is about 5 to 10 times higher than its affinity to TfR.

[0192] As evidenced by the fact that dual transporters containing a TfR-binding domain and a CD98hc-binding domain possess similar properties to dual transporters containing a TfR-binding domain and a MOG-binding domain, it is understood that the above properties of dual transporters are likely to apply to other BBB transporter combinations with a second protein (e.g., GLUT1, IGF1R, MFSD2A, and CA-IV).

[0193] As described herein, a dual transporter or its components may be fused with a therapeutic peptide via a linker. The linker may be, but is not limited to, a peptide linker (e.g., a hinge region). The peptide linker may be configured such that the therapeutic peptide and the Fc polypeptide or Fc dimer are rotatable with respect to each other and / or resistant to digestion by proteases. In some embodiments, the linker may be a mobile linker containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters. For example, the linker may be a Gly-Ser repeat or (Gly) m (Ser) n It may have repeats such as repeats. The linker may be of any appropriate length. The peptide linker may contain at least two or three amino acids, for example, 5-50, 5-25, 5-15, 5-10, 10-50, 10-25, or 10-15 amino acids.

[0194] In some embodiments, the first binding region is linked to a therapeutic molecule. In some embodiments, the second binding region is linked to a therapeutic molecule. In some embodiments, both the first and second binding regions are linked to a therapeutic molecule. In some embodiments, the first binding region is linked to a first therapeutic molecule, and the second binding region is linked to a second therapeutic agent.

[0195] In some embodiments, the dual transporter, or its components, are linked to or fused with peptides or proteins useful for protein purification (e.g., polyhistidine, epitope tags (e.g., FLAG tags, c-Myc tags, hemagglutinin tags, etc.), glutathione S transferase, thioredoxin, protein A, protein G, or maltose-binding proteins). The peptides or proteins may be linked to or fused with the dual transporter or its components via cleavable linkers (e.g., protease cleavage sites such as cleavage sites for factor Xa or thrombin).

[0196] A. Transferrin receptor (TfR) binding domain The transferrin receptor (TfR) (differentiation antigen group 71 (also known as CD71)) binds to transferrin (Tf) and plays a crucial role in iron uptake into cells through interaction with iron-bound transferrin. TfR is highly expressed in brain capillary endothelial cells (BCECs) that form the blood-brain barrier (BBB). TfR is a 90 kDa type II transmembrane glycoprotein consisting of 760 amino acids and is found on the cell surface as a dimer (180 kDa) linked by disulfide bonds. The TfR1 monomer consists of a large extracellular C-terminal domain of 671 amino acids containing the Tf binding site, a transmembrane domain (28 amino acids), and an intracellular N-terminal domain (61 amino acids). The C-terminal extracellular domain contains three N-linked glycosylation sites at asparagine residues 251, 317, and 727, and one O-linked glycosylation site at threonine 104, all of which are necessary for proper receptor function.

[0197] A TfR-binding domain (a means for binding to TfR) is a molecule such as a polypeptide, a larger polypeptide, or a protein region or domain that specifically binds to TfR, such as human TfR.

[0198] In some embodiments, the TfR binding region binds to the apical domain of TfR, which contains residues 189–383 of human TfR. In some embodiments, the TfR binding region binds to an epitope containing position 208 of the full-length human TfR sequence. In some embodiments, the TfR binding region binds to the apical domain of TfR in an epitope containing positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and / or 294 of the full-length human TfR sequence (SEQ ID NO: 4).

[0199] In some embodiments, binding of the TfR-binding region to TfR does not inhibit the binding of transferrin to TfR. In some embodiments, binding of the dual transporter to TfR does not inhibit the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, the binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0200] 1. Modified CH3 peptide TfR binding domain of Group I a) Group Ia In some embodiments, the TfR binding region containing a modified Fc polypeptide (e.g., a modified CH3 peptide) includes four, five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 384, 386, 387, 388, 389, 413, 415, 416, and 421, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from Y at position 384; T at position 386; E at position 387; W at position 388; V, S, or A at position 389; T or S at position 413; E at position 415; E at position 416; and F at position 421.

[0201] In some embodiments, the TfR binding region comprising a modified Fc polypeptide (e.g., a modified CH3 peptide) includes four, five, six, seven, eight, or nine substitutions selected from Y at position 384; T at position 386; E at position 387; W at position 388; V, S, or A at position 389; T or S at position 413; E at position 415; E at position 416; and F at position 421.

[0202] In some embodiments, the TfR binding region containing a modified Fc polypeptide (e.g., a modified CH3 peptide) includes Y at position 384; T at position 386; E at position 387; W at position 388; V, S, or A at position 389; T or S at position 413; E at position 415; E at position 416; and F at position 421.

[0203] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or an Fc polypeptide (e.g., a modified CH3 peptide), includes Y at position 391, K at position 392, S at position 424, and S at position 426.

[0204] b) Group Ib In some embodiments, the TfR binding region containing a modified Fc polypeptide (e.g., a modified CH3 peptide) includes five, six, seven, eight, nine, ten, eleven, twelfth, tenth, eleventh, twelfth, twelfth, twelfth, twelfth, and twelfth substitutions at a series of amino acid positions consisting of 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are: L, Q, S, V, W, or Y at position 380; F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; D, E, I, or V at position 387; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; A, D, E, F, G, H, K, L, Q, R, S, T, V, or Y at position 390; 39 The following are selected for 1st place: F, Q, S, T, or V; 392nd place: Q, F, or H; 413th place: A, E, H, I, L, P, S, or T; 414th place: R, G, or P; 415th place: D, E, G, T, P, Q, or R; 416th place: D, E, N, or T; 421st place: F, H, K, W, or Y; 424th place: T, W, E, or K; and 426th place: C, P, M, W, or G.

[0205] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide), includes F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; and F, H, K, W, or Y at position 421. In some embodiments, the TfR binding region further includes V at position 387.

[0206] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; and F, H, K, W, or Y at position 421, as well as one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions at a series of amino acid positions consisting of 380, 387, 390, 391, 392, 413, 414, 415, 416, 424, and 426, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from L, Q, S, V, W, or Y at position 380; D, E, I, or V at position 387; A, D, E, F, G, H, K, L, Q, R, S, T, V, or Y at position 390; F, Q, S, T, or V at position 391; Q, F, or H at position 392; A, E, H, I, L, P, S, or T at position 413; R, G, or P at position 414; D, E, G, T, P, Q, or R at position 415; D, E, N, or T at position 416; T, W, E, or K at position 424; and C, P, M, W, or G at position 426.

[0207] In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes substitutions of F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; and F, H, K, W, or Y at position 421, as well as one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions selected from the following: at position 380 L, Q, S, V, W, or Y; D, E, I, or V at 387th place; A, D, E, F, G, H, K, L, Q, R, S, T, V, or Y at 390th place; F, Q, S, T, or V at 391st place; Q, F, or H at 392nd place; A, E, H, I, L, P, S, or T at 413th place; R, G, or P at 414th place; D, E, G, T, P, Q, or R at 415th place; D, E, N, or T at 416th place; T, W, E, or K at 424th place; and C, P, M, W, or G at 426th place.

[0208] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; V at position 387; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; and F, H, K, W, or Y at position 421, as well as one, two, three, four, five, six, seven, eight, nine, or ten substitutions selected from the following: at position 380 L, Q, S, V, W, or Y; F, H, I, L, N, P, T, or V at rank 386; A, D, E, F, G, H, K, L, Q, R, S, T, V, or Y at rank 390; F, Q, S, T, or V at rank 391; Q, F, or H at rank 392; A, E, H, I, L, P, S, or T at rank 413; R, G, or P at rank 414; D, E, G, T, P, Q, or R at rank 415; D, E, N, or T at rank 416; T, W, E, or K at rank 424; and C, P, M, W, or G at rank 426.

[0209] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or an Fc polypeptide (e.g., a modified CH3 peptide), includes Y at position 391, K at position 392, S at position 424, and S at position 426.

[0210] In some embodiments, the TfR binding region containing the modified CH3 peptide includes four, five, six, seven, eight, nine, ten, eleven, t, two, three, and sixteen amino acid substitutions at a series of amino acid positions consisting of 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426, where the amino acid at each of positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 may be any of the amino acids shown in Table 1.

[0211] In some embodiments, the TfR binding domain contains a modified CH3 peptide having one of the amino acid substitutions from the clones shown in Tables 3-5. The affinity (Kd) for human and TfR for the shown TfR binding domains is shown in Table 20. [Table 1]

[0212] In some embodiments, the TfR binding region containing the modified CH3 peptide has at least 85%, at least 90%, at least 95%, or 100% identity with any of the amino acid sequences of SEQ ID NOs. 6-14 and 124 and 125, clone CH3C.35.23.2 with a knob mutation, clone CH3C.35.23.2 with a knob mutation and an LALA mutation, clone CH3C.35.23.2 with a knob mutation and an LALAPG mutation, clone CH3C.35.23.3 with a knob mutation and an LALA mutation, clone CH3C.35.23.3 with a knob mutation and an LALA mutation, and clone CH3C.35.23.3 with a knob mutation and an LALAPG mutation. This includes clone CH3C.35.23.3 having a knob mutation, clone CH3C.35.23.4 having a knob mutation and an LALA mutation, clone CH3C.35.23.4 having a knob mutation and an LALAPG mutation, clone TV35.d1.9 having a knob mutation, clone TV35.d1.9 having a knob mutation and an LALA mutation, clone TV35.d1.9 having a knob mutation and an LALAPG mutation, clone TV35.d1.10 having a knob mutation, clone TV35.d1.10 having a knob mutation and an LALA mutation, and clone TV35.d1.10 having a knob mutation and an LALAPG mutation. Any of the above may contain a hole mutation instead of a knob mutation.

[0213] In some embodiments, the TfR binding region containing the modified CH3 peptide comprises a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with amino acids 111-217 of any of SEQ ID NOs. 6-14 and 124 and 125. [Table 2-1] [Table 2-2] [Table 2-3]

[0214] c) Group Ic In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises 6, 7, 8, 9, and 10 substitutions at a series of amino acid positions consisting of 384, 386, 387, 388, 389, 390, 391, 413, 416, and 421, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from L, M, V, W, or Y at position 384; F, H, L, P, or T at position 386; E or V at position 387; W at position 388; A, G, S, or V at position 389; A, D, E, F, G, H, K, L, Q, S, or T at position 390; F, Q, S, T, or V at position 391; A, E, L, P, S, or T at position 413; D, E, N, or T at position 416; and F, H, W, or Y at position 421.

[0215] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises L, M, V, W, or Y at position 384; F, H, L, P, or T at position 386; W at position 388; A, G, S, or V at position 389; D, E, N, or T at position 416; and F, H, W, or Y at position 421. In some embodiments, the TfR binding region further comprises V at position 387.

[0216] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes L, M, V, W, or Y at position 384; F, H, L, P, or T at position 386; W at position 388; A, G, S, or V at position 389; D, E, N, or T at position 416; and F, H, W, or Y at position 421; as well as one, two, three, or four substitutions at a series of amino acid positions consisting of 387, 390, 391, and 413, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from E or V at position 387; A, D, E, F, G, H, K, L, Q, S, or T at position 390; F, Q, S, T, or V at position 391; and A, E, L, P, S, or T at position 413.

[0217] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes L, M, V, W, or Y at position 384; F, H, L, P, or T at position 386; W at position 388; A, G, S, or V at position 389; D, E, N, or T at position 416; and F, H, W, or Y at position 421; as well as one, two, three, or four substitutions selected from: E or V at position 387; A, D, E, F, G, H, K, L, Q, S, or T at position 390; F, Q, S, T, or V at position 391; and A, E, L, P, S, or T at position 413.

[0218] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes L, M, V, W, or Y at position 384; F, H, L, P, or T at position 386; V at position 387; W at position 388; A, G, S, or V at position 389; D, E, N, or T at position 416; and F, H, W, or Y at position 421; as well as one, two, or three substitutions selected from: A, D, E, F, G, H, K, L, Q, S, or T at position 390; F, Q, S, T, or V at position 391; and A, E, L, P, S, or T at position 413.

[0219] In some embodiments, the TfR binding region contains a modified CH3 peptide having one of the amino acid substitutions from the clones shown in Table 3. [Table 3-1] [Table 3-2]

[0220] d) Group ID In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes eight, nine, ten, or eleven substitutions at a series of amino acid positions consisting of 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421, where the positions are determined by reference to EU numbering. In some embodiments, the substitutions are selected from W or L at position 380; F or Y at position 384; T at position 386; E at position 387; W at position 388; S, A, or V at position 389; S at position 390; S or T at position 413; E at position 415; E at position 416; and F at position 421.

[0221] In some embodiments, the TfR-binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) comprises F or Y at position 384; T at position 386; E at position 387; W at position 388; S, A, or V at position 389; S or T at position 413; E at position 416; and F at position 421.

[0222] In some embodiments, the TfR-binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) comprises F or Y at position 384; T at position 386; E at position 387; W at position 388; S, A, or V at position 389; S or T at position 413; E at position 416; and F at position 421; and one or two substitutions at a series of amino acid positions consisting of 390 and 415, the positions being determined by reference to EU numbering. In some embodiments, the substitutions are selected from S at position 390 and E at position 415.

[0223] In some embodiments, the TfR-binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) comprises F or Y at position 384; T at position 386; E at position 387; W at position 388; S, A, or V at position 389; S or T at position 413; E at position 416; and F at position 421; and one or two substitutions selected from the following: S at position 390 and E at position 415.

[0224] In some embodiments, the TfR-binding region comprises a modified CH3 peptide having the amino acid substitutions of any of the clones shown in Table 4.

Table 4-1

Table 4-2

Table 4-3

[0225] e) Group Ie In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises six, seven, eight, nine, ten, eleven, twelfth, or thirteen substitutions at a series of amino acid positions consisting of 380, 384, 386, 387, 388, 389, 390, 391, 413, 414, 415, 416, 421, 424, and 426, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from W, L, S, V, or Y at position 380; Y, F, M, P, V, or W at position 384; T, N, or V at position 386; E, I, or V at position 387; W at position 388; S, A, I, T, or V at position 389; S, R, or T at position 390; S, T, or H at position 413; E, D, G, T, P, Q, or R at position 415; E at position 416; F, H, K, or Y at position 421; T or W at position 424; and C, P, M, or W at position 426.

[0226] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes Y, F, M, P, V, or W at position 384; T, N, or V at position 386; W at position 388; S, A, I, T, or V at position 389; S, T, or H at position 413; and F, H, K, or Y at position 421.

[0227] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes Y, F, M, P, V, or W at position 384; T, N, or V at position 386; W at position 388; S, A, I, T, or V at position 389; S, T, or H at position 413; and F, H, K, or Y at position 421; as well as one, two, three, four, five, six, or seven substitutions at a series of amino acid positions consisting of 380, 387, 390, 415, 416, 424, and 426, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from W, L, S, V, or Y at position 380; E, I, or V at position 387; S, R, or T at position 390; E, D, G, T, P, Q, or R at position 415; E at position 416; T or W at position 424; and C, P, M, or W at position 426.

[0228] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes Y, F, M, P, V, or W at position 384; T, N, or V at position 386; W at position 388; S, A, I, T, or V at position 389; S, T, or H at position 413; and F, H, K, or Y at position 421; as well as one, two, three, four, five, six, or seven substitutions selected from the following: W, L, S, V, or Y at position 380; E, I, or V at position 387; S, R, or T at position 390; E, D, G, T, P, Q, or R at position 415; E at position 416; T or W at position 424; and C, P, M, or W at position 426.

[0229] f) Group If In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 384, 386, 387, 389, 390, 391, 413, 416, and 421, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from V, Y, L, or W at position 384; T, L, H, P, or F at position 386; V or E at position 387; A, S, V, or G at position 389; A, D, E, G, H, L, Q, T, or V at position 390; T, F, Q, or V at position 391; L, S, E, A, or P at position 413; E, D, T, or N at position 416; and W, Y, H, or F at position 421.

[0230] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes V, Y, L, or W at position 384; T, L, H, P, or F at position 386; A, S, V, or G at position 389; L, S, E, A, or P at position 413; E, D, T, or N at position 416; and W, Y, H, or F at position 421.

[0231] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes V, Y, L, or W at position 384; T, L, H, P, or F at position 386; A, S, V, or G at position 389; L, S, E, A, or P at position 413; E, D, T, or N at position 416; and W, Y, H, or F at position 421; as well as one, two, or three substitutions at a series of amino acid positions consisting of 387, 390, and 391, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from V or E at position 387; A, D, E, G, H, L, Q, T, or V at position 390; and T, F, Q, or V at position 391.

[0232] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes V, Y, L, or W at position 384; T, L, H, P, or F at position 386; A, S, V, or G at position 389; L, S, E, A, or P at position 413; E, D, T, or N at position 416; and W, Y, H, or F at position 421; as well as one, two, or three substitutions selected from: V or E at position 387; A, D, E, G, H, L, Q, T, or V at position 390; and T, F, Q, or V at position 391.

[0233] In some embodiments, the TfR-binding domain contains a modified CH3 peptide having one of the amino acid substitutions among the clones shown in Table 5. The affinity (Kd) to human TfR for the shown TfR-binding domains is shown in Table 5. [Table 5-1] [Table 5-2]

[0234] 2. TfR binding domain of modified CH3 peptide in Group II a) Group IIa In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) is located at 378, 380, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 414, 417, 419, 420, 421, 422, 424, 426, 427, 428, 429, 433, 434 This includes six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 amino acid substitutions at a series of amino acid positions consisting of 437, 438, 439, 440, 442, and 443 and 426, the positions of which are determined by reference to EU numbering. In some embodiments, substitutions are E, L, or I at position 378; N, R, Y, I, S, or F at position 380; F at position 382; Y, A, G, T, or deletion at position 383; A, D, E, F, G, or T at position 384; A, D, or N at position 385; A, G, K, N, S, or Y at position 386; G, I, K, N, R, S, or T at position 387; L, Q, or D at position 388; I, P, Q, R, S, or T at position 389; G, T, Y, or L at position 390; and at position 391. The following are selected: S, T, I, L, or P; P at position 414; K at position 417; P at position 419; R or Q at position 420; A, F, G, S, or Y at position 421; L at position 422; A at position 424; E at position 426; E at position 427; E at position 428; G at position 429; E at position 433; G at position 434; D at position 437; Y at position 438; D, E, or S at position 439; L at position 440; G or W at position 442; and E or Y at position 443.

[0235] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) includes F at position 382, ​​L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440.

[0236] In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) is F at position 382, ​​L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440; as well as 378, 380, 383, 384, 385, 386, 387, 388, 389, 390, 391, 414 This includes one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 substitutions at a series of amino acid positions consisting of 417, 419, 420, 421, 427, 428, 429, 433, 434, 437, 439, 442, and 443, the positions of which are determined by reference to EU numbering. In some embodiments, substitutions are E, L, or I at position 378; N, R, Y, I, S, or F at position 380; Y, A, G, T, or deletion at position 383; A, D, E, F, G, or T at position 384; A, D, or N at position 385; A, G, K, N, S, or Y at position 386; G, I, K, N, R, S, or T at position 387; L, Q, or D at position 388; I, P, Q, R, S, or T at position 389; and at position 390. Selected from G, T, Y, or L; S, T, I, L, or P at position 391; P at position 414; K at position 417; P at position 419; R or Q at position 420; A, F, G, S, or Y at position 421; E at position 427; E at position 428; G at position 429; E at position 433; G at position 434; D at position 437; D, E, or S at position 439; G or W at position 442; and E or Y at position 443.

[0237] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) comprises F at position 382, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440; and one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or twenty-five substitutions selected from: E, L, or I at position 378; N, R, Y, I, S, or F at position 380; Y, A, G, T, or deletion at position 383; A, D, E, F, G, or T at position 384; A, D, or N at position 385; A, G, K, N, S, or Y at position 386; G, I, K, N, R, S, or T at position 387; L, Q, or D at position 388; I, P, Q, R, S, or T at position 389; G, T, Y, or L at position 390; S, T, I, L, or P at position 391; P at position 414; K at position 417; P at position 419; R or Q at position 420; A, F, G, S, or Y at position 421; E at position 427; E at position 428; G at position 429; E at position 433; G at position 434; D at position 437; D, E, or S at position 439; G or W at position 442; and E or Y at position 443.

[0238] In some embodiments, the TfR binding region containing the modified CH3 peptide is located at six, seven, eight, nine, ten, eleven, twelfth The amino acids at positions 4, 25, 26, 27, 28, 29, 30, or 31 include amino acid substitutions, and the amino acids at each of the positions 378, 380, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 414, 417, 419, 420, 421, 422, 424, 426, 427, 428, 429, 433, 434, 437, 438, 439, 440, 442, and 443 may be any of the amino acids shown in Table 6.

[0239] In some embodiments, the TfR binding region includes a modified CH3 peptide having one of the amino acid substitutions from the clones shown in Table 7.

[0240] In some embodiments, the TfR binding region includes a modified CH3 peptide having at least 85%, at least 90%, at least 95%, or 100% identical amino acids to any of the clones shown in Table 8.

[0241] Table 9 shows the affinity (Kd) for human TfR for the TfR-binding domains shown. [Table 6] [Table 7] [Table 8-1] [Table 8-2] [Table 9]

[0242] b) Group IIb In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes substitutions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 at a series of amino acid positions consisting of 378, 380, 382, ​​384, 386, 387, 388, 389, 390, 391, 414, 417, 421, 422, 424, 426, 437, 438, 439, 440, and 442, where the positions are determined by reference to EU numbering. In some embodiments, the substitution is selected from E, L, or I at position 378; N, R, Y, I, S, or F at position 380; F at position 382; G, F, D, or E at position 384; A, S, or N at position 386; I, R, T, K, N, or S at position 387; L at position 388; Q, T, I, S, or P at position 389; G, T, Y, or L at position 390; S, T, I, L, or P at position 391; P at position 414; K at position 417; A, F, G, S, or Y at position 421; L at position 422; A at position 424; E at position 426; D at position 437; Y at position 438; D, E, or S at position 439; L at position 440; and W at position 442.

[0243] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; G, F, D, or E at position 384; I, R, T, K, N, or S at position 387; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440.

[0244] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; G, F, D, or E at position 384; I, R, T, K, N, or S at position 387; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; as well as one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, or 13 substitutions at a set of amino acid positions consisting of 378, 380, 386, 388, 389, 390, 391, 414, 417, 421, 437, 439, and 442, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from E, L, or I at position 378; N, R, Y, I, S, or F at position 380; A, S, or N at position 386; L at position 388; Q, T, I, S, or P at position 389; G, T, Y, or L at position 390; S, T, I, L, or P at position 391; P at position 414; K at position 417; A, F, G, S, or Y at position 421; D at position 437; D, E, or S at position 439; and W at position 442.

[0245] In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) may be F at position 382; G, F, D, or E at position 384; I, R, T, K, N, or S at position 387; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; and one, two, three, four, five, six, seven, eight, nine, ten, 11, 12 selected from the following, and This includes 13 substitutions: E, L, or I at position 378; N, R, Y, I, S, or F at position 380; A, S, or N at position 386; L at position 388; Q, T, I, S, or P at position 389; G, T, Y, or L at position 390; S, T, I, L, or P at position 391; P at position 414; K at position 417; A, F, G, S, or Y at position 421; D at position 437; D, E, or S at position 439; and W at position 442.

[0246] c) Group IIc In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes six, seven, eight, nine, ten, or eleven substitutions at a series of amino acid positions consisting of 382, ​​384, 385, 386, 387, 389, 422, 424, 426, 438, and 440, where the positions are determined by reference to EU numbering. In some embodiments, the substitutions are selected from F at position 382; N, F, or D at position 384; A at position 385; S, G, or A at position 386; K or T at position 387; S or T at position 389; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440.

[0247] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440.

[0248] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) includes F at position 382; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; as well as one, two, three, four, or five substitutions at a series of amino acid positions consisting of 384, 385, 386, 387, and 389, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from N, F, or D at position 384; A at position 385; S, G, or A at position 386; K or T at position 387; and S or T at position 389.

[0249] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) comprises F at position 382; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; and one, two, three, four, or five substitutions selected from: N, F, or D at position 384; A at position 385; S, G, or A at position 386; K or T at position 387; and S or T at position 389.

[0250] d) Group IId In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), includes substitutions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 at a series of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 388, 389, 419, 420, 421, 422, 424, 426, 427, 428, 429, 438, 440, 442, and 443, where the positions are determined by reference to EU numbering. In some embodiments, the substitution is selected from F at position 382; Y, A, G, T or deletion at position 383; D, E, G, or T at position 384; D, A, or N at position 385; S, N, A, K, Y, or G at position 386; K or G at position 387; L, Q, or D at position 388; T or R at position 389; P at position 419; R or Q at position 420; G at position 421; L at position 422; A at position 424; E at position 426; E at position 427; E at position 428; G at position 429; Y at position 438; L at position 440; G at position 442; and E or Y at position 443.

[0251] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; D, E, G, or T at position 384; S, N, A, K, Y, or G at position 386; K or G at position 387; T or R at position 389; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440.

[0252] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; D, E, G, or T at position 384; S, N, A, K, Y, or G at position 386; K or G at position 387; T or R at position 389; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; as well as one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions at a series of amino acid positions consisting of 383, 385, 388, 419, 420, 421, 427, 428, 429, 442, and 443, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from Y, A, G, T, or deletion at position 383; D, A, or N at position 385; L, Q, or D at position 388; P at position 419; R or Q at position 420; G at position 421; E at position 427; E at position 428; G at position 429; G at position 442; and E or Y at position 443.

[0253] In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) is characterized by F at position 382; D, E, G, or T at position 384; S, N, A, K, Y, or G at position 386; K or G at position 387; T or R at position 389; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440. Furthermore, it includes one, two, three, four, five, six, seven, eight, nine, ten, or eleven selected from the following: Y, A, G, T or missing at position 383; D, A, or N at position 385; L, Q, or D at position 388; P at position 419; R or Q at position 420; G at position 421; E at position 427; E at position 428; G at position 429; G at position 442; and E or Y at position 443.

[0254] e) Group IIe In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), includes seven, eight, nine, ten, eleven, twelfth In some embodiments, the substitution is selected from F, N, or Y at position 380; F at position 382; A, Y, or deletion at position 383; D, E, or G at position 384; D, A, or N at position 385; N, A, G, or S at position 386; G, I, K, or R at position 387; L, Q, or D at position 388; R, S, or T at position 389; L at position 422; A at position 424; E at position 426; E at position 433; G at position 434; Y at position 438; and L at position 440.

[0255] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; G, I, K, or R at position 387; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440.

[0256] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) includes F at position 382; G, I, K, or R at position 387; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; as well as one, two, three, four, five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 380, 383, 384, 385, 386, 388, 389, 433, and 434, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from F, N, or Y at position 380; A, Y, or deletion at position 383; D, E, or G at position 384; D, A, or N at position 385; N, A, G, or S at position 386; L, Q, or D at position 388; R, S, or T at position 389; E at position 433; and G at position 434.

[0257] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes F at position 382; G, I, K, or R at position 387; L at position 422; A at position 424; E at position 426; Y at position 438; and L at position 440; and one, two, three, four, five, six, seven, eight, or nine substitutions selected from: F, N, or Y at position 380; A, Y, or deletion at position 383; D, E, or G at position 384; D, A, or N at position 385; N, A, G, or S at position 386; L, Q, or D at position 388; R, S, or T at position 389; E at position 433; and G at position 434.

[0258] 3. TfR binding domain of modified CH3 peptide in Group III In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises four, five, six, seven, eight, nine, ten, eleven, twelfth, twelve, thirteenth, fif In some embodiments, substitutions are D, E, F, H, N, Q, S, V, or Y at position 378; A, D, F, H, I, K, L, M, Q, S, T, or Y at position 380; G at position 382; T at position 383; A, E, F, H, I, K, L, Q, S, V, or Y at position 384; I, T, or V at position 385; A, H, N, S, T, or V at position 386; A, F, H, K at position 421 , L, M, Q, S, T, V, or Y; A, F, H, I, K, L, R, T, or Y at rank 422; A, G, or P at rank 424; A, I, L, T, or V at rank 426; A or L at rank 428; S at rank 434; I, F, L, V, or Y at rank 438; A, G, I, M, N, P, T, or V at rank 440; and selected from A, K, M, R, T, or V at rank 442.

[0259] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; A, E, F, H, I, K, L, Q, S, V, or Y at position 384; I, T, or V at position 385; and I, F, L, V, or Y at position 438.

[0260] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve substitutions at a series of amino acid positions consisting of 378, 380, 383, 386, 421, 422, 424, 426, 428, 434, 440, and 442, where the positions are determined by reference to EU numbering. In some embodiments, the substitution is selected from D, E, F, H, N, Q, S, V, or Y at position 378; A, D, F, H, I, K, L, M, Q, S, T, or Y at position 380; T at position 383; A, H, N, S, T, or V at position 386; A, F, H, K, L, M, Q, S, T, V, or Y at position 421; A, F, H, I, K, L, R, T, or Y at position 422; A, G, or P at position 424; A, I, L, T, or V at position 426; A or L at position 428; S at position 434; A, G, I, M, N, P, T, or V at position 440; and A, K, M, R, T, or V at position 442.

[0261] In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; A, E, F, H, I, K, L, Q, S, V, or Y at position 384; I, T, or V at position 385; and I, F, L, V, or Y at position 438, along with one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, or 16 substitutions selected from: D, E, F, H, N, Q, S, V, or Y at position 378; A, D, F, H, I, K, L, M, Q, S, T, or Y at rank 380; T at rank 383; A, H, N, S, T, or V at rank 386; A, F, H, K, L, M, Q, S, T, V, or Y at rank 421; A, F, H, I, K, L, R, T, or Y at rank 422; A, G, or P at rank 424; A, I, L, T, or V at rank 426; A or L at rank 428; S at rank 434; A, G, I, M, N, P, T, or V at rank 440; and A, K, M, R, T, or V at rank 442.

[0262] In some embodiments, the TfR binding region containing the modified CH3 peptide has four, five, six, seven, eight, nine, ten, eleven, t, two, three, four, and sixteen amino acid substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 421, 422, 424, 426, 428, 434, 438, 440, and 442, where the amino acid at each of the positions 378, 380, 382, ​​383, 384, 385, 386, 421, 422, 424, 426, 428, 434, 438, 440, and 442 may be any of the amino acids shown in Table 10.

[0263] In some embodiments, the TfR-binding domain contains a modified CH3 peptide having one of the amino acid substitutions among the clones shown in Tables 11 and 12. The affinity (Kd) to TfR in humans and cynomolgus monkeys for the shown TfR-binding domains is shown in Table 11. [Table 10] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 12]

[0264] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), includes four, five, six, seven, eight, nine, ten, or eleven substitutions at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 422, 424, 426, 436, 438, and 440, where the positions are determined by reference to EU numbering. In some embodiments, the substitution is selected from K, L, I, Q, V, F, or M at position 380; G at position 382; A, E, or L at position 384; V at position 385; A, M, S, or V at position 386; I, H, R, or L at position 422; P or A at position 424; T, I, or V at position 426; F at position 436; L, V, I, or Y at position 438; and M, G, A, T, or V at position 440.

[0265] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or an Fc polypeptide (e.g., a modified CH3 peptide), includes G at position 382; V at position 385; and L, V, I, or Y at position 438.

[0266] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; V at position 385; and L, V, I, or Y at position 438, as well as one, two, three, four, five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 380, 384, 386, 422, 424, 426, 436, and 440, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from K, L, I, Q, V, F, or M at position 380; A, E, or L at position 384; A, M, S, or V at position 386; I, H, R, or L at position 422; P or A at position 424; T, I, or V at position 426; F at position 436; and M, G, A, T, or V at position 440.

[0267] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) comprises G at position 382; V at position 385; and L, V, I, or Y at position 438, and one, two, three, four, five, six, seven, eight, or nine substitutions selected from: K, L, I, Q, V, F, or M at position 380; A, E, or L at position 384; A, M, S, or V at position 386; I, H, R, or L at position 422; P or A at position 424; T, I, or V at position 426; F at position 436; and M, G, A, T, or V at position 440.

[0268] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) includes G at position 382; A, E, or L at position 384; V at position 385; P or A at position 424; T, I, or V at position 426; L, V, I, or Y at position 438; and M, G, A, T, or V at position 440.

[0269] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; A, E, or L at position 384; V at position 385; P or A at position 424; T, I, or V at position 426; L, V, I, or Y at position 438; and M, G, A, T, or V at position 440, as well as one, two, three, or four substitutions at a series of amino acid positions consisting of 380, 386, 422, and 436, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from K, L, I, Q, V, F, or M at position 380; A, M, S, or V at position 386; I, H, R, or L at position 422; and F at position 436.

[0270] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; A, E, or L at position 384; V at position 385; P or A at position 424; T, I, or V at position 426; L, V, I, or Y at position 438; and M, G, A, T, or V at position 440, along with one, two, three, or four substitutions selected from: K, L, I, Q, V, F, or M at position 380; A, M, S, or V at position 386; I, H, R, or L at position 422; and F at position 436.

[0271] In some embodiments, the TfR binding region contains a modified CH3 peptide having one of the amino acid substitutions from the clones shown in Table 13. [Table 13]

[0272] In some embodiments, the TfR binding region containing a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes four, five, six, seven, eight, nine, ten, or eleven substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​384, 385, 386, 422, 424, 426, 438, and 440, where the positions are determined by reference to EU numbering. In some embodiments, the substitutions are selected from D, Y, H, S, V, F, or Y at position 378; F, Y, or D at position 380; G at position 382; A, E, or L at position 384; V at position 385; A or S at position 386; F, L, or I at position 422; P or A at position 424; I at position 426; I or Y at position 438; and V, G, or T at position 440.

[0273] In some embodiments, the TfR binding region, which includes a modified antibody heavy chain constant domain or an Fc polypeptide (e.g., a modified CH3 peptide), comprises G at position 382; V at position 385; and I or Y at position 438.

[0274] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; V at position 385; and I or Y at position 438; as well as one, two, three, four, five, six, seven, or eight substitutions at a series of amino acid positions consisting of 378, 380, 384, 386, 422, 424, 426, and 440, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from D, Y, H, S, V, F, or Y at position 378; F, Y, or D at position 380; A, E, or L at position 384; A or S at position 386; F, L, or I at position 422; P or A at position 424; I at position 426; and V, G, or T at position 440.

[0275] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) comprises G at position 382; V at position 385; and I or Y at position 438; and one, two, three, four, five, six, seven, or eight substitutions selected from: D, Y, H, S, V, F, or Y at position 378; F, Y, or D at position 380; A, E, or L at position 384; A or S at position 386; F, L, or I at position 422; P or A at position 424; I at position 426; and V, G, or T at position 440.

[0276] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; A, E, or L at position 384; V at position 385; P or A at position 424; I at position 426; and I or Y at position 438.

[0277] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide) includes G at position 382; A, E, or L at position 384; V at position 385; P or A at position 424; I at position 426; I or Y at position 438; and one, two, three, or four substitutions at a series of amino acid positions consisting of 378, 382, ​​383, 389, 421, 440, and 442, the positions of which are determined by reference to EU numbering. In some embodiments, the substitutions are selected from D, Y, H, S, V, F, or Y at position 378; F, Y, or D at position 380; A or S at position 386; and F, L, or I at position 422.

[0278] In some embodiments, the TfR binding region comprising a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) includes G at position 382; A, E, or L at position 384; V at position 385; P or A at position 424; I at position 426; I or Y at position 438; and one, two, three, four, five, six, or seven substitutions selected from: D, Y, H, S, V, F, or Y at position 378; F, Y, or D at position 380; A or S at position 386; and F, L, or I at position 422.

[0279] In some embodiments, a TfR binding region containing a modified CH3 peptide having one of the amino acid substitutions among the clones shown in Table 14. [Table 14]

[0280] 4. TfR binding region of group IV In some embodiments, the TfR binding region includes an anti-TfR antibody antigen-binding domain. The anti-TfR antibody antigen-binding domain may include an antibody, Fab (containing F(ab')2), scFab, Fv fragment, scFv, or nanobody. Examples of anti-TfR antibody antigen-binding domains include: 17H10 anti-TfR Fab or scFv; 17H10.1 anti-TfR Fab or scFv; JC-141 anti-TfR antibody; JC-141 anti-TfR Fab; JC-141 anti-TfR scFv; anti-TfR antibody, Fab, scFab, Fv fragment, or scFv (WO2016208695) having the CDR1, CDR2, and CDR3 sequences of the heavy and light chains of the JR-141 antibody; JC-171 anti-TfR antibody; JC-171 anti-TfR Fab; JC-171 anti-TfR scFv;Anti-TfR antibody, Fab, scFab, Fv fragment, or scFv (WO2018124121) having the CDR1, CDR2, and CDR3 sequences of the heavy and light chains of JR-171 antibody; "Brain Shuttle" (BS) anti-TfR Fab;Anti-TfR antibody, Fab, scFab, Fv fragment, or scFv (WO2018210898, WO2015101588, and WO2014033074) having the CDR1, CDR2, and CDR3 sequences of the heavy and light chains of BS anti-TfR Fab; 13E4v2ii anti-TfR antibody; 13E4v2ii anti-TfR Fab; 13E4v2ii anti-TfR Anti-TfR antibody, Fab, scFab, Fv fragment, or scFv(WO2020132584);TfR12 anti-TfR scFv;TfR12 anti-TfR scFv;Anti-TfR antibody, Fab, scFab, Fv fragment, or scFv(WO2021 / 205358);TfR13 anti-TfR scFv;or Tfr13 anti-TfR Examples include, but are not limited to, anti-TfR antibodies, Fab, scFab, Fv fragments, or scFv(WO2021 / 205358) (sequence shown in Table 15), which have the CDR1, CDR2, and CDR3 sequences of the heavy and light chains of scFv.

[0281] Further anti-TfR antibodies are described in WO2021 / 205358, and the double-bonded molecules of the present invention include TfR1, TfR2, TfR3, TfR4, TfR5, TfR6, TfR7, TfR8, TfR9, TfR10, TfR11, TfR12, TfR13, TfR14, TfR15, TfR16, TfR17, TfR18, TfR19, TfR20, TfR21, T It may include any antibody-antigen-binding domain (as described in the same document) having one of the following CDRs or variable regions: fR22, TfR23, TfR24, TfR25, TfR26, TfR27, TfR28, TfR29, TfR30, TfR31, TfR32, TfR33, TfR34, TfR35, TfR36, TfR37, and TfR38. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7] [Table 15-8] [Table 15-9]

[0282] Further anti-TfR antibodies are known in the art and are available from various commercial sources. In some embodiments, the anti-TfR antibody or the TfR-binding fragment of the anti-TfR antibody binds to the apical domain of TfR. In some embodiments, the binding of the anti-TfR antibody or the TfR-binding fragment of the anti-TfR antibody to TfR does not inhibit the binding of transferrin to TfR. Examples of anti-TfR antibodies include, but are not limited to, B3 / 25, RBC4, 7579, E2.3, A27.15, D65.30, D2C, ch128.1Av, ch128.1 / IgG3, ch128.1 / IgG1, hu128.1 (Candelaria et al. Front).Immunol.12 (17 March 2021), 2021), and Ri7,8D3 (Weber et al. Cell Reports 22:149-162, 2018). Exemplary anti-TfR antibodies are also described in U.S. Patent Publications US2018282408A1, US2020071413A1, US20210138083A1, US20190092870A1, and US20130028891 (each of which is incorporated herein by reference).

[0283] An exemplary anti-TfR vNAR is described in WO2022 / 103769.

[0284] Brain shuttles containing anti-TfR or other anti-BBB protein binding domains are described in WO2014 / 033074 and WO2015 / 101588 (each of which is incorporated herein by reference).

[0285] Antibody-antigen-binding domains that specifically bind to BBB proteins can be identified using methods available in the art for producing and / or identifying antibodies that specifically bind to target proteins. Such methods include, but are not limited to, immunization, phage display, and ribosome display.

[0286] In some embodiments, the TfR binding region includes a fibronectin type III domain peptide modified to specifically bind to TfR, for example, one of the domains described in WO2021 / 076546 and WO2022 / 221505 (each of which is incorporated herein by reference).

[0287] In some embodiments, the TfR binding domain includes a bicyclic peptide that specifically binds to TfR, for example, one of the bicyclic peptides described in WO2022 / 101633 (incorporated herein by reference).

[0288] The TfR-binding region may originate from proteins known to bind to TfR, such as Plasmodium vivax reticulocyte-binding protein 2b (PvRBP2b), or from viral proteins known to bind to TfR, such as arenavirus proteins (e.g., Machupovirus, Sabiavirus, Juninvirus, Guanalitovirus, or Chaparevirus).

[0289] In some embodiments, the TfR binding region comprises an engineered polypeptide. The engineered polypeptide may be an antigen-binding region of a polypeptide (e.g., an antibody Fc polypeptide) or an anti-TfR antibody, which has been modified to alter its affinity for TfR. The engineered peptide may be identified or produced using methods available in the art for identifying or producing peptides that have affinity for a known target (e.g., TfR). Such methods include, but are not limited to, phage displays, yeast displays (e.g., yeast surface displays), and directed evolution methods, as well as combinations thereof.

[0290] In some embodiments, the TfR binding domain binds to human TfR with an affinity of approximately 15 nM to approximately 6000 nM (e.g., approximately 15 nM, approximately 50 nM, approximately 100 nM, approximately 200 nM, approximately 300 nM, approximately 400 nM, approximately 500 nM, approximately 600 nM, approximately 700 nM, approximately 800 nM, approximately 900 nM, approximately 1000 nM, approximately 1500 nM, approximately 2000 nM, approximately 2500 nM, approximately 3000 nM, approximately 3500 nM, approximately 4000 nM, approximately 4500 nM, approximately 5000 nM, approximately 5500 nM, or approximately 6000 nM). In some embodiments, the TfR binding domain binds to human TfR with an affinity of approximately 100 nM to approximately 1000 nM or approximately 100 nM to approximately 600 nM. In some embodiments, the TfR binding domain binds to human TfR with an affinity of approximately 600 nM to approximately 6000 nM or approximately 600 to approximately 1000 nM. In some embodiments, the TfR binding domain binds to human TfR with an affinity of approximately 200 nM to approximately 1000 nM, approximately 300 nM to approximately 1000 nM, approximately 400 nM to approximately 1000 nM, or approximately 500 to approximately 1000 nM. In some embodiments, the TfR binding domain binds to human TfR with an affinity of approximately 100 nM to approximately 1000 nM (for example, approximately 100 nM, approximately 150 nM, approximately 200 nM, approximately 250 nM, approximately 300 nM, approximately 350 nM, approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, approximately 800 nM, approximately 850 nM, approximately 900 nM, approximately 950 nM, or approximately 1000 nM). In some embodiments, the TfR binding domain binds to human TfR with affinity of about 600 nM to about 1000 nM (e.g., about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, or about 1000 nM). In some embodiments, the TfR binding domain exhibits cynomolgus monkey (cyno) cross-reactivity. In some embodiments, the TfR binding region binds to cynomolgus monkey TfR with an affinity of 80 nM to 5 μM (e.g., 80 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM).

[0291] B.CD98hc binding region: The CD98hc binding domain (means for binding to CD98hc) is a molecule such as a polypeptide or a larger polypeptide or protein region or domain that specifically binds to CD98hc, such as human TfR.

[0292] In some embodiments, the CD98hc binding region includes a modified antibody heavy chain constant domain or an Fc polypeptide (e.g., a modified CH3 peptide). In some embodiments, the CD98hc-bound Fc-Fab fusion includes a modified CH3 peptide.

[0293] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 382, ​​384, 385, 387, 422, 424, 426, 438, and 440, the positions of which are determined by reference to EU numbering. In some embodiments, the substitution is selected from the group consisting of R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; N, L, Y, R, F, G, S, D, or T at position 387; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; I, V, F, N, P, or S at position 438; and K, T, P, I, or F at position 440.

[0294] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, where the positions are determined by reference to EU numbering. In some embodiments, the substitution is S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, or T at position 387; and at position 389. Selected from T, Y, or F at position 421; D, E, or Q at position 422; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

[0295] 1. Modified CH3 peptide CD98hc binding domain of Group I In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide, includes at least 11, 12, 13, 14, or 15 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442, where the positions are determined according to EU numbering. In some embodiments, substitutions are: S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; V, L, A, I, F, Y, S, T, H, R, or E at position 386; L or I at position 387; D, Q, A, T, H, or V at position 389; 3 Selected from T, V, or A at rank 91; E, Q, or A at rank 421; L, M, I, T, or P at rank 422; A at rank 424; N at rank 426; L, T, P, YF, I, A, K, H, or W at rank 428; S at rank 434; L, V, H, F, P, R, or W at rank 436; F or W at rank 438; L, P, E, N, V, A, I, or D at rank 440; P at rank 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at rank 442.

[0296] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide, includes at least 13 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442, where the positions are determined according to EU numbering. In some embodiments, substitutions are: S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; V, L, A, I, F, Y, S, T, H, R, or E at position 386; L or I at position 387; D, Q, A, T, H, or V at position 389; 3 Selected from T, V, or A at rank 91; E, Q, or A at rank 421; L, M, I, T, or P at rank 422; A at rank 424; N at rank 426; L, T, P, YF, I, A, K, H, or W at rank 428; S at rank 434; L, V, H, F, P, R, or W at rank 436; F or W at rank 438; L, P, E, N, V, A, I, or D at rank 440; P at rank 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at rank 442.

[0297] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 15-30, wherein the modified constant domain is S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; V, L, A, I, F, Y, S, T, H, R, or E at position 386; 38 This includes at least 11, 12, 13, 14, or 15 substitutions at a series of amino acid positions consisting of L or I at position 7; D, Q, A, T, H, or V at position 389; T, V, or A at position 391; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, YF, I, A, K, H, or W at position 428; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442, the positions being determined according to EU numbering.

[0298] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 15-30, wherein the modified constant domain comprises S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; and V, L, A, I, F, Y, S, T, H, R at position 386. or E; L or I at position 387; D, Q, A, T, H, or V at position 389; T, V, or A at position 391; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, YF, I, A, K, H, or W at position 428; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442, the positions being determined according to EU numbering.

[0299] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of any one of the sequences from SEQ ID NOs. 15-30, wherein the modified constant domain includes S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; and V, L, A, I, F, Y, S, T, H, R at position 386. This includes substitutions of at least 11, 12, 13, 14, or 15 amino acid positions, the following sequence of positions: E; L or I at position 387; D, Q, A, T, H, or V at position 389; T, V, or A at position 391; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, YF, I, A, K, H, or W at position 428; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442, the positions of which are determined according to EU numbering.

[0300] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of any one of the sequences of SEQ ID NOs. 15-30, wherein the modified constant domain includes S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; and V, L, A, I, F, Y, S at position 386. The substitutions include at least 13 amino acid positions, comprising: T, H, R, or E; L or I at position 387; D, Q, A, T, H, or V at position 389; T, V, or A at position 391; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, YF, I, A, K, H, or W at position 428; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442, the positions of which are determined according to EU numbering.

[0301] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes one of the amino acids 111-217 from SEQ ID NOs. 15-30.

[0302] In some embodiments, the CD98hc binding region containing a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide includes one of the sequence numbers 15 to 30. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4]

[0303] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), includes substitutions of 11, 12, 13, 14, or 15 at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

[0304] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain or Fc polypeptide (e.g., a modified CH3 peptide), comprises at least 13 or at least 14 substitutions at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

[0305] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 15-30, wherein the modified constant domain comprises substitutions at 11, 12, 13, 14, or 15 at a series of amino acid positions consisting of L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

[0306] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 15-30, wherein the modified constant domain comprises at least 13 or at least 14 substitutions at a series of amino acid positions consisting of L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

[0307] In some embodiments, the polypeptide comprises a modified constant domain (e.g., a modified CH3 peptide) having at least 85%, at least 90%, or at least 95% sequence identity with amino acids 111–217 of the sequences of SEQ ID NOs. 15–30, wherein the modified constant domain comprises substitutions of 11, 12, 13, 14, or 15 at a series of amino acid positions consisting of L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

[0308] In some embodiments, the polypeptide comprises a modified constant domain (e.g., a modified CH3 peptide) having at least 85%, at least 90%, or at least 95% sequence identity with amino acids 111–217 of the sequences of SEQ ID NOs. 15–30, wherein the modified constant domain comprises at least 13 or at least 14 substitutions at a series of amino acid positions consisting of L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

[0309] In some embodiments, the CD98hc binding region containing the modified CH3 peptide is (a) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place, (b) L at 380th, N at 382nd, R at 384th, F at 385th, V at 386th, L at 387th, E at 421st, I at 422nd, A at 424th, N at 426th, Y at 428th, F at 438th, N at 440th, and A at 442nd, (c) L at 380th place, N at 382nd place, Q at 384th place, Y at 385th place, E at 386th place, L at 387th place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place, (d) L at 380th place, N at 382nd place, H at 384th place, Y at 385th place, E at 386th place, L at 387th place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place, (e) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place, (f) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, E at 421st place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place. (g) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, E at 421st place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, and N at 440th place, (h) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and R at 442nd place, (i) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and H at 442nd place, (j) L at 380th, N at 382nd, R at 384th, F at 385th, V at 386th, L at 387th, I at 422nd, A at 424th, N at 426th, Y at 428th, R at 436th, F at 438th, N at 440th, and R at 442nd, (k) L at 380th, N at 382nd, H at 384th, Y at 385th, E at 386th, L at 387th, I at 422nd, A at 424th, N at 426th, Y at 428th, F at 438th, N at 440th, and A at 442nd. (l) L at 380th place, N at 382nd place, Q at 384th place, F at 385th place, H at 386th place, L at 387th place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and L at 442nd place, (m) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, T at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place. (n) L at 380th place, N at 382nd place, R at 384th place, F at 385th place, V at 386th place, L at 387th place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and K at 442nd place, (o) L at 380th, N at 382nd, R at 384th, F at 385th, V at 386th, L at 387th: I at 422nd, A at 424th, N at 426th, Y at 428th, W at 436th, F at 438th, N at 440th, and R at 442nd, or (p) Including L at 380th place, N at 382nd place, Q at 384th place, Y at 385th place, L at 386th place, L at 387th place, E at 421st place, I at 422nd place, A at 424th place, N at 426th place, Y at 428th place, F at 438th place, N at 440th place, and A at 442nd place, The location is determined according to the EU numbering system.

[0310] In some embodiments, the CD98hc binding region containing the modified CH3 peptide includes the following amino acid sequences: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 135.

[0311] In some embodiments, the CD98hc binding region containing the modified CH3 peptide is (i) A first amino acid sequence of LX1NX2X3X4X5L (Sequence ID 31), where X1 is any amino acid, X2 is R, H, or Q, X3 is F or Y, X4 is V, L, I, F, Y, or E, and X5 is any amino acid. (ii) A second amino acid sequence of X1X2X3AX4X5X6X7 (Sequence ID 32), wherein X1 is E, N, Q, or A, X2 is I, V, T, or P, X3 and X4 are any amino acid, X5 is N or S, X6 is any amino acid, and X7 is Y or W, and (iii) A third amino acid sequence of X1X2X3X4NX5X6 (Sequence ID 33) wherein X1 is Y, R, or W, X2 is any amino acid, X3 is F or W, X4 and X5 are any amino acids, and X6 is A, Q, K, R, H, M, or S.

[0312] 2. Modified CH3 peptide CD98hc binding domain of Group II In some embodiments, the CD98hc binding region containing a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide includes eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, where the positions are determined according to EU numbering. In some embodiments, the substitution is S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, or T at position 387; and at position 389. Selected from T, Y, or F at position 421; D, E, or Q at position 422; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

[0313] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide includes at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, where the positions are determined according to EU numbering. In some embodiments, the substitution is S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, or T at position 387; and at position 389. Selected from T, Y, or F at position 421; D, E, or Q at position 422; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

[0314] In some embodiments, the CD98hc binding region containing a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide contains a sequence having at least 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain has S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; and at position 387 The substitutions include at least eight, nine, ten, eleven, twelve, thirteen, or nineteen substitutions at a series of amino acid positions consisting of N, L, Y, R, F, G, S, D, or T at position 389; T, Y, or F at position 421; D, E, or Q at position 422; I, K, L, R, T, F, or H at position 424; V, W, G, L, I, P, or Y at position 426; D, A, Q, W, L, or P at position 428; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

[0315] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, 90%, or 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain includes S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; and T, P, E, K, A, V, D, T at position 386. This includes at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of F; N, L, Y, R, F, G, S, D, or T at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

[0316] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; and T, P, E, K, A, V, D, T at position 386. , or F; N, L, Y, R, F, G, S, D, or T at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442, comprising at least eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at a series of amino acid positions.

[0317] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; and T, P, E, K at position 386. The substitutions include at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of A, V, D, T, or F; N, L, Y, R, F, G, S, D, or T at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

[0318] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes amino acids 111-217 of either SEQ ID NO: 34 or SEQ ID NO: 35.

[0319] In some embodiments, the CD98hc binding region containing a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide includes either SEQ ID NO: 34 or SEQ ID NO: 35.

[0320] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes eight, nine, ten, eleven, twelve, or thirteen substitutions at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440.

[0321] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes substitutions of 11, 12, or 13 at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440.

[0322] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; and F, K, D, M, I, N, Y, L at position 385. or H; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440, comprising 8, 9, 10, 11, 12, or 13 substitutions at a series of amino acid positions.

[0323] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; and F, K, D, M, I, N at position 385. This includes substitutions of 11, 12, or 13 at a series of amino acid positions consisting of Y, L, or H; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440.

[0324] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of either sequence SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain has D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; and F, K, D at position 385. The substitutions include eight, nine, ten, eleven, twelve, or thirteen amino acid positions consisting of M, I, N, Y, L, or H; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440.

[0325] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of either sequence SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; and F at position 385. The substitutions include 11, 12, or 13 at a series of amino acid positions consisting of K, D, M, I, N, Y, L, or H; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440.

[0326] In some embodiments, the CD98hc binding region containing a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide includes eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; K at position 385; P at position 386; Y at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I at position 422; V at position 424; D at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

[0327] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide includes at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; K at position 385; P at position 386; Y at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I at position 422; V at position 424; D at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

[0328] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain has S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; K at position 385; and at position 386. This includes substitutions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids at a series of amino acid positions consisting of P; Y at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I at position 422; V at position 424; D at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

[0329] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; K at position 385; P at position 386; Y at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I at position 422; V at position 424; D at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

[0330] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, comprises a sequence having at least 85%, 90%, or 95% sequence identity with amino acids 111-217 of the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain has S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; K at position 385; and at position 386. This includes substitutions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids at a series of amino acid positions consisting of P at position 387; Y at position 389; T, Y, or F at position 421; D, E, or Q at position 422; I at position 424; V at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

[0331] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, comprises a sequence having at least 85%, 90%, or 95% sequence identity with amino acids 111-217 of the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain has S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; and K at position 385. The substitutions include at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of P at position 386; Y at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I at position 422; V at position 424; D at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

[0332] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes eight, nine, ten, eleven, twelfth, or fifteen substitutions at a series of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the substitution is selected from R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0333] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or Fc polypeptide includes at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 436, 438, and 440, the positions determined according to EU numbering. In some embodiments, the substitutions are selected from R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0334] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises eight, nine, ten, eleven, twelfth, or fifteen substitutions at a series of amino acid positions consisting of R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0335] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0336] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity to amino acids 111-217 of the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises eight, nine, ten, 11, 12, 13, 14, or 15 substitutions at a series of amino acid positions consisting of R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0337] In some embodiments, the CD98hc binding region comprising a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide comprises a sequence having at least 85%, at least 90%, or at least 95% sequence identity with amino acids 111-217 of the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, wherein the modified constant domain comprises at least 11, at least 12, or at least 13 substitutions at a series of amino acid positions consisting of R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0338] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes amino acids 111-217 of SEQ ID NO: 34 or SEQ ID NO: 35.

[0339] In some embodiments, the CD98hc binding region, which includes a modified antibody heavy chain constant domain (e.g., a modified CH3 peptide) or an Fc polypeptide, includes the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35. [Table 17]

[0340] In some embodiments, the CD98hc binding region containing the modified CH3 peptide includes (a) R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, F at position 436, I at position 438, and K at position 440, or (b) R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440, the positions of which are determined according to EU numbering.

[0341] In some embodiments, the CD98hc binding region containing the modified CH3 peptide is (i) A first amino acid sequence of X1X2YKPYX3T (Sequence ID 54), where X1 is E or R, X2 is S or T, and X3 is any amino acid. (ii) A second amino acid sequence of X1X2X3VX4DX5X6 (Sequence ID 55), wherein X1 is N or D, X2 is V or I, X3, X4, and X5 are any amino acid, and X6 is M or L, and (iii) A third amino acid sequence of X1X2IX3X4 (Sequence ID 56) comprising a third amino acid sequence in which X1 is Y or F, X2 and X3 are any amino acid, and X4 is S or K.

[0342] Further CH3 domains modified to specifically bind to CD98hc are described in PCT / US2022 / 053220.

[0343] 3. CD98hc binding region of Group III In some embodiments, the CD98hc binding region includes an anti-CD98hc antibody antigen-binding domain. The anti-CD98hc antibody antigen-binding domain may include an antibody, Fab (containing F(ab')2), scFab, Fv fragment, scFv, or nanobody. Anti-CD98hc antibody antigen-binding domains include: 1C03 anti-CD98hc VHH (e.g., 1C03-4 or 1C03-5 anti-CD98hc VHH); CD98hc2 anti-CD98hc nanobody; anti-CD98hc heavy chain antibody or nanobody having the CDR1, CDR2, and CDR3 sequences of the CD98hc2 anti-CD98hc nanobody; CD98hc4 anti-CD98hc scFv; anti-CD98hc antibody, Fab, scFab, Fv fragment, or scFv having the CDR1, CDR2, and CDR3 sequences of the heavy and light chains of CD98hc4 anti-CD98hc scFv; CD98hc5 anti-CD98hc scFv; or CD98hc5 anti-CD98hc Examples of anti-CD98hc antibodies, Fab, scFab, Fv fragments, or scFv (as disclosed in WO2021 / 205361, sequences shown in Table 18), having the heavy and light chain CDR1, CDR2, and CDR3 sequences of scFv, include, but are not limited to, these. The dual transporter of the present invention may include an antigen-binding domain having one of the CDR chains or variable chains of the antibodies CD98hc1, CD98hc2, CD98hc3, CD98hc4, CD98hc5, CD98hc6, CD98hc7, CD98hc8, CD98hc9, CD98hc10, CD98hc11, CD98hc12, CD98hc13, CD98hc14, and CD98hc15 as described in WO2021 / 205361 (incorporated herein by reference). [Table 18-1] [Table 18-2]

[0344] Further anti-CD98hc antibodies are known in the art and are available from various commercial sources. Exemplary anti-CD98hc antibodies include, but are not limited to, IGN523 (Hayes GM et al. Int J Cancer 2015 137(3):710-720). Exemplary anti-CD98hc antibodies are also described in the following patent publications: WO2008 / 017828, WO2013 / 078377, WO2021 / 205361, WO2022 / 252167, WO2015 / 146132, US20130052197, and US7943745 (each of which is incorporated herein by reference).

[0345] Brain shuttles containing anti-CD98hc antibodies are described in Pornnoppadol G et al. “Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs” bioRxiv April 29, 2023 (doi:10.1101 / 2023.04.29.538811.Preprint) (incorporated herein by reference).

[0346] Anti-CD98hc vNARs are described in WO2021 / 205361, WO2019 / 246288, and WO2023 / 023166.

[0347] Antibody-antigen-binding domains that specifically bind to BBB proteins can be identified using methods available in the art for producing and / or identifying antibodies that specifically bind to target proteins. Such methods include, but are not limited to, immunization, phage display, and ribosome display.

[0348] In some embodiments, the CD98hc binding region includes a heavy chain complementarity-determining region 3 (CDRH3) manipulated within the antibody constant domain as described in WO2023 / 087017.

[0349] In some embodiments, the CD98hc binding domain includes a fibronectin type III domain peptide modified to specifically bind to CD98hc.

[0350] In some embodiments, the CD98hc binding domain includes a bicyclic peptide that specifically binds to CD98hc.

[0351] In some embodiments, the CD98hc binding domain binds to human CD98hc with an affinity of about 15 nM to less than 6000 nM, or about 15 nM to less than 5000 nM (e.g., about 15 nM, about 50 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM, about 1500 nM, about 2000 nM, about 2500 nM, about 3000 nM, about 3500 nM, about 4000 nM, about 4500 nM, about 5000 nM, about 5500 nM, or about 6000 nM). In some embodiments, the CD98hc binding domain binds to human CD98hc with affinity of about 100 nM to about 500 nM (e.g., about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, or about 500 nM). In some embodiments, the CD98hc binding domain exhibits cynomolgus monkey (cyno) cross-reactivity. In some embodiments, the CD98hc binding region binds to cynomolgus monkey CD98hc with an affinity of approximately 80 nM to approximately 5 μM (e.g., 80 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM).

[0352] C.MOG binding polypeptide: The MOG-binding region (means for binding to MOG) is a molecule such as a polypeptide or a larger polypeptide or protein region or domain that specifically binds to human MOGs such as MOG.

[0353] In some embodiments, the MOG binding region includes an anti-MOG antibody antigen-binding domain. The anti-MOG antibody antigen-binding domain may include an antibody, Fab(containing F(ab')2), scFab, Fv fragment, scFv, or nanobody. The anti-MOG antibody antigen-binding domain may be an anti-MOG antibody as described in WO2018123979 (e.g., SEQ ID NO: 9), WO2023035002 (e.g., see paragraph

[0032] ), CN117683131B (e.g., see paragraphs

[0006] ,

[0008] , or see Nakano R et al ("A new technology for increasing therapeutic protein levels in the brain over extended periods". PLoS One. 2019 Apr 12;14(4):e0214404.) (each of which is incorporated herein by reference)), or WO2018123979, WO2023035002, CN117683131B, or Nakano R et al. The anti-MOG antibodies described in al include anti-MOG, Fab, scFab, Fv fragments, or scFv, each having the heavy and light chain CDR1, CDR2, and CDR3 sequences of any of the anti-MOG antibodies described in al. Further anti-MOG antibodies are known in the art and are available from various commercial sources.

[0354] In some embodiments, the MOG binding region binds to human MOG with an affinity of approximately 15 nM to less than 6000 nM, or approximately 15 nM to less than 5000 nM (e.g., approximately 15 nM, approximately 50 nM, approximately 100 nM, approximately 200 nM, approximately 300 nM, approximately 400 nM, approximately 500 nM, approximately 600 nM, approximately 700 nM, approximately 800 nM, approximately 900 nM, approximately 1000 nM, approximately 1500 nM, approximately 2000 nM, approximately 2500 nM, approximately 3000 nM, approximately 3500 nM, approximately 4000 nM, approximately 4500 nM, approximately 5000 nM, approximately 5500 nM, or approximately 6000 nM). In some embodiments, the MOG binding domain binds to human MOG with affinity of approximately 100 nM to approximately 500 nM (e.g., approximately 100 nM, approximately 150 nM, approximately 200 nM, approximately 250 nM, approximately 300 nM, approximately 350 nM, approximately 400 nM, approximately 450 nM, or approximately 500 nM). In some embodiments, the MOG binding domain exhibits cynomolgus monkey (cyno) cross-reactivity. In some embodiments, the MOG binding region binds to cynomolgus monkey MOG with an affinity of approximately 80 nM to approximately 5 μM (e.g., 80 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM).

[0355] D. Additional polypeptide modifications Any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides, may further include additional mutations, for example, to promote heterodimer formation, modulate effector function, extend serum half-life and / or stability, affect glycosylation, and / or reduce immunogenicity in humans.

[0356] 1. Polypeptide modification for heterodimerization Any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides, may further contain one or more mutations that promote heterodimer formation and prevent homodimer formation. These modifications are useful, for example, when it is desired to form a bispecific antibody or a heteromeric heavy chain dimer or heteromeric Fc dimer. Heterodimeric Fc dimer formation can be used, for example, to form an Fc dimer having one Fc polypeptide that binds to a BBB transporter protein (e.g., TfR or CD98hc) and a second Fc polypeptide that binds to a different second protein, which is a BBB surface protein or a brain-retaining protein, or to form an Fc dimer that is monovalent with respect to binding to a BBB transporter protein or a second protein. In some embodiments, the Fc dimer binds to both TfR and CD98hc.

[0357] Exemplary mutations that promote Fc heterodimer formation include knob and hole mutations. The knob-into-holes method generally involves introducing a projection ("knob") at the interface of a polypeptide (e.g., Fc polypeptide) and a corresponding cavity ("hole") into the interface of a second polypeptide (e.g., Fc polypeptide), so that the projection can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The projection is constructed by replacing amino acids with smaller side chains from the interface of the first polypeptide (e.g., Fc polypeptide) with amino acids with larger side chains (e.g., Tyr or Trp). A complementary cavity of the same or similar size as the projection is created by replacing amino acids with larger side chains with amino acids with smaller side chains (e.g., Ala or Thr) within the interface of the second polypeptide (e.g., Fc polypeptide). In some embodiments, such additional mutations are located within the polypeptide (e.g., Fc polypeptide) in a position that does not negatively affect the binding of the polypeptide to the BBB transport protein or a second protein (e.g., CD98hc or TfR).

[0358] For example, the knob-and-hole method for dimerization may include replacing the native Thr at position 366 of one polypeptide in the dimer (e.g., Fc polypeptide) with Trp to form a knob (i.e., the T366W knob mutation), and replacing the native Tyr at position 407 of the other polypeptide in the dimer (e.g., Fc polypeptide) with Val to form a hole (i.e., the Y407V hole mutation). The other polypeptide (e.g., Fc polypeptide) may further include substitution of the native Thr at position 366 with Ser and substitution of the native Leu at position 368 with Ala (i.e., the T366S and L368A hole mutations). In some embodiments, one polypeptide in the dimer (e.g., Fc polypeptide) has the T366W knob mutation, and the other polypeptide (e.g., Fc polypeptide) has the Y407V hole mutation, typically accompanied by the T366S and L368A hole mutations. All positions are numbered according to EU numbering.

[0359] The knob-into-hole method (e.g., T366W knob substitution in one polypeptide (e.g., Fc polypeptide) and hole substitutions of T366S, L368A, and Y407V in the other polypeptide (e.g., Fc polypeptide)) can be used with any of the antibodies, heavy chain dimers, or dual transporters containing Fc dimers that include one of the described CD98hc-conjugated Fc polypeptides and / or TfR-conjugated Fc polypeptides.

[0360] In some embodiments, the dual transporter comprises a first Fc polypeptide having a CH3 peptide modified to bind to a BBB transport protein (e.g., TfR or CD98hc), and a second Fc polypeptide having a CH3 peptide modified to bind to a different second protein, which is either a BBB surface protein or a brain-retaining protein. In some embodiments, the dual transporter comprises a first Fc polypeptide having a CH3 peptide modified to bind to a BBB transport protein or a second protein, and a second Fc polypeptide that does not have a CH3 peptide modified to bind to a BBB protein transport protein or a second protein. In some embodiments, the first Fc polypeptide contains a knob mutation (e.g., T366W), and the second Fc polypeptide contains a whole mutation (e.g., T366S, L368A, and Y407V). In some embodiments, the first Fc polypeptide contains a whole mutation (e.g., T366S, L368A, and Y407V), and the second Fc polypeptide contains a knob mutation (e.g., T366W).

[0361] In some embodiments, one or both polypeptides present in a polypeptide dimer (e.g., an Fc polypeptide dimer) (e.g., an Fc polypeptide dimer) may be manipulated to include other modifications that promote heterodimerization, such as electrostatic manipulation of contact residues within a naturally charged CH3-CH3 interface, or hydrophobic patch modifications.

[0362] 2. Polypeptide modification to adjust effector function Any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides, may further contain one or more mutations that reduce or eliminate effector function. Reducing or eliminating effector function includes reducing or eliminating the ability of an Fc polypeptide or dimer to induce specific biological functions upon binding of the Fc polypeptide or Fc dimer to an Fc receptor expressed on effector cells that mediate effector 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. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0363] In some embodiments, any of the antibodies or Fc polypeptides described herein may contain a CH2 peptide having one or more mutations that reduce effector function. The mutations that reduce effector function may be, but are not limited to, mutations at positions 234, 235, and 390 according to EU numbering. The mutations may be present in the single heavy chain of the antibody, the single Fc polypeptide of the Fc dimer, both heavy chains of the antibody, or both Fc polypeptides of the Fc dimer.

[0364] In some embodiments, any of the antibodies or Fc polypeptides described herein may comprise a CH2 peptide having A at position 234, A at position 235; G at position 329 ("P329G" or "PG" mutation); S at position 329 ("P329S" or "PS" mutation); A at position 234 and A at position 235 ("LALA" mutation); A at position 234, A at position 235, and G at position 329 ("LALA PG" mutation); or A at position 234, A at position 235, and S at position 329 ("LALA PS" as used herein).

[0365] In some embodiments, any of the described antibodies or Fc dimers may contain, in both CH2 peptides, A at position 234, A at position 235; G at position 329 ("P329G" or "PG" mutation); S at position 329 ("P329S" or "PS" mutation); A at position 234 and A at position 235 ("LALA" mutation); A at position 234, A at position 235, and G at position 329 ("LALA PG" mutation); or A at position 234, A at position 235, and S at position 329 ("LALA PS" as used herein).

[0366] In some embodiments, any of the antibodies or Fc dimers described herein may contain, in a single CH2 peptide, A at position 234, A at position 235; G at position 329 ("P329G" or "PG" mutation); S at position 329 ("P329S" or "PS" mutation); A at position 234 and A at position 235 ("LALA" mutation); A at position 234, A at position 235, and G at position 329 ("LALA PG" mutation); or A at position 234, A at position 235, and S at position 329 ("LALA PS" as used herein).

[0367] Additional mutations that modulate effector function include, but are not limited to, (a) substitutions of the native P at position 329 with G, A, S, or R, or substitutions of amino acid residues large enough to inhibit the formation of the Fc / Fcγ receptor interface between P329 of Fc and Trp87 and Trp110 of FcγRIII, and / or (b) substitutions of one or more of S228P, E233P, L235E, N297A, N297D, or P331S, according to the EU numbering scheme.

[0368] Substitution combinations that adjust the effector function include, but are not limited to, the following, according to the EU numbering scheme: L234A, L235A, and P329G of human IgG1; S228P and L235E of human IgG4; L234A and G237A of human IgG1; L234A, L235A, and G237A of human IgG2; V234A and G237A of human IgG2; L235A, G237A, and E318A of human IgG4; and S228P and L236E of human IgG4.

[0369] 3. Polypeptide modification to extend the serum half-life. Any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides, may further contain one or more mutations that alter their serum half-life. In some embodiments, any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides, may further contain one or more mutations that improve or prolong their serum half-life.

[0370] Exemplary mutations that improve serum half-life include, but are not limited to, M428L substitutions, N434S substitutions, M428L and N434S substitutions ("LS" substitutions), N434S substitutions, N434A substitutions, M428L substitutions, M252Y substitutions, S254T substitutions, T256E substitutions, or combinations of M252Y, S254T, and T256E substitutions, as numbered according to the EU numbering scheme.

[0371] One or more mutations that improve the serum half-life may be present in the single heavy chain of the antibody, the single Fc polypeptide of the Fc dimer, both heavy chains of the antibody, or both Fc polypeptides of the Fc dimer.

[0372] 4. Polypeptide with the C-terminal lysine residue removed. The C-terminal lysine residue (e.g., the lysine residue at position 447 according to EU numbering) can be removed from any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides. The C-terminal lysine residue is highly conserved in immunoglobulins across many species and can be completely or partially removed by cellular mechanisms during protein production. Removal of the C-terminal lysine from Fc polypeptides can improve protein stability. In some embodiments, any of the antibodies or Fc polypeptides described herein, including those containing modified CH3 peptides, may contain a C-terminal lysine deletion. In some embodiments, any heavy chain of any of the described antibodies or any of the described Fc polypeptides may be produced without the C-terminal lysine. In some embodiments, the C-terminal lysine can be removed from any of the antibodies or Fc polypeptides described herein. The C-terminal lysine can be removed from a single heavy chain of an antibody, from a single Fc polypeptide of an Fc dimer, from both heavy chains of an antibody, or from both Fc polypeptides of an Fc dimer.

[0373] III. Manufacturing method A method for identifying a dual transporter is described, comprising: (a) providing a first polypeptide comprising a first binding domain that specifically binds to a BBB transporter protein; and (ii) a second polypeptide comprising a second binding domain that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transporter protein; (b) forming a single molecule comprising the first polypeptide and the second polypeptide; and (c) measuring the brain concentration of the single molecule at a predetermined time, and identifying the single molecule as a dual transporter if it is present in the brain at a higher concentration than a corresponding control molecule that binds only to the BBB transporter protein or the second protein. The single molecule may be peripherally administered to an animal prior to step (c). The animal may be, but is not limited to, a mouse, rat, or a non-human primate. High concentrations present in the brain may result in increased transport rates to the brain and / or increased retention in the brain (i.e., extended brain exposure or brain exposure time) compared to the corresponding control molecule that binds only to the BBB transport protein or only to a second protein. The BBB transport protein may, but is not limited to, the TfR of CD98hc. In some embodiments, the BBB transport protein is the TfR.The second set of proteins includes TfR, CD98hc, large neutral amino acid transporter small subunit 1 (CD98 light chain), GLUT1, MFSD2A, CA-IV, low-density lipoprotein receptor, IGF1R, insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 50A, basidine, leptin receptor, claudin-5, P-selectin, and lactoferrin. The second protein may be, but is not limited to, a receptor, a folate receptor, a sodium-dependent lysophosphatidylcholine cotransporter 1, a solute transporter organic anion transporter family member 1C1, a sodium-coupled neutral amino acid transporter 5, an LDL receptor-related protein 8, a high-affinity cationic amino acid transporter 1, a sodium chloride-dependent taurine transporter, an insulin-like growth factor-binding protein 7, a solute transporter family member 40 1, a zinc transporter 6, a heparin-binding epidermal growth factor-like growth factor, and MOG. In some embodiments, the second protein is CD98hc. In some embodiments, the second protein is MOG. In some embodiments, the second protein is selected from the group consisting of GLUT1, MFSD2A, CA-IV, and IGF1R.

[0374] The first binding region may be any polypeptide known to bind to a BBB transport protein, including either a TfR or CD98hc binding region as disclosed or described herein. The second binding region may be any polypeptide known to bind to a second protein, including either a TfR or CD98hc binding region as disclosed or described herein. In some embodiments, the first binding region binds to TfR.

[0375] In some embodiments, the method further includes determining the binding affinity of a first binding region to a BBB transporter protein and the binding affinity of a second binding region to a second protein. In some embodiments, the method further includes determining the binding affinity of a single molecule to the BBB transporter protein, the second protein, or both the BBB transporter protein and the second protein. Binding affinity or binding strength may be determined using methods available in the art for determining the binding affinity of a molecule to a cell, protein, or cell surface protein. Such methods include, but are not limited to, surface plasmon resonance, biolayer interferometry, ELISA, and flow cytometry. Binding of the first binding region, the second binding region, or a single molecule (e.g., a dual transporter) may be measured on the surface of a cell expressing the BBB transporter protein, the second protein, or both the BBB transporter protein and the second protein. In some embodiments, affinity is measured using surface plasmon resonance (SPR) (e.g., using the Biacore® system), coupled equilibrium exclusion (e.g., using the KinExA® system), and / or biolayer interferometry (e.g., using the ForteBio® Octet® platform).

[0376] A method for determining whether a dual transporter is increasing and / or prolonging exposure in the brain of an animal is described, comprising: (a) providing a single molecule comprising: (i) a first binding region that specifically binds to a BBB transporter protein; and (ii) a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is a second binding region distinct from that of the BBB transporter protein; (b) peripherally administering the molecule to an animal; and (c) measuring the concentration of the molecule in the brain to determine whether the molecule is present in the brain at a higher concentration than a corresponding control molecule that binds to only the BBB transporter protein or only the second protein. Measuring the concentration of the molecule in the brain may involve measuring the concentration of the molecule in the brain at one or more predetermined time intervals. Higher concentrations in the brain may result from increased transport rates to the brain and / or increased retention in the brain (i.e., prolonged brain exposure or brain exposure time) compared to a corresponding control molecule that binds to only the BBB transporter protein or only the second protein. The BBB transport protein may, but is not limited to, a TfR of CD98hc. In some embodiments, the BBB transport protein is a TfR.The second set of proteins includes TfR, CD98hc, large neutral amino acid transporter small subunit 1 (CD98 light chain), GLUT1, MFSD2A, CA-IV, low-density lipoprotein receptor, IGF1R, insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 50A, basidine, leptin receptor, claudin-5, P-selectin, and lactoferrin. The second protein may be, but is not limited to, a receptor, a folate receptor, a sodium-dependent lysophosphatidylcholine cotransporter 1, a solute transporter organic anion transporter family member 1C1, a sodium-coupled neutral amino acid transporter 5, an LDL receptor-related protein 8, a high-affinity cationic amino acid transporter 1, a sodium chloride-dependent taurine transporter, an insulin-like growth factor-binding protein 7, a solute transporter family member 40 1, a zinc transporter 6, a heparin-binding epidermal growth factor-like growth factor, and MOG. In some embodiments, the second protein is CD98hc. In some embodiments, the second protein is MOG. In some embodiments, the second protein is selected from the group consisting of GLUT1, MFSD2A, CA-IV, and IGF1R.

[0377] The first binding region may be any polypeptide known to bind to a BBB transport protein, including either a TfR or CD98hc binding region as disclosed or described herein. The second binding region may be any polypeptide known to bind to a second protein, including either a TfR or CD98hc binding region as disclosed or described herein.

[0378] In some embodiments, the method further includes determining the binding affinity of a first binding region to a BBB transporter protein and the binding affinity of a second binding region to a second protein. In some embodiments, the method further includes determining the binding affinity of a single molecule to the BBB transporter protein, the second protein, or both the BBB transporter protein and the second protein. Binding affinity or binding strength may be determined using methods available in the art for determining the binding affinity or binding strength of a molecule to a cell, protein, or cell surface protein. Such methods include, but are not limited to, surface plasmon resonance, biolayer interferometry, ELISA, and flow cytometry. Binding of the first binding region, the second binding region, or a single molecule (e.g., a dual transporter) may be measured on the surface of a cell expressing the BBB transporter protein, the second protein, or both the BBB transporter protein and the second protein. In some embodiments, affinity is measured using surface plasmon resonance (SPR) (e.g., using the Biacore® system), coupled equilibrium exclusion (e.g., using the KinExA® system), and / or biolayer interferometry (e.g., using the ForteBio® Octet® platform).

[0379] A method for producing a dual transporter is described, comprising: (a) providing a first polypeptide comprising a first binding region that specifically binds to a BBB transporter protein; and (ii) a second polypeptide comprising a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transporter protein; and (b) forming a single molecule comprising the first polypeptide and the second polypeptide, thereby forming a dual transporter, wherein the dual transporter has increased and / or extended brain exposure in animals compared to a corresponding control molecule that binds to either the BBB transporter protein or the second protein alone. In some embodiments, the first binding region specifically binds to TfR. In some embodiments, the first binding region specifically binds to CD98hc. In some embodiments, the second binding region specifically binds to CD98hc. In some embodiments, the second binding region specifically binds to MOG. In some embodiments, the second binding domain specifically binds to a protein selected from the group consisting of GLUT1, MFSD2A, CA-IV, and IGF1R.

[0380] The first binding region may be any polypeptide known to bind to a BBB transport protein, including either a TfR or CD98hc binding region (means for binding to TfR or CD98hs, respectively) as disclosed or described herein. The second binding region may be any polypeptide known to bind to a second protein, including either a TfR, CD98hc, or MOG binding region (means for binding to TfR, CD98hs, or MOG, respectively) as disclosed or described herein.

[0381] The first and second polypeptides may be synthesized or encoded by one or more nucleic acids. One or more nucleic acids may be expressed in a host cell (one or more), and the coding polypeptide(s) may be purified from the host cell using methods available in the art for producing proteins from the host cell. The host cell may be a bacterial cell, a eukaryotic cell, an insect cell, or a mammalian cell.

[0382] IV. Nucleic acids, vectors, and host cells The dual transporters described herein can be prepared using recombinant methods. Therefore, isolated nucleic acids containing sequences or portions thereof encoding any of the dual transporters described herein can be readily produced using methods available in the art. Host cells into which nucleic acids can be introduced and which can be used to replicate polypeptide-coding nucleic acids and / or express polypeptides are also available in the art. The host cells may be, but are not limited to, prokaryotic or eukaryotic cells. Eukaryotic cells may be, but are not limited to, yeast cells, insect cells, or mammalian cells (e.g., human cells).

[0383] The nucleic acid encoding the dual transporter or a portion thereof may be DNA, RNA, cDNA, mRNA, single-stranded, double-stranded, linear, or circular.

[0384] A dual transporter may contain two or more (e.g., three) polypeptides, each of which may be encoded by a distinct nucleic acid sequence. These distinct nucleic acid sequences may reside on the same plasmid or vector, or on different plasmids or vectors. If present on the same plasmid or vector, the distinct nucleic acid sequences can be expressed from a single promoter or different promoters. Methods for expressing nucleic acids encoding distinct polypeptides from a single promoter are known in the art and include, but are not limited to, the use of 2A elements and intrasequence ribosome entry sites.

[0385] Nucleic acids encoding dual transporters or portions thereof may be provided as plasmids or vectors. Plasmids or vectors can be used to replicate nucleic acids or to promote nucleic acid expression. Plasmids or vectors may be, but are not limited to, viral vectors, phagemids, yeast chromosome vectors, or non-episomal mammalian vectors.

[0386] In some embodiments, a nucleic acid encoding a dual transporter or a portion thereof is functionally ligated to one or more regulatory sequences in an expression construct. The expression construct can be adapted for polypeptide expression in a dual transporter-producing system. Such a system may be, but is not limited to, a mammalian cell expression system, an insect cell expression system, a yeast cell expression system, or a bacterial cell expression system.

[0387] Plasmids and other vectors can be used as expression media for the production of recombinant polypeptides. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pETC-derived plasmids for expression in prokaryotic cells such as E. coli. Vectors derived from pcDNAI / amp, pcDNAEneo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-l) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVLl392, pVLl393, and pVL94l), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors. Further expression systems include those for adenoviruses, adeno-associated viruses, and other viruses.

[0388] Expression vectors for expressing dual transporters or portions thereof, or plasmids or vectors containing nucleic acids, can be transformed, transfected, or transduced into host cells. Host cells may be, but are not limited to, mammalian cells, yeast cells, insect cells, prokaryotic cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, NSO cells, YO cells, HEK293 cells, COS cells, Vero cells, or HeLa cells. Host cells containing expression vectors can be cultured under appropriate conditions to enable the expression of dual transporter proteins or portions thereof.

[0389] Dual transporters can be produced by culturing host cells containing one or more nucleic acids encoding the dual transporter, expressing the dual transporter, and isolating the expressed dual transporter from the culture.

[0390] V. Formulations Any of the dual transporters described may be prepared, provided, or formulated as a salt, mixed salt, or free acid. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts.

[0391] Any of the dual transporters described may be provided or formulated as a pharmaceutical composition. The pharmaceutical composition or drug contains a pharmaceutically effective amount of at least one of the dual transporters described, and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance other than the active pharmaceutical ingredient (API, therapeutic product (e.g., dual transporter)) that is intentionally included in the pharmaceutical composition. Excipients do not exhibit, or are not intended to exhibit, a therapeutic effect at the intended dose. Excipients may act to (a) assist in the processing of the API during manufacturing, (b) protect, assist, or improve the stability, bioavailability, or patient tolerability of the API, (c) assist in product identification, and / or (d) improve the overall safety, efficacy, or other properties of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0392] Examples of additives include, but are not limited to, absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, fluidizing agents, wetting agents, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, media, water repellents, and wetting agents.

[0393] The carrier may be, but is not limited to, a solvent or dispersion medium containing, for example, water, physiological saline, phosphate-buffered saline, Ringer's solution, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The carrier may also contain auxiliary agents or additives such as preservatives, wetting agents, emulsifiers, and dispersants. The carrier may also contain isotonic agents such as sugars, polyhydric alcohols, and sodium chloride.

[0394] The pharmaceutical composition may contain other further components commonly used in pharmaceutical compositions. Such further components may include, but are not limited to, antipruritic agents, astringents, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.).

[0395] Pharmacopoeia means that a property and / or substance is acceptable to a subject from a pharmacological / toxicological standpoint. The expression "pharmacopoeia" means that a molecule, composition, or property is physiologically acceptable to administration to a subject and does not typically produce allergic reactions or other harmful or toxic reactions. In some embodiments, a pharmacopoeia is approved by a federal or state regulatory authority, or is listed in the United States Pharmacopeia, or is listed in another generally recognized pharmacopoeia for use in animals, more specifically in humans.

[0396] In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. The additional active pharmaceutical ingredients may, but are not limited to, small molecule drugs.

[0397] Dual transporters or pharmaceutical compositions containing dual transporters can be formulated as liquid formulations or as solid formulations (powder or lyophilized formulations, e.g., lyophilized cake or powder).

[0398] In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

[0399] As disclosed above, dual transporters or pharmaceutical compositions containing dual transporters can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, dual transporters can be formulated into preparations by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, for example, vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acids, or esters of propylene glycol, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives, as desired. In some embodiments, polypeptides can be formulated in aqueous solutions, preferably physiologically compatible buffers, for example, Hanks' solution, Ringer's solution, or physiological salt buffer. The formulations for injection can be provided in unit dosage forms (e.g., ampoules or multi-dose containers) with added preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0400] Pharmaceutical compositions intended for in vivo administration are typically sterilized. Sterilization can be carried out by methods known in the art (e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation).

[0401] The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is well within the skill of those skilled in the art.

[0402] kit In some embodiments, a kit comprising the dual transporter described herein is provided. In some embodiments, the kit is intended for use in the prevention or treatment of neurological disorders of the central nervous system (CNS).

[0403] The dual transporters described herein and the pharmaceutical compositions containing the dual transporters disclosed herein may be packaged or included in kits, containers, packs, or dispensers. The dual transporters and the pharmaceutical compositions containing the dual transporters may be packaged in pre-filled syringes or vials. Any of the dual transporters or pharmaceutical compositions containing the dual transporters described herein may be formulated or packaged in single-dose or multi-dose form. Any of the dual transporters identified herein or pharmaceutical compositions containing the dual transporters described herein may be formulated for repeated doses.

[0404] In some embodiments, the kit further includes one or more additional therapeutic agents. For example, in some embodiments, the kit includes the dual transporter described herein and further includes one or more additional therapeutic agents for use in treating neurological disorders or conditions of the CNS.

[0405] The kit or package may further contain instructions. Instructions include documentation describing the relevant substances or methods relating to the kit. Instructions may include one or more of the following: background information, a list of ingredients and information on how to obtain them (e.g., purchase information), a simple or detailed protocol for using the kit, troubleshooting guidance, references, technical support, indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the drug, and any other relevant documentation. Instructions may be attached to the kit or provided as a separate component, either in paper or electronic form. Instructions may include notices in the format prescribed by the government agency that regulates the manufacture, use, or sale of the drug or biological product, and such notices may reflect the government agency's approval of manufacture, use, or sale for human administration.

[0406] VI. Treatment method The described dual transporters may be used to deliver therapeutic agents into the target brain, i.e., across the blood-brain barrier. In some embodiments, the described dual transporters may be used to deliver therapeutic agents via the BBB to extracellular targets on or near astrocytes, microglia, oligodendrocytes, or cancer cells. The described dual transporters may be used to provide uptake and delivery into the brain to specific extracellular targets or neurotumor targets within the brain. The described dual transporters may be used to promote or increase the transcytosis of therapeutic agents across the blood-brain barrier.

[0407] Examples of therapeutic agents include ABCA1, ABCA7, ADAM17, ALK, alpha-synuclein or its derivatives or fragments, amyloid-beta peptide or its derivatives or fragments, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD25, CD30, CD33, CD37, CD39, CD44v6, CD46, CD56 (NCAM), CD73, CD79b, CDH6 (cadherin 6), CEACAM5 (CD66E), CR1, and EGFR. viii, EGFR, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, HLA-DR1, HLA-DR5, huntingtin, IGFR1, IL1RAP, IL-34, KIT, LIV1A, LRRC15 (leucine-rich repeat 15), MET, MS4A4A, MS4A4E, MS4A6A, NaPi2B, TAR This could be DNA-binding protein 43 (TDP-43) or its derivatives or fragments, PDL1, PILRA, PMEL17, PRAME, PSMA, pTau, PTK7 (CCK4; colon cancer kinase), RON, ROR1, Siglec11, SORL1, Tau or its derivatives or fragments, TF (tissue factor), transthyretin, TREM2, TREML2, TROP2, or a drug that targets tumor cells.

[0408] The described dual transporters may be administered to subjects to treat diseases or conditions of the brain or CNS. After administration to a subject, the described dual transporters cross the blood-brain barrier (BBB) ​​into the brain.

[0409] A method for treating a subject suffering from a neurological disorder or condition of the central nervous system (CNS) is described, comprising administering to the subject a dual transporter linked to a therapeutic agent or a composition containing a dual transporter linked to a therapeutic agent. The neurological disorder or condition of the CNS may be, but is not limited to, a neurodegenerative disease, Alzheimer's disease, or an infection.

[0410] A method for treating a neurological disorder or condition of the central nervous system (CNS) is described, comprising administering a dual transporter or a composition containing a dual transporter to a subject. In some embodiments, the subject has, has been diagnosed with, or is at high risk of developing a neurological disorder or condition of the CNS. The neurological disorder or condition of the CNS may be, but is not limited to, neurodegenerative diseases, Alzheimer's disease, cognitive impairment, memory loss, or dementia.

[0411] The description includes a method for delaying or preventing one or more symptoms or pathological conditions associated with a neurological disorder or condition of the central nervous system (CNS) in a subject, the method comprising administering a dual transporter or a composition containing a dual transporter to the subject.

[0412] Dual transporters can be administered parenterally, intravenously, intrathecally, epidurally, or intravenously. Administration may be by bolus injection, infusion, or a combination thereof. Infusion may be administered over approximately 10 to 30 minutes, or over at least 1 to 4 hours or longer.

[0413] Dual transporters can be administered in combination with one or more additional therapeutic agents.

[0414] In some embodiments, the method further comprises administering one or more additional therapeutic agents to a target. For example, in some embodiments for treating diseases of the brain or central nervous system, the method may include administering neuroprotective agents (e.g., anticholinergics, dopamine agonists, glutamatergics, histone deacetylase (HDAC) inhibitors, cannabinoids, caspase inhibitors, melatonin, anti-inflammatory agents, hormones (e.g., estrogen or progesterone), or vitamins) to a target. In some embodiments, the method may include administering agents (e.g., antidepressants, dopamine agonists, or antipsychotics) to a target for use in treating cognitive or behavioral symptoms of neurological disorders. [Examples]

[0415] Example 1. TV for use in a dual transporter CD98hc Discovery and characterization of clones. TV from LLB1 and LLB2 families CD98hc The operation and characterization of the variants are described in PCT / US2022 / 053220 and herein.

[0416] LLB1 and LLB2 clones were selected for incorporation into CD98hc based on their dual transporter affinity to it. TV for human CD98hc CD98hc The affinity of the variant was evaluated by surface plasmon resonance (SPR) using a Biacore® 8K instrument. These measurements showed that the indicated CD98hc-bound domain (TV) CD98hc The CD98hc molecule was incorporated into a single Fc peptide of an anti-DNP antibody in the absence of a second (e.g., TfR) binding domain of the BBB. A set of clones encompassing a broad affinity range was selected to evaluate how CD98hc affinity affects the uptake and biodistribution of the dual transporter into the brain. Exemplary clones and their affinities to human CD98hc are shown below. [Table 19]

[0417] Example 2. TV for use in a dual transporter TfR Discovery and characterization of clones. TV from the TV42 family TfR The manipulation and characterization of variants (modified CH3 regions that specifically bind to TfR) are described in PCT / US2022 / 053220 (incorporated herein by reference) and herein (e.g., modified CH3 peptide TfR binding regions of Group II). TV from the TV35 family TfR The manipulation and characterization of the variants are described in WO2018 / 152326 (incorporated herein by reference), Kariolis et al., Sci Transl Med 12(545):eaay1359, 2020 (incorporated herein by reference), and herein (e.g., modified CH3 peptide TfR binding region of Group I). TV from further families TfR The manipulation and characterization of the variants are described in PCT / US2022 / 053220 and PCT / US2022 / 053234 (each of which is incorporated herein by reference) and herein (e.g., modified CH3 peptide TfR binding domains of Group III).

[0418] TV35 and TV42 clones were selected for incorporation into TfR based on their dual transporter affinity to TfR. TV for human TfR TfR The affinity of the variant was evaluated by surface plasmon resonance (SPR) using a Biacore® 8K instrument. These measurements showed that the indicated TfR-binding domain (TV) TfR The TfR was incorporated into a single Fc peptide of an anti-DNP antibody in the absence of a second (e.g., CD98hc) binding domain of the BBB. A set of clones encompassing a broad affinity range was selected to evaluate how TfR affinity affects the uptake and biodistribution of the dual transporter into the brain. Exemplary clones and their affinities to human TfR are shown below. [Table 20]

[0419] Any of the above-described modified CH3 TfR binding regions may further contain additional mutations to promote heterodimer formation (e.g., knob mutations or hole mutations), modulate effector function (e.g., LALA mutations or LALAPG mutations), extend serum half-life and / or stability, affect glycosylation, and / or reduce immunogenicity in humans.

[0420] Example 3. Design, expression, and characterization of a dual transporter clone. A dual transporter was constructed by creating a heterodimeric Fc domain, and one of the Fc polypeptides of the Fc dimer was converted to TfR(TV). TfR The second Fc polypeptide of the Fc dimer is manipulated to bind to CD98hc(TV CD98hc The molecules were manipulated to bind to ). Knob-into-hole mutations were incorporated to promote proper heterodimer pairing while reducing or inhibiting homodimer species formation. A wide variety of TVs derived from multiple TV families were used to evaluate the effects of binding affinity, epitope, and orientation on brain uptake and CNS biodistribution. TfR Array and TV CD98hc We examined the arrays in combination.

[0421] TV TfR Variants and TV CD98hc Using a variant, the heavy chain of the antibody transport vehicle (ATV) was generated by fusing the manipulated Fc domain to the Fab that binds to the hapten, dinitrophenyl (DNP; Figure 1). Knob mutations were used in the TV. TfR While adding to the ATV heavy chain, hole mutations are added to TV CD98hc It was incorporated into the ATV heavy chain. In both cases, a mutation (LALAPG) was included to reduce binding to FcγR in vivo and weaken the effector function.

[0422] The heavy chain sequence and corresponding light chain of anti-DNP Fab were cloned into a mammalian expression vector along with the CMV promoter and secretion-related signal peptides. TfR Heavy chain, Hall TV CD98hc The heavy chain and anti-DNP light chain were co-transfected into Expi293 cells. After 5 days of culture, the supernatant was collected, filtered, and the dual transporter was purified by protein A affinity chromatography, and subsequently by size exclusion chromatography as needed. Co-expressed NOV TV TfR and Hall TV CD98hc We investigated permutations of dual transporters by modifying the combinations.

[0423] The affinity of dual transporters for human TfR and human CD98hc was evaluated by surface plasmon resonance (SPR) using a Biacore® 8K instrument. To investigate the importance of relative affinity for TfR and CD98hc, different TVs from both the TV35 and TV42 families were used. TfR The variant combinations include various TVs from the LLB1 and LLB2 families. CD98hc Clones were combined. Representative dual transporter variants and their affinities to TfR and CD98hc are shown below. [Table 21A-1] [Table 21A-2]

[0424] In the case of any of the dual transporters described above, the modified CH3 peptide TfR binding region may have a hole mutation, and the modified CH3 peptide CD98 binding region may have a knob mutation. Furthermore, in the case of any of the dual transporters described above, the modified CH3 peptide may or may not contain LALA mutations and / or PG mutations.

[0425] Example 4. Cell binding of dual transporters. Dual transporters were tested for their ability to bind to both TfR and CD98hc on the cell surface of model BBB cells. HEK293T cells expressing both human CD98hc and human TfR were seeded at 40,000 cells per well in a 96-well plate in standard growth medium (DMEM (GIBCO® 11995073) + 10% FBS (VWR89510-188) + 1× Pen / strep (Gibco 15140122)). After approximately 24 hours, the dual transporters were diluted in standard growth medium warmed to 37°C. Old medium was removed from the cells, and the diluted molecules were added to the cells. The cells were incubated at 37°C for 45 minutes. The cells were washed with PBS and then sterilized with 4% PFA (Electron Microscopy Sciences Cells were fixed in 15714-S for 10 minutes. They were washed with PBS and then blocked for 30 minutes in PBS containing 5% BSA and 0.3% Triton® X100. Cells were stained for at least 30 minutes with anti-human IgG-AlexaFluor488 (1:1000; Jackson Immuno Research 109-545-003), cell mask (1:10,000; Thermo H32721), and DAPI (1:2000; Thermo D1306), diluted in PBS containing 1% BSA and 0.3% Triton® X100. Cells were washed with PBS, imaged with Opera Phenix, and images were analyzed with Harmony software.

[0426] Dual transporter is ATV TfR and ATV CD98hc Compared to the other method, it was found to bind to cells more readily at a given concentration, demonstrating the dual transporter's ability to associate with both targets on the cell surface (Figures 4 and 5).

[0427] Example 5. Production of monoclonal antibodies against the human transferrin receptor. Balb / c and SJL mice were immunized with recombinant human TfR extracellular domains via the intraperitoneal route using complete / incomplete Freund's adjuvant. After multiple boost cycles, mice were sacrificed and spleens and lymph nodes were collected. Single cells were fused to mouse myeloma cells using PEG, and the fused hybridomas were seeded into 96-well tissue culture plates and grown in HAT selective medium. After approximately two weeks of selection and growth, the supernatant was screened by ELISA for binding to recombinant human and cynomolgus monkey TfR proteins, and then screened for binding to CHO cells overexpressing either full-length human or cynomolgus monkey TfR, which did not bind to parental CHO cells. Recombinant hybridomas selectively bound to cell-expressed TfR proteins were subcloned and sequenced using 5'-RACE.

[0428] The variable heavy chain domain and variable light chain domain of TfR conjugates identified from hybridoma screening were subcloned into the constant heavy chain domain or constant kappa light chain domain of human IgG1 containing the LALA mutation, respectively. The expression plasmids contained a CMV promoter to induce expression and a signal peptide for secretion. The heavy chain plasmids and light chain plasmids were cotransfected into Expi293 cells. The supernatant was collected after 5 days, and monoclonal antibodies were purified by protein A chromatography. Alanine scan variants, rationally designed variants, and humanized variants of these antibodies were constructed, expressed, and purified using the same method.

[0429] The affinity of humans and cynomolgus monkeys for the TfR apical domain was measured using Biacore.

[0430] Example 6. Development of the anti-human CD98hc VHH domain The camelid VHH domain that binds to human CD98hc was generated by immunizing two llamas with the extracellular domains of human and cynomolgus monkey CD98hc (antigen). The llamas were immunized at week 0 with 0.5 mg of antigen supplemented with complete Freund's adjuvant, and then boosted at weeks 2, 4, 8, and 12 with 0.5 mg of antigen supplemented with incomplete Freund's adjuvant. At weeks 10 and 12, 500 mL of production hemorrhage in whole blood was collected for PBMC isolation.

[0431] PBMC cells were isolated from fresh blood within 4 hours of collection. Total RNA was isolated using the RNeasy Maxi kit (Qiagen), cDNA was reverse transcribed, and a VHH library was generated from it. The library DNA was cloned into a phagemid vector, and the ligated vector was transformed into TG1 cells. The resulting library had a size of 1.8 × 10⁶. 9 It was presumed that this was the case.

[0432] VHH clones exhibiting cross-reactivity binding to human and cynomolgus monkey CD98hc were isolated from the phage library by three consecutive rounds of panning. To prevent enrichment of nonspecifically binding clones, the phage library was first pre-absorbed onto a BSA-coated plate in each round. Subsequently, the library was panned against an isomixture of human and cynomolgus monkey CD98hc at a total antigen concentration of 10 μg / mL.

[0433] Following a third round of panning, 94 clones were screened to evaluate binding to human and cynomolgus monkey CD98hc. Periplasmic fractions containing the VHH domain were prepared, and the clones were evaluated for binding by ELISA, yielding 10 unique VHH sequences, which were selected for further characterization.

[0434] The selected VHH domain was cloned into a mammalian expression vector along with the CMV promoter and a signal peptide that enables secretion. Plasmid DNA encoding the VHH Fc fusion was transfected into Expi293 cells. After 5 days of culture, the supernatant was collected and filtered, and the VHH Fc fusion was purified by protein A affinity chromatography, and subsequently by size exclusion chromatography if necessary. Binding of the VHH domain to human and cynomolgus monkey CD98hc was evaluated by surface plasmon resonance (SPR) using a Biacore® 8K instrument. Clones 1C03-4 showed cross-reactive binding.

[0435] Example 7. Design of a Dual Transporter Molecules that bind to both TfR and CD98hc were designed using two different formats (Figures 2A-D). In the first format, a bispecific antibody was constructed using an antibody framework, with one Fab arm binding to TfR (e.g., 17H10 or 17H10.1) and the other Fab arm replaced with an anti-CD98hc VHH domain (e.g., 1C03-4 or 1C03-5) to provide the Fab-Fc / VHH-Fc format. In the second format, an antibody with anti-DNP Fab as a scaffold was used, and binding to TfR and CD98hc was combined by adding antibody fragments to the C-terminus of each heavy chain. One heavy chain had an anti-TfR scFv fused to its C-terminus, and the other heavy chain had an anti-CD98hc VHH domain attached to its C-terminus (format of antibody fused to C-terminal VHH and scFv). A mobile G4S peptide linker can be used to link the antibody heavy chain to the fused antibody fragment. In either structure, knob-into-hole mutations can be incorporated to promote proper heterodimer pairing while reducing or inhibiting homodimer formation. Dual transporters in antibody frameworks may contain mutations in their Fc polypeptides (e.g., LALAPG mutations) to reduce binding to FcγR and attenuate effector function in vivo.

[0436] Four affinity variants for each structure are generated as outlined in Table 22 below. [Table 22] The measured affinity for the TfR and CD98 binding domains for each target was weaker than that previously measured for the TfR and CD98 binding domains alone, in relation to the VHH-Fc / Fab-Fc format and the format of the scFv and VHH domains linked to the full-length antibody.

[0437] Example 8: TfR at a dose of 8.50 mg / kg mu / hu Characterization of the PK of dual transporters in KI;CD98mu / hu mice. The peripheral and brain pharmacokinetic (PK) profiles of dual transporter variants were studied in transgenic mice (TfR) possessing humanized transferrin receptor and humanized CD98hc. mu / hu KI;CD98hc mu / hu Evaluation was performed in KI mice. These mice expressed (a) a (knock-in) TfR protein in which the natural apical domain was replaced with a human apical domain at the endogenous locus, and (b) CD98hc in which the extracellular domain of CD98hc was humanized at the endogenous locus. The mice were given dual TV, ATV at 50 mg / kg (Figures 6 and 7), 25 mg / kg, and 10 mg / kg. TfR ATV CD98hcAlternatively, control IgG was administered intravenously. Single-binding ATVs served as a control to establish a baseline for PK profiles when TfR and CD98hc bound independently (Figures 6, 7, 8). Blood was collected by cardiac puncture at the time points shown in the figures (in-vivo bleeding was collected by submandibular hemorrhage), and mice were perfused with PBS at terminal time. Brain tissue was homogenized using a Qiagen TissueLyser in 10 times the tissue weight of lysis buffer containing 1% NP-40 along with a protease inhibitor in PBS. Blood was collected in EDTA tubing to prevent coagulation, and plasma was separated by centrifugation at 14000 rpm for 7 minutes. Total huIgG concentrations in plasma and brain were quantified using a standard anti-human IgG sandwich format ELISA. In short, the plates were coated overnight at 4°C with a sodium bicarbonate solution (Sigma#C3041-50CAP) containing 1 μg / mL of donkey anti-human IgG (JIR#709-006-098) while gently agitated. The plates were then washed three times with washing buffer (PBS + 0.05% Tween® 20). Assay standards and samples were diluted with PBS + 0.05% Tween® 20 + 1% BSA (10 mg / mL). Standard curve preparations ranged from 0.003 to 10 nM (BLQ < 0.03 nM). The standard solutions and diluted samples were incubated at room temperature for 2 hours with agitation. After incubation, the plates were washed three times with washing buffer. The detection antibody, goat anti-human IgG (JIR#109-036-098), was diluted with blocking buffer (PBS + 0.05% Tween® 20 + 5% BSA (50 mg / mL)) to a final concentration of 0.02 μg / mL, and the plate was incubated at room temperature for 1 hour with agitation. After three final washes, the plate was prepared by adding the TMB substrate and incubated for 5–10 minutes. The reaction was quenched by adding 4N H2SO4, and the absorbance was read using a 450 nM absorbance standard. Following our initial analysis, it was determined that in this assay, dual TV would yield more accurate quantification using a dual TV standard. The graph in Figure 7 shows the updated measurements of the sample, also shown in Figure 6.

[0438] Dual transporters that associate with both CD98hc and TfR exhibit both higher maximal brain concentrations and / or prolonged brain exposure compared to their corresponding single-binding ATVs. For example, at a dose of 50 mg / kg, the maximal brain concentration for a single ATV conjugated with TfR (TV35.23.4) was approximately 20 nM, while that for a single ATV conjugated with CD98hc (LLB2) was approximately 25 nM. In contrast, the corresponding dual construct showed a much greater effect than additive, with a maximal brain concentration exceeding approximately 100 nM (upgrading from the previously determined highest concentration of 70 nM). This effect, exceeding additive, persisted for at least one week. This effect is even more dramatic considering that the peripheral concentrations of the dual transporters were significantly lower than those of either single TV at all time points. [Table 23A] [Table 23B]

[0439] Example 9. The pharmacokinetic (PK) profiles in plasma, brain, kidney, and bone marrow of dual transporter variants with different TfR affinity and CD98hc affinity were examined. mu / hu KI;CD98hc mu / hu Evaluation was performed using KI mice (Figures 9-12). Mice were given 25 mg / kg of dual TV and ATV. TfR ATV CD98hcAlternatively, control IgG was administered intravenously. huIgG concentrations in plasma, kidney, and bone marrow were measured as described above. Brain concentrations were quantified using the standard anti-human IgG sandwich format electrochemiluminescence immunoassay (ECLIA) on the Meso Scale Discovery (MSD) platform. Briefly, 1% casein-based PBS blocking buffer (Thermo Scientific, 37528) was added to an MSD GOLD96 well small spot streptavidin-coated microtiter plate (Meso Scale Discovery, L45SA) and incubated for approximately 1 hour. After the blocking and washing steps of the plate, the assay plate was coated with biotinylated goat anti-human IgG (SouthernBiotech 2049-08) at a working concentration of 0.5 μg / mL and incubated for 1–2 hours. Subsequently, the test sample was diluted (1:100 MRD in 0.5% casein-based PBS assay buffer) and added to the assay plate. After 1-2 hours of incubation in the capture step, pre-adsorbed secondary ruthenium-labeled (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, R32AJ) was added to the assay plate at 0.5 μg / mL (standard solution) and incubated for approximately 1 hour. Subsequently, assay read buffer (1× MSD Read Buffer T, R92TC) was added to generate an electrochemiluminescence (ECL) assay signal expressed in ECL units (ECLU). All assay reaction steps were performed at ambient temperature with shaking on a plate shaker (if necessary). Brain lysate sample concentrations were calculated from the standard curve and fitted to a weighted 4-parameter nonlinear logistic regression.

[0440] Early brain concentrations of the dual transporter correlated with stronger TfR affinity, while higher dual transporter concentrations at later time points correlated with stronger CD98hc affinity. Specific brain exposure dynamics can be optimized by modulating transport (e.g., TfR) and retention (e.g., CD98c) affinities to induce different amounts of brain uptake and retention. Increasing TfR affinity can increase BBB transport; however, increasing TfR affinity may also lead to a decrease in brain retention.

[0441] Peripheral distribution to the bone marrow correlated with TfR affinity and was substantially unaffected by CD98hc affinity. Similarly, renal localization correlated with CD98hc affinity and was only slightly reduced with strong TfR affinity. These data suggest that the peripheral distribution of the dual transporter reflects the distribution of each target it is combined with.

[0442] Example 10. After intravenous administration of 25 mg / kg of a dual transporter with different TfR affinity and CD98hc affinity, mice were perfused with PBS and the half-brain was fixed overnight by immersion (drop) in 4% PFA. Sagittal brain sections (40 μm) were cut using a microtome (MultiBrain® Technology by NeuroScience Associates) and blocked at room temperature for 2 hours in 5% BSA + 0.3% Triton® X-100. The sections were probed overnight at 4°C with goat anti-huIgG-Alexa-647 (Jackson ImmunoResearch 709-606-149, 1:500), rabbit anti-Aquaporin4 (Millipore, AB2218, 1:500), and mouse anti-NeuN (Millipore MAB377, 1:500). Subsequently, the sections were incubated overnight at 4°C with donkey-anti-rabbit-488 (Thermofisher A-21206, 1:500) and goat-anti-mouse IgG1 (Thermo Fisher A-21124, 1:500). The sections were washed and mounted in ProLong Glass (ThermoFisher P36982). Images of the entire sagittal section were captured using a Zeiss Axioscan Z1 with a 20x objective lens, and representative confocal images of the cortex were acquired using a Leica SP8 Lightning confocal microscope with a 25x objective lens.

[0443] Sagittal images were registered in a brain atlas using rigid, affine, and bspline warps in itk-elastix v0.19.1 (K.Ntatsis et al. "itk-elastix: Medical image registration in Python", Proceedings of the 22nd Python in Science Conference, pp.101 - 105, 2023). Then, the data were quantified using the regionprops tool in scikit-image v0.22.0 (Stefan van der Walt S et al. "scikit-image: Image processing in Python. PeerJ 2:e453(2014)), and the mean huIgG intensities across various brain regions are shown in Figure 13. These data indicate that dual TV provides a relatively enhanced distribution to brainstem regions (medulla and pons) that is broadly distributed across brain regions and not observed with TV coupled only with TfR or CD98hc.

[0444] In cortical confocal images, the vascular system was segmented using bright, large aquaporin 4-positive regions, and NeuN-positive regions were segmented as neurons using a custom code constructed with the filtering and thresholding functions of ndimage as described in scipy v1.11.4 (Pauli Virtanen P et al. “SciPy 1.0: Fundamental Algorithms for Scientific Computing in Python”. Nature Methods, 17(3), 261-272 (2020)). Tissue outside the vascular region was designated as parenchyma, and non-neuronal parenchymal regions were defined as non-vascular and non-neuronal regions. The mean huIgG intensity for each of these segments is shown in Figure 14. Consistent with PK measurements, strong TfR affinity correlated with brighter anti-huIgG staining on day 1 post-administration, and stronger CD98hc affinity correlated with brighter anti-huIgG staining...

Claims

1. (a) A first binding region that specifically binds to a blood-brain barrier (BBB) ​​transport protein, wherein the BBB transport protein is a transferrin receptor (TfR) or CD98hc, (b) A dual transporter comprising a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transport protein.

2. The second protein mentioned above includes TfR, CD98hc, large neutral amino acid transporter small subunit 1 (CD98 light chain), glucose transporter 1 (GLUT1), major promoter superfamily domain-containing protein 2A (MFSD2A), carbonic anhydrase IV (CA-IV), low-density lipoprotein receptor, insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 50A, basidine, leptin receptor, and claudin. A dual transporter according to claim 1, selected from the group consisting of -5, P-selectin, lactoferrin receptor, folate receptor, sodium-dependent lysophosphatidylcholine cotransporter 1, solute transporter organic anion transporter family member 1C1, sodium-conjugated neutral amino acid transporter 5, LDL receptor-related protein 8, high affinity cationic amino acid transporter 1, sodium chloride-dependent taurine transporter, insulin-like growth factor-binding protein 7, solute transporter family 40 member 1, zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and myelin oligodendrocyte glycoprotein (MOG).

3. The dual transporter according to claim 1, wherein the second protein is a brain-retained protein.

4. The dual transporter according to claim 3, wherein the brain-retained protein is CD98hc, MOG, GLUT1, IGF1R, CA-IV, or MFSD2A.

5. The dual transporter according to any one of claims 1 to 4, wherein the transferrin receptor is human TfR1.

6. The dual transporter according to any one of claims 1 to 4, wherein the CD98hc is human CD98hc.

7. (a) The BBB transport protein is human TfR1 and the second protein is human CD98hc, (b) The BBB transport protein is human TfR1 and the second protein is human MOG, (c) The BBB transport protein is human TfR1 and the second protein is human GLUT1, (d) The BBB transport protein is human TfR1 and the second protein is human IGF1R, (d) The BBB transport protein is human TfR1 and the second protein is human CA-IV, or (d) The dual transporter according to claim 2, wherein the BBB transport protein is human TfR1 and the second protein is human MFSD2A.

8. The dual transporter according to any one of claims 1 to 7, wherein the second protein undergoes receptor-mediated transcytosis.

9. The first bonding region described above is (a) A first peptide that specifically binds to the BBB transport protein, (b) A first antibody-antigen binding domain that specifically binds to the BBB transport protein, (c) A first Fc polypeptide modified to specifically bind to the BBB transport protein, (d) A first CH3 peptide modified to specifically bind to the BBB transport protein, (e) A first fibronectin type III domain peptide modified to specifically bind to the BBB transport protein, or (f) The dual transporter according to any one of claims 1 to 8, comprising a first bicyclic peptide that specifically binds to the BBB transport protein.

10. The dual transporter according to claim 9, wherein the first antibody-antigen binding domain comprises an antibody, a single-chain antibody, an F(ab')2 fragment, a Fab fragment, a single-chain Fab (scFab), an Fv fragment, a single-chain variable fragment (scFv), or an antibody variable domain (nanobody) consisting only of a heavy chain.

11. The second bonding region is (a) A second peptide that specifically binds to the second protein, (b) A second antibody-antigen binding domain that specifically binds to the second protein, (c) A second Fc polypeptide modified to specifically bind to the second protein, (d) A second CH3 peptide modified to specifically bind to the second protein, (e) A second fibronectin type III domain peptide modified to specifically bind to the second protein, or (f) The dual transporter according to any one of claims 1 to 10, comprising a second bicyclic peptide that specifically binds to the second protein.

12. The dual transporter according to claim 11, wherein the second antibody-antigen binding domain comprises an antibody, a single-chain antibody, an F(ab')2 fragment, a Fab fragment, a single-chain Fab (scFab), an Fv fragment, a single-chain variable fragment (scFv), or an antibody variable domain (nanobody) consisting only of a heavy chain.

13. The dual transporter according to any one of claims 1 to 12, wherein the first binding region comprises a first antibody-antigen binding domain that specifically binds to the BBB transport protein, and the second binding region comprises a second antibody-antigen binding domain that specifically binds to the second protein.

14. The dual transporter according to claim 13, comprising a bispecific antibody selected from the group consisting of a full-length IgG bispecific antibody, a bispecific heavy chain antibody, and a bispecific F(ab')2, the bispecific antibody comprising the first antibody-antigen binding domain and the second antibody-antigen binding domain.

15. The dual transporter according to claim 13, wherein the first binding region comprises a Fab fragment, scFab, Fv fragment, scFv, or nanobody, and the second binding region comprises a Fab fragment, scFab, Fv fragment, scFv, or nanobody.

16. (a) The first binding region includes a Fab fragment, and the second binding region includes a Fab fragment, (b) The first binding region includes scFab, and the second binding region includes scFab, (c) The first binding region includes an Fv fragment, and the second binding region includes an Fv fragment, (d) The first bonding region includes scFv and the second bonding region includes scFv, or (e) The dual transporter according to claim 15, wherein the first binding region comprises a nanobody and the second binding region comprises a nanobody.

17. (a) The first binding region comprises the first antibody-antigen binding domain, and the second binding region comprises the second peptide, the second Fc polypeptide, the second CH3 peptide, the second fibronectin type III domain peptide, or the second bicyclic peptide, or (b) The dual transporter according to claim 11 or 12, wherein the first binding region comprises the first peptide, the first Fc polypeptide, the first CH3 peptide, the first fibronectin type III domain peptide, or the first bicyclic peptide, and the second binding region comprises the second antibody-antigen binding domain.

18. (a) The first binding region comprises the first Fc polypeptide, and the second binding region comprises the second peptide, the second antibody-antigen binding domain, the second Fc polypeptide, the second CH3 peptide, the second fibronectin type III domain peptide, or the second bicyclic peptide, (b) The first binding region comprises the first CH3 peptide, and the second binding region comprises the second peptide, the second antibody-antigen binding domain, the second Fc polypeptide, the second CH3 peptide, the second fibronectin type III domain peptide, or the second bicyclic peptide, (c) The first binding region comprises the first peptide, the first antibody-antigen binding domain, the first Fc polypeptide, the first CH3 peptide, the first fibronectin type III domain peptide, or the first bicyclic peptide, and the second binding region comprises the second Fc polypeptide, or (d) The dual transporter according to claim 11 or 12, wherein the first binding region comprises the first peptide, the first antibody-antigen binding domain, the first Fc polypeptide, the first CH3 peptide, the first fibronectin type III domain peptide, or the first bicyclic peptide, and the second binding region comprises the second CH3 peptide.

19. (a) The first binding region comprises the first Fc polypeptide, the second binding region comprises the second Fc polypeptide, and optionally the first Fc polypeptide and the second Fc polypeptide form an Fc dimer. (b) The first binding region contains the first CH3 peptide, and the second binding region contains the second CH3 polypeptide, (c) The first binding region contains the first fibronectin type III domain peptide, and the second binding region contains the second fibronectin type III domain peptide, or (d) The dual transporter according to claim 11 or 12, wherein the first binding region comprises the first bicyclic peptide and the second binding region comprises the second bicyclic peptide.

20. The dual transporter according to any one of claims 1 to 19, wherein the first binding region is connected to the second binding region.

21. (a) The first binding region forms a dimer with the second binding region, (b) The first binding region is linked to a polypeptide that forms a dimer with the second binding region, (c) The second binding region is linked to a polypeptide that forms a dimer with the first binding region, or (d) The dual transporter according to any one of claims 1 to 19, wherein the first binding region is linked to a first polypeptide, and the second binding region is linked to a second polypeptide, so that the first polypeptide and the second polypeptide form a dimer.

22. The dual transporter according to any one of claims 1 to 19, wherein the first coupling region, the second coupling region, or both the first coupling region and the second coupling region are connected to a scaffold.

23. The dual transporter according to claim 22, wherein the scaffold comprises an antibody or a fragment thereof.

24. The aforementioned scaffold contains an antibody, (a) The first binding region is linked to the heavy chain of the antibody and the second binding region is linked to the heavy chain of the antibody, or both the first and second binding regions are linked to the heavy chain of the antibody, and optionally the first binding region is linked to the C-terminus of the heavy chain of the antibody and / or the second binding region is linked to the C-terminus of the heavy chain of the antibody. (b) The first binding region is linked to the Fc region of the antibody and the second binding region is linked to the Fc region of the antibody, or both the first and second binding regions are linked to the Fc region of the antibody, and optionally the first binding region is linked to the C-terminus of the Fc region of the antibody and / or the second binding region is linked to the C-terminus of the Fc region of the antibody. (c) The first binding region is linked to the light chain of the antibody and the second binding region is linked to the light chain of the antibody, or both the first and second binding regions are linked to the light chain of the antibody, and optionally the first binding region is linked to the C-terminus of the light chain of the antibody and / or the second binding region is linked to the C-terminus of the light chain of the antibody, or (d) The dual transporter according to claim 23, wherein the first binding region is linked to the light chain of the antibody and the second binding region is linked to the heavy chain of the antibody, or the first binding region is linked to the heavy chain of the antibody and the second binding region is linked to the light chain of the antibody.

25. The dual transporter according to claim 24, wherein the first binding region comprises a Fab fragment, scFab, Fv fragment, scFv, or nanobody, and the second binding region comprises a Fab fragment, scFab, Fv fragment, scFv, or nanobody.

26. The dual transporter according to claim 24 or 25, wherein the scaffold is a full-length IgG antibody that binds to a therapeutic intracerebral target, and the first and second binding regions are each bound to the respective C-terminuses of the heavy chain of the full-length IgG antibody.

27. The dual transporter according to claim 26, wherein the first and second binding regions are each an antibody-antigen binding domain.

28. The dual transporter according to claim 27, wherein each antibody-antigen binding domain independently comprises a Fab fragment or scFab.

29. The dual transporter according to claim 27, wherein the first and second binding regions are each scFv.

30. The dual transporter according to any one of claims 1 to 29, wherein the dual transporter binds to the BBB transport protein with a higher affinity than the dual transporter binds to the second protein, and optionally, the dual transporter binds to the BBB transport protein with an affinity of about 50 nM to about 1000 nM, and binds to the second protein with an affinity about 5 to about 10 times lower than its affinity to the BBB transport protein.

31. The dual transporter according to any one of claims 1 to 29, wherein the dual transporter binds to the BBB transport protein with a lower affinity than the dual transporter binds to the second protein, and optionally, the dual transporter binds to the BBB transport protein with an affinity of about 50 nM to about 6000 nM, and binds to the second protein with an affinity about 5 to about 10 times higher than its affinity to the BBB transport protein.

32. The dual transporter according to any one of claims 1 to 29, wherein the first binding region has a moderate or weak affinity for the BBB transport protein, and the second binding region has a strong affinity for the second protein.

33. The dual transporter according to any one of claims 24 to 29, wherein the first and second binding regions bind to hTfR1 and human CD98hc, respectively.

34. The dual transporter according to claim 33, wherein the first binding region includes a CDR or variable chain region of a brain shuttle anti-TfR antibody, a TfR12 anti-TfR antibody, a TfR13 anti-TfR scFv, a 13E4v2ii anti-TfR antibody, a JC-141 anti-TfR antibody, a JC-171 anti-TfR antibody, a 17H10 anti-TfR Fab or scFv, or a 17H10.1 anti-TfR Fab or scFv.

35. The dual transporter according to any one of claims 33 or 34, wherein the second binding region includes a CDR or variable chain region of 1C03-4 anti-CD98hc VHH, 1C03-5 anti-CD98hc VHH, CD98hc2 anti-CD98hc nanobody, CD98hc4 anti-CD98hc scFv, or CD98hc5 anti-CD98hc scFv.

36. The dual transporter according to any one of claims 23 to 25, wherein the scaffold comprises a full-length antibody directed toward a therapeutic intracerebral target, and the first and second binding regions are each linked to the C-terminus of the light chain of the full-length antibody.

37. The dual transporter according to any one of claims 23 to 25, wherein the antibody comprises a bispecific antibody.

38. The dual transporter according to any one of claims 23 to 37, wherein the antibody specifically binds to a therapeutic target.

39. The aforementioned dual transport molecule includes a bispecific antibody. (a) The bispecific antibody binds specifically to the BBB transport protein and specifically to the therapeutic target, and the second binding region is linked to the bispecific antibody, or (b) The dual transporter according to any one of claims 23 to 25 and 37 to 38, wherein the bispecific antibody specifically binds to the second protein and specifically binds to a therapeutic target, and the first binding region is linked to the bispecific antibody.

40. The dual transporter according to any one of claims 1 to 13, wherein the dual transport molecule comprises a triplicate antibody, a triplicate antibody-like molecule, a quadruplicate antibody, or a quadruplicate antibody-like molecule.

41. The dual transporter according to any one of claims 1 to 13, wherein the dual transport molecule comprises DVD-Ig, the first Fab arm of DVD-Ig comprises a first Fv fragment that specifically binds to a first target protein and a second Fv fragment that specifically binds to the BBB transport protein, and the second Fab arm of DVD-Ig comprises a third Fv fragment that specifically binds to a second target protein and a fourth Fv fragment that specifically binds to the second protein.

42. The first binding region comprises the first Fc polypeptide, the second binding region comprises the second Fc polypeptide, the first Fc polypeptide and the second Fc polypeptide form an Fc dimer, the Fc dimer is linked to one or more Fabs, one or more F(ab')2s, one or more scFabs, one or more scFvs, or one or more nanobodies, optionally the Fabs, F(ab') ２ scFab, scFv, or nanobody, therapeutic Fab, F(ab') ２ The dual transporter according to claim 19, comprising scFab, scFv, or nanobody.

43. The dual transporter according to any one of claims 1 to 42, wherein the dual transport molecule is linked to a therapeutic agent.

44. The dual transporter according to claim 18 or 19, wherein the dual transport molecule comprises a therapeutic antibody comprising a first Fc polypeptide or CH3 peptide modified to specifically bind to the BBB transport protein, and a second Fc polypeptide or CH3 peptide modified to specifically bind to the second protein.

45. The dual transporter according to any one of claims 1 to 4, 6 to 19, and 44, wherein the BBB transport protein comprises TfR.

46. The dual transporter according to claim 45, wherein the first binding region binds to the apical domain of TfR.

47. The dual transporter according to claim 46, wherein the first binding region binds to TfR without inhibiting the binding of transferrin to TfR.

48. The dual transporter according to any one of claims 45 to 47, wherein the first binding region includes a first antibody-antigen binding domain that specifically binds to TfR.

49. The dual transporter according to claim 48, wherein the first antibody-antigen binding domain comprises a CDR sequence of JC-141 anti-TfR antibody, JC-171 anti-TfR antibody, brain shuttle anti-TfR antibody, 13E4v2ii anti-TfR antibody, TfR12 anti-TfR antibody, TfR13 anti-TfR antibody, 17H10 anti-TfR Fab or scFv, or 17H10.1 anti-TfR Fab or scFv.

50. The dual transporter according to claim 49, wherein the first antibody-antigen binding domain comprises an antibody-antigen binding domain from the JC-141 anti-TfR antibody, the JC-171 anti-TfR antibody, the brain shuttle anti-TfR antibody, the 13E4v2ii anti-TfR antibody, the TfR12 anti-TfR antibody, the TfR13 anti-TfR antibody, the 17H10 anti-TfR Fab or scFv, or the 17H10.1 anti-TfR Fab or scFv.

51. The dual transporter according to any one of claims 45 to 47, wherein the first binding region comprises a first Fc polypeptide or a first CH3 peptide modified to specifically bind to TfR.

52. The dual transporter according to any one of claims 45 to 51, wherein the first binding region specifically binds to TfR with an affinity of less than about 100 nM.

53. The dual transporter according to any one of claims 45 to 51, wherein the first binding region specifically binds to TfR with an affinity of about 100 nM to about 1000 nM or about 100 nM to about 600 nM.

54. The dual transporter according to any one of claims 45 to 51, wherein the first binding region specifically binds to TfR with an affinity of about 600 nM to about 6000 nM or about 600 nM to about 1000 nM.

55. The dual transporter according to claim 51, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises five, six, seven, eight, nine, ten, eleven, twelfth, twelfth, and sixteenth substitutions at a series of amino acid positions consisting of 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426, wherein the positions are determined by reference to EU numbering.

56. The substitutions include L, Q, S, V, W, or Y at position 380; F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; D, E, I, or V at position 387; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; A, D, E, F, G, H, K, L, Q, R, S, T, V, or Y at position 390; and F, Q, S, T at position 391. The dual transporter according to claim 55, wherein is selected from V; Q, F, or H at position 392; A, E, H, I, L, P, S, or T at position 413; R, G, or P at position 414; D, E, G, T, P, Q, or R at position 415; D, E, N, or T at position 416; F, H, K, W, or Y at position 421; T, W, E, or K at position 424; and C, P, M, W, or G at position 426.

57. The dual transporter according to claim 46, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises F, L, M, P, V, W, Y, or I at position 384; F, H, I, L, N, P, T, or V at position 386; W, F, or Y at position 388; A, G, I, S, T, or V at position 389; and F, H, K, W, or Y at position 421.

58. The first CH3 peptide, modified to specifically bind to TfR, has L, Q, S, V, W, or Y at position 380; D, E, I, or V at position 387; A, D, E, F, G, H, K, L, Q, R, S, T, V, or Y at position 390; F, Q, S, T, or V at position 391; Q, F, or H at position 392; and A, E, H, I, L, P, S, or T at position 413. The dual transporter according to claim 57, further comprising one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions selected from: R, G, or P at position 414; D, E, G, T, P, Q, or R at position 415; D, E, N, or T at position 416; T, W, E, or K at position 424; and C, P, M, W, or G at position 426.

59. The dual transporter according to claim 51, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises four, five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 384, 386, 387, 388, 389, 413, 415, 416, and 421, the positions being determined by reference to EU numbering.

60. The dual transporter according to claim 59, wherein the substitution is selected from Y at position 384; T at position 386; E at position 387; W at position 388; V, S, or A at position 389; T or S at position 413; E at position 415; E at position 416; and F at position 421.

61. The dual transporter according to claim 60, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises Y at position 384; T at position 386; E at position 387; W at position 388; V, S, or A at position 389; T or S at position 413; E at position 415; E at position 416; and F at position 421.

62. The dual transporter according to any one of claims 55 to 61, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises Y at position 391, K at position 392, S at position 424, and S at position 426.

63. The dual transporter according to any one of claims 55 to 61, wherein the first CH3 peptide modified to specifically bind to TfR comprises (a) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with any amino acid sequence among SEQ ID NOs. 6-14 and 124 and 125, or (b) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with amino acids 111-217 among any amino acids among SEQ ID NOs. 6-14 and 124 and 125.

64. The first CH3 peptide, modified to specifically bind to TfR, is 378, 380, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 414, 417, 419, 420, 421, 422, 424, 426, 427, 428, 429, 433, 434, 437, 438, 439, 440, 442, and 443 The dual transporter according to claim 51, comprising six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 amino acid substitutions at a series of amino acid positions consisting of , and 426, wherein the positions are determined by reference to EU numbering.

65. The substitution is E, L, or I at position 378; N, R, Y, I, S, or F at position 380; F at position 382; Y, A, G, T, or deletion at position 383; A, D, E, F, G, or T at position 384; A, D, or N at position 385; A, G, K, N, S, or Y at position 386; G, I, K, N, R, S, or T at position 387; L, Q, or D at position 388; I, P, Q, R, S, or T at position 389; G, T, Y, or L at position 390; S, T, I, L, or P at position 391; 41 A dual transporter according to claim 64, selected from P at position 4; K at position 417; P at position 419; R or Q at position 420; A, F, G, S, or Y at position 421; L at position 422; A at position 424; E at position 426; E at position 427; E at position 428; G at position 429; E at position 433; G at position 434; D at position 437; Y at position 438; D, E, or S at position 439; L at position 440; G or W at position 442; and E or Y at position 443.

66. The dual transporter according to claim 65, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises F at position 382, ​​L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440.

67. The first CH3 peptide, modified to specifically bind to TfR, has E, L, or I at position 378; N, R, Y, I, S, or F at position 380; Y, A, G, T, or deletion at position 383; A, D, E, F, G, or T at position 384; A, D, or N at position 385; A, G, K, N, S, or Y at position 386; G, I, K, N, R, S, or T at position 387; L, Q, or D at position 388; I, P, Q, R, S, or T at position 389; G, T, Y, or L at position 390; S, T, I, L, or P at position 391; P at position 414; 41 The dual transporter according to claim 66, further comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, twelve, thirteenth, fif

68. The dual transporter according to any one of claims 64 to 67, wherein the first CH3 peptide modified to specifically bind to TfR comprises (a) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with any amino acid sequence of SEQ ID NOs. 57-64 and 126, or (b) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with amino acids 111-217 of any amino acid sequence of SEQ ID NOs. 57-64 and 126.

69. The dual transporter according to claim 51, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, or 16 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 421, 422, 424, 426, 428, 434, 438, 440, and 442, wherein the positions are determined by reference to EU numbering.

70. The substitution is D, E, F, H, N, Q, S, V, or Y at position 378; A, D, F, H, I, K, L, M, Q, S, T, or Y at position 380; G at position 382; T at position 383; A, E, F, H, I, K, L, Q, S, V, or Y at position 384; I, T, or V at position 385; A, H, N, S, T, or V at position 386; A, F, H, K, L, M, Q, S, T, V, or The dual transporter according to claim 69, selected from Y; A, F, H, I, K, L, R, T, or Y at position 422; A, G, or P at position 424; A, I, L, T, or V at position 426; A or L at position 428; S at position 434; I, F, L, V, or Y at position 438; A, G, I, M, N, P, T, or V at position 440; and A, K, M, R, T, or V at position 442.

71. The dual transporter according to claim 70, wherein the first CH3 peptide, modified to specifically bind to TfR, comprises G at position 382; A, E, F, H, I, K, L, Q, S, V, or Y at position 384; I, T, or V at position 385; and I, F, L, V, or Y at position 438.

72. The first CH3 peptide, modified to specifically bind to TfR, has D, E, F, H, N, Q, S, V, or Y at position 378; A, D, F, H, I, K, L, M, Q, S, T, or Y at position 380; T at position 383; A, H, N, S, T, or V at position 386; A, F, H, K, L, M, Q, S, T, V, or Y at position 421; A, F, H, I, K, L, R, T, or Y at position 422; and at position 424. The dual transporter according to claim 71, further comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, t, t, th

73. The dual transporter according to any one of claims 1 to 72, wherein the second protein comprises CD98hc.

74. The dual transporter according to claim 73, wherein the second binding region includes a second antibody-antigen binding domain that specifically binds to CD98hc.

75. The dual transporter according to claim 74, wherein the second antibody-antigen binding domain comprises a CDR sequence of 1C03-4 anti-CD98hc VHH, 1C03-5 anti-CD98hc VHH, CD98hc2 anti-CD98hc nanobody, CD98hc4 anti-CD98hc scFv, or CD98hc5 anti-CD98hc scFv.

76. The dual transporter according to claim 75, wherein the second antibody-antigen binding domain comprises the 1C03-4 anti-CD98hc VHH, the 1C03-5 anti-CD98hc VHH, the CD98hc2 anti-CD98hc nanobody, the CD98hc4 anti-CD98hc scFv, or the CD98hc5 anti-CD98hc scFv.

77. The dual transporter according to claim 73, wherein the second binding region comprises a second Fc polypeptide or a second CH3 peptide modified to specifically bind to CD98hc.

78. The dual transporter according to any one of claims 73 to 77, wherein the second binding region specifically binds to CD98hc with an affinity of about 15 nM to less than about 5000 nM, or about 100 nM to about 500 nM.

79. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 382, ​​384, 385, 387, 422, 424, 426, 438, and 440, the positions being determined according to EU numbering.

80. The dual transporter according to claim 79, wherein the substitution is selected from R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; N, L, Y, R, F, G, S, D, or T at position 387; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; I, V, F, N, P, or S at position 438; and K, T, P, I, or F at position 440.

81. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises at least five, six, seven, eight, or nine substitutions at a series of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, wherein the positions are determined according to EU numbering.

82. The substitutions are: S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, or T at position 387; T, Y, or F at position 389; 421 A dual transporter according to claim 81, selected from D, E, or Q at position 422; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

83. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises at least 11, 12, 13, 14, or 15 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442, wherein the positions are determined according to EU numbering.

84. The substitutions are: S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; V, L, A, I, F, Y, S, T, H, R, or E at position 386; L or I at position 387; D, Q, A, T, H, or V at position 389; T, V, or A at position 391; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, Y at position 428 A dual transporter according to claim 83, selected from F, I, A, K, H, or W; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442.

85. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises substitutions of 11, 12, 13, 14, or 15 at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442, wherein the positions are determined according to EU numbering.

86. The dual transporter according to claim 85, wherein the substitution is selected from L at position 380; N at position 382; R, H, or Q at position 384; F or Y at position 385; V, L, I, F, Y, or E at position 386; L at position 387; E, Q, or A at position 421; I, T, or P at position 422; A at position 424; N at position 426; Y or W at position 428; R or W at position 436; F or W at position 438; N at position 440; and A, Q, K, R, H, or M at position 442.

87. The dual transporter according to any one of claims 69 to 72, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises (a) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with any amino acid sequence of SEQ ID NOs. 15 to 30, or (b) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with amino acids 111 to 217 of any amino acid sequence of SEQ ID NOs. 15 to 30.

88. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, wherein the positions are determined according to EU numbering.

89. The substitutions are: S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, or T at position 387; T, Y, or F at position 389; 421 A dual transporter according to claim 88, selected from D, E, or Q at position 422; I, K, L, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L or Y at position 428; S at position 434; F at position 436; I, V, F, N, P, or S at position 438; K, T, P, I, or F at position 440; and Q or M at position 442.

90. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises eight, nine, ten, eleven, twelfth, or thirteenth substitutions at a series of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440, the positions being determined according to EU numbering.

91. The dual transporter according to claim 90, wherein the substitution is selected from D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440.

92. The dual transporter according to claim 77 or 78, wherein the second CH3 peptide, modified to specifically bind to CD98hc, comprises eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at a series of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, wherein the positions are determined according to EU numbering.

93. The dual transporter according to claim 92, wherein the substitution is selected from S or V at position 378; D at position 380; R at position 382; T at position 383; Y at position 384; K at position 385; P at position 386; Y at position 387; T, Y, or F at position 389; D, E, or Q at position 421; I at position 422; V at position 424; D at position 426; L or Y at position 428; S at position 434; F at position 436; I or V at position 438; K at position 440; and Q or M at position 442.

94. The dual transporter according to any one of claims 88 to 93, wherein the first CH3 peptide modified to specifically bind to TfR comprises (a) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence of SEQ ID NO: 34 or 35, or (b) a polypeptide having at least 85%, at least 90%, at least 95%, or 100% identity with amino acids 111 to 217 of SEQ ID NO: 34 or 35.

95. The dual transporter according to any one of claims 1 to 94, wherein the dual transporter comprises an Fc polypeptide having one or more mutations that (a) promote heterodimer formation, (b) regulate effector function, or (c) extend serum half-life.

96. The dual transporter according to any one of claims 1 to 32 and 36 to 72, wherein the first binding region specifically binds to TfR and the second binding region specifically binds to CD98hc.

97. A dual transporter according to any one of claims 1 to 9, 11, 18 to 24, 30 to 32, 36 to 38, 42 to 47, 51, and 55 to 73, wherein the first binding region comprises a first Fc polypeptide or CH3 peptide modified to specifically bind to TfR, and the second binding region comprises a second Fc polypeptide or CH3 peptide modified to specifically bind to CD98hc.

98. The dual transporter according to any one of claims 77 to 97, wherein the first Fc polypeptide and the second Fc polypeptide form an Fc dimer.

99. The dual transporter according to claim 98, wherein the dual transporter comprises an antibody comprising the first Fc polypeptide and the second Fc polypeptide.

100. A dual transporter according to any one of claims 1 to 13, 16, 22 to 25, 30 to 38, 43, 45 to 50, and 73 to 76, wherein the first binding region comprises an anti-TfR antibody antigen-binding domain linked to the C-terminus of the first light chain of the antibody, and the second binding region comprises an anti-CD98hc antibody antigen-binding domain linked to the C-terminus of the second light chain of the antibody.

101. A dual transporter according to any one of claims 1 to 13, 16, 22 to 36, 37 to 38, 43, 45 to 50, and 73 to 76, wherein the first binding region comprises an anti-TfR antibody antigen-binding domain linked to the C-terminus of the first heavy chain of the antibody, and the second binding region comprises an anti-CD98hc antibody antigen-binding domain linked to the C-terminus of the second heavy chain of the antibody.

102. The dual transporter according to claim 100 or 101, wherein the antibody comprises a monospecific antibody, a bispecific antibody, a monospecific therapeutic antibody, or a bispecific therapeutic antibody.

103. The dual transporter according to any one of claims 100 to 102, wherein the first binding region comprises a Fab fragment, scFab, Fv fragment, scFv, or nanobody, and the second binding region comprises a Fab fragment, scFab, Fv fragment, scFv, or nanobody.

104. The dual transporter according to any one of claims 1 to 16, 22 to 36, 43, 45 to 50, and 73 to 76, wherein the dual transporter comprises a bispecific antibody, one Fab arm of the antibody specifically binds to TfR, and the other Fab arm of the antibody specifically binds to CD98hc.

105. The dual transporter according to any one of claims 1 to 16, 22 to 25, 30 to 35, 37 to 40, 46 to 50, and 73 to 76, wherein the dual transporter comprises a bispecific antibody, one Fab arm of the antibody specifically binds to TfR or CD98hc, and the other Fab arm of the antibody specifically binds to a target protein.

106. (a) The first binding region comprises a first fibronectin type III domain peptide modified to specifically bind to TfR, and the second binding region comprises a second fibronectin type III domain peptide modified to specifically bind, wherein the first and second binding regions are linked to an antibody, or (b) The dual transporter according to any one of claims 1 to 12, 19, and 30 to 33, wherein the first binding region comprises a first bicyclic peptide that specifically binds to TfR, and the second binding region comprises a second bicyclic peptide that specifically binds to CD98hc, and the first and second binding regions are linked to an antibody.

107. The dual transporter, (a) an anti-TfR monospecific or bispecific antibody linked to the second binding region, (b) A dual transporter according to any one of claims 1 to 18, 20 to 25, 30 to 35, 46 to 50, and 73 to 76, comprising an anti-CD98hc monospecific or bispecific antibody linked to the first binding region.

108. A dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region has a moderate or weak affinity for TfR, and the second binding region has a strong affinity for CD98hc.

109. (a) The first binding region binds to TfR with an affinity of about 1000 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 1000 nM to about 6000 nM, (b) The first binding region binds to TfR with an affinity of about 900 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 900 nM to about 6000 nM, (c) The first binding region binds to TfR with an affinity of about 1000 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 300 nM to about 900 nM, (d) The first binding region binds to TfR with an affinity of about 900 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 300 nM to about 900 nM, (e) The first binding region binds to TfR with an affinity of about 1000 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of less than about 250 nM, or (f) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 900 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to about 250 nM.

110. (a) The first binding region binds to TfR with an affinity of about 300 nM to about 900 nM, and the second binding region binds to CD98hc with an affinity of about 1000 nM to about 6000 nM, (b) The first binding region binds to TfR with an affinity of about 300 nM to about 900 nM, and the second binding region binds to CD98hc with an affinity of about 300 nM to about 900 nM, (c) The first binding region binds to TfR with an affinity of about 300 nM to about 900 nM, and the second binding region binds to CD98hc with an affinity of less than about 250 nM, or (d) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 300 nM to about 900 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to about 250 nM.

111. (a) The first binding region binds to TfR with an affinity of less than approximately 250 nM, and the second binding region binds to CD98hc with an affinity of approximately 1000 nM to approximately 6000 nM, (b) The first binding region binds to TfR with an affinity of less than approximately 250 nM, and the second binding region binds to CD98hc with an affinity of approximately 300 nM to approximately 900 nM, (c) The first binding region binds to TfR with an affinity of about 50 nM to about 250 nM, and the second binding region binds to CD98hc with an affinity of about 300 nM to about 900 nM, (d) The first binding region binds to TfR with an affinity of less than approximately 250 nM, and the second binding region binds to CD98hc with an affinity of less than approximately 250 nM, or (e) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 50 nM to about 250 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to about 250 nM.

112. (a) The first binding region binds to TfR with an affinity of about 300 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 1000 nM to about 6000 nM, (b) The first binding region binds to TfR with an affinity of about 300 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 900 nM to about 6000 nM, (c) The first binding region binds to TfR with an affinity of about 300 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 300 nM to about 900 nM, (d) The first binding region binds to TfR with an affinity of about 300 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of less than about 250 nM, (e) The first binding region binds to TfR with an affinity of about 300 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to 250 nM, or (f) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 300 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to 900 nM.

113. (a) The first binding region binds to TfR with an affinity of about 50 nM to about 1000 nM, and the second binding region binds to CD98hc with an affinity of about 1 nM to about 500 nM, (b) The first binding region binds to TfR with an affinity of about 100 nM to about 600 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to about 300 nM, or (c) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 100 nM to about 400 nM, and the second binding region binds to CD98hc with an affinity of about 50 nM to about 200 nM.

114. (a) The first binding region binds to TfR with an affinity of about 250 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 1 nM to about 250 nM, (b) The first binding region binds to TfR with an affinity of about 250 nM to about 1000 nM, and the second binding region binds to CD98hc with an affinity of about 1 nM to about 100 nM, or (c) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 50 nM to about 250 nM, and the second binding region binds to CD98hc with an affinity of about 1 nM to about 100 nM.

115. (a) The first binding region binds to TfR with an affinity of about 1000 nM to about 6000 nM, and the second binding region binds to CD98hc with an affinity of about 1 nM to about 500 nM. (b) The first binding region binds to TfR with an affinity of about 250 nM to about 1000 nM, and the second binding region binds to CD98hc with an affinity of about 250 nM to about 1000 nM. (c) The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the first binding region binds to TfR with an affinity of about 50 nM to about 250 nM, and the second binding region binds to CD98hc with an affinity of about 500 nM to about 6000 nM.

116. The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the dual transporter binds to TfR with an affinity of about 50 nM to about 1000 nM and to CD98hc with an affinity about 5 to about 10 times lower than the affinity to TfR.

117. The dual transporter according to any one of claims 1 to 29, 33 to 51, 55 to 77, and 79 to 107, wherein the dual transporter binds to TfR with an affinity of about 50 nM to about 6000 nM and to CD98hc with an affinity about 5 to about 10 times higher than the affinity to TfR.

118. The dual transporter according to any one of claims 1 to 117, wherein the dual transporter can be actively transported across the blood-brain barrier.

119. The dual transporter according to any one of claims 1 to 117, wherein the dual transporter can promote the transport of a therapeutic agent across the blood-brain barrier.

120. The aforementioned therapeutic agent may be ABCA1, ABCA7, ADAM17, ALK, alpha-synuclein or its derivatives or fragments, amyloid-beta peptide or its derivatives or fragments, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD25, CD30, CD33, CD37, CD39, CD44v6, CD46, CD56 (NCAM), CD73, CD79b, CDH6 (cadherin 6), CEACAM5 (CD66E), CR1, EGFR viiii, EGFR, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, HLA-DR1, HLA-DR5, Huntinthin, IGFR1, IL1RAP, IL-34, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, MS4A4A, MS4A4E, MS4A6A, NaPi2B, TAR The dual transporter according to claim 119, which targets DNA-binding protein 43 (TDP-43) or its derivatives or fragments, PDL1, PILRA, PMEL17, PRAME, PSMA, pTau, PTK7 (CCK4; colon cancer kinase), RON, ROR1, Siglec11, SORL1, Tau or its derivatives or fragments, TF (tissue factor), transthyretin, TREM2, TREML2, TROP2, or tumor cells.

121. A nucleic acid encoding a dual transporter according to any one of claims 1 to 120.

122. A vector comprising the nucleic acid described in claim 121.

123. A host cell expressing the dual transporter according to any one of claims 1 to 120.

124. The host cell according to claim 110, wherein the cell comprises the nucleic acid according to claim 121 or the vector according to claim 121.

125. A method for producing a dual transporter, (a) Culturing a host cell containing one or more nucleic acids encoding a dual transporter according to any one of claims 1 to 120 under conditions in which the dual transporter encoded by the nucleic acid or vector is expressed, and (b) The method comprising isolating the dual transporter from the culture.

126. A composition comprising the dual transporter according to any one of claims 1 to 120.

127. A pharmaceutical composition comprising a dual transporter according to any one of claims 1 to 120 or the composition according to claim 126, and a pharmaceutically acceptable excipient.

128. A method for delivering a molecule to the brain of a target, comprising administering to the target a dual transporter according to any one of claims 1 to 120, a composition according to claim 126, or a pharmaceutical composition according to claim 127, wherein the dual transporter is linked to the molecule.

129. The method according to claim 128, wherein the molecule comprises a therapeutic agent.

130. A method for delivering a molecule into the brain of a target via a blood-brain barrier (BBB), comprising administering to the target a dual transporter according to any one of claims 1 to 120, a composition according to claim 126, or a pharmaceutical composition according to claim 127, wherein the molecule is linked to the dual transporter.

131. The method according to claim 130, wherein the molecule comprises a therapeutic agent.

132. The aforementioned therapeutic agent may be ABCA1, ABCA7, ADAM17, ALK, alpha-synuclein or its derivatives or fragments, amyloid-beta peptide or its derivatives or fragments, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD25, CD30, CD33, CD37, CD39, CD44v6, CD46, CD56 (NCAM), CD73, CD79b, CDH6 (cadherin 6), CEACAM5 (CD66E), CR1, EGFR viiii, EGFR, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, HLA-DR1, HLA-DR5, Huntinthin, IGFR1, IL1RAP, IL-34, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, MS4A4A, MS4A4E, MS4A6A, NaPi2B, TAR The method according to claim 130 or 131, which targets DNA-binding protein 43 (TDP-43) or its derivatives or fragments, PDL1, PILRA, PMEL17, PRAME, PSMA, pTau, PTK7 (CCK4; colon cancer kinase), RON, ROR1, Siglec11, SORL1, Tau or its derivatives or fragments, TF (tissue factor), transthyretin, TREM2, TREML2, TROP2, or tumor cells.

133. A method for treating a central nervous system (CNS) disease or condition in a subject, comprising administering to the subject a dual transporter according to any one of claims 1 to 120, the composition according to claim 126, or the pharmaceutical composition according to claim 127.

134. The method according to claim 133, wherein the disease or condition of the CNS includes a neurological disease or condition, a neurodegenerative disease or condition, cancer, or an infection.

135. The method according to claim 133, wherein the subject is suffering from or has been diagnosed with a neurological disease or condition, a neurodegenerative disease or condition, cancer of the CNS or brain, or an infection of the CNS or brain.

136. A method for manufacturing a dual transporter, (a) (i) a first polypeptide comprising a first binding region known to specifically bind to a BBB transport protein, and (ii) a second polypeptide comprising a second binding region known to specifically bind to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transport protein, and (b) Forming a single molecule comprising the first polypeptide and the second polypeptide, thereby forming the dual transporter, The method wherein the dual transporter has increased and / or prolonged brain exposure in animals compared to a corresponding control molecule that binds only the BBB transport protein or only the second protein.

137. The method according to claim 136, wherein the BBB transport protein is TfR or CD98hc.

138. The method according to claim 136, wherein the BBB transport protein is TfR.

139. The second protein mentioned above includes TfR, CD98hc, large neutral amino acid transporter small subunit 1 (CD98 light chain), GLUT1, MFSD2A, CA-IV, low-density lipoprotein receptor, IGF1R, insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 50A, basidine, leptin receptor, claudin-5, P-selectin, lactoferrin receptor, and folic acid receptor. The method according to any one of claims 136 to 138, selected from the group consisting of body, sodium-dependent lysophosphatidylcholine cotransporter 1, solute transporter organic anion transporter family member 1C1, sodium-conjugated neutral amino acid transporter 5, LDL receptor-related protein 8, high affinity cationic amino acid transporter 1, sodium chloride-dependent taurine transporter, insulin-like growth factor binding protein 7, solute transporter family 40 member 1, zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and MOG.

140. The method according to claim 138 or 139, wherein the second protein is CD98hc.

141. The method according to claim 140, wherein the first binding domain includes a first binding domain as described in any one of claims 9 to 10, 13 to 29, 34, and 45 to 72, and the second binding domain includes a second binding domain as described in any one of claims 11 to 29, 35, and 73 to 94.

142. The method according to claim 136 or 137, wherein the second binding region specifically binds to CD98hc.

143. (a) (i) a first polypeptide comprising a first binding region known to specifically bind to a BBB transport protein, and (ii) a second polypeptide comprising a second binding region known to specifically bind to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transport protein. (b) To form a single molecule encompassing the first polypeptide and the second polypeptide, (c) A method for identifying a dual transporter, comprising measuring the concentration of the single molecule in the brain at a predetermined time, and identifying the single molecule as a dual transporter if it is present in the brain at a higher concentration than a corresponding control molecule that binds only to the BBB transporter protein or the second protein.

144. The method according to claim 143, further comprising peripherally administering the single molecule to an animal prior to step (c).

145. The method according to claim 144, wherein the animal is a non-human animal, and optionally the animal is a mouse, a rat, or a non-human primate.

146. The method according to any one of claims 143 to 145, wherein higher concentrations in the brain include an increased rate of transport to the brain or an extended brain exposure.

147. The method according to any one of claims 143 to 146, further comprising determining the affinity or binding strength of the first binding region to the BBB transport protein and determining the affinity or binding strength of the second binding region to the second protein.

148. The method according to any one of claims 146 and 147, wherein the BBB transport protein is TfR or CD98hc.

149. The method according to claim 148, wherein the BBB transport protein is TfR.

150. The second protein mentioned above is TfR, CD98hc, CD98lc, GLUT1, MFSD2A, CA-IV, low-density lipoprotein receptor, IGF1R, insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 2, insulin receptor, cell cycle regulatory protein 50A, transmembrane protein 50A, basidine, leptin receptor, claudin-5, P-selectin, lactoferrin receptor, folate receptor, sodium-dependent lysophosphate The method according to any one of claims 143 to 149, selected from the group consisting of sphatidylcholine cotransporter 1, solute transporter organic anion transporter family member 1C1, sodium-conjugated neutral amino acid transporter 5, LDL receptor-related protein 8, high affinity cationic amino acid transporter 1, sodium chloride-dependent taurine transporter, insulin-like growth factor-binding protein 7, solute transporter family 40 member 1, zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and MOG.

151. The method according to claim 149 or 150, wherein the second protein is CD98hc.

152. The method according to claim 151, wherein the first binding domain includes a first binding domain as described in any one of claims 9 to 10, 13 to 29, 34, and 45 to 72, and the second binding domain includes a second binding domain as described in any one of claims 11 to 29, 35, and 73 to 94.

153. The method according to any one of claims 143 to 152, further comprising measuring the binding of the single molecule to the BBB transport protein and / or the second protein.

154. The method according to claim 153, wherein the coupling is measured using surface plasmon resonance, ELISA, or flow cytometry.

155. The method according to claim 153 or 154, wherein the binding is measured on the surface of a cell expressing the BBB transport protein, the second protein, or both on its surface.

156. A method for improving the delivery of a therapeutic agent to the central nervous system, wherein the therapeutic agent is linked to a delivery vehicle having a first binding domain that specifically binds to a BBB transport protein, the method comprising linking the delivery vehicle to a second binding domain that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transport protein.

157. A method for improving the delivery of a delivery vehicle having a first binding domain that specifically binds to a BBB transport protein to the central nervous system, comprising linking the delivery vehicle to a second binding domain that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is different from the BBB transport protein.

158. The method according to claim 156 or 157, wherein the BBB transport protein is TfR or CD98hc.

159. The method according to claim 156 or 157, wherein the BBB transport protein is TfR and the second protein is CD98hc.

160. A method for improving the delivery of a therapeutic agent to the central nervous system, wherein the therapeutic agent is linked to a polypeptide that specifically binds to TfR, comprising linking the polypeptide that specifically binds to TfR to a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is not TfR.

161. A method for improving the delivery of a therapeutic agent to the central nervous system, wherein the therapeutic agent is linked to a polypeptide that specifically binds to CD98hc, the method comprising linking the polypeptide that specifically binds to CD98hc to a second binding region that specifically binds to a second protein, wherein the second protein is expressed on the luminal surface of the BBB or is a brain-retained protein, and the second protein is not CD98hc.

Images