Transferrin receptor binding protein

Abstract

The present disclosure provides antibodies and antigen-binding fragments that target the transferrin receptor, as well as the use of these antibodies and antigen-binding fragments as carriers for delivering therapeutic molecules across the blood-brain barrier.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application No. 22306784.4, filed December 5, 2022. The disclosure of that priority application is incorporated herein by reference in its entirety.

[0002] Sequence Listing

[0001] This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The electronic copy of the Sequence Listing (created on December 3, 2023) is titled 122548.WO005.xml and is 92,235 bytes in size. [Background technology]

[0003] Transferrin receptor 1 (TfR), also known as CD71, is a ubiquitously expressed transmembrane glycoprotein involved in the cellular uptake of iron. TfR uptakes iron via receptor-mediated endocytosis of the iron-binding protein, transferrin. TfR is highly expressed by brain capillary endothelial cells that form the blood-brain barrier (BBB) ​​and transports iron across the BBB via transcytosis. Therefore, TfR has been explored as a potential target for molecular shuttles designed to transport macromolecular drugs across the BBB (see, for example, Bourassa et al., Mol Pharm. (2019) 16(2):583-94).

[0004] There are several notable challenges in using TfR as a target for molecular shuttles. First, the presence of high blood levels of transferrin may require the shuttle to compete with transferrin for binding to TfR. Other challenges include specificity for brain tissue, potential lysosomal degradation, and significant transport to the brain parenchyma (Pulgar, Front Neurosci. (2019) 12: doi10.3389 / fnins.2018.01019). There remains a need for TfR-targeted molecular shuttles with sufficient brain specificity and efficient uptake. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides an anti-human transferrin receptor (TfR) antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR (HCDR)1 comprising GYTFTRYY (SEQ ID NO: 26), or GYTFTRYW (SEQ ID NO: 27), or DYTFTRYW (SEQ ID NO: 5), an HCDR2 comprising IDPSVSET (SEQ ID NO: 28) or IDPSVSEC (SEQ ID NO: 6), and an HCDR3 comprising SQIRLPYYYAMDS (SEQ ID NO: 7); and a light chain CDR (LCDR)1 comprising QDISSF (SEQ ID NO: 29) or QDINSF (SEQ ID NO: 9), an LCDR2 comprising YTS (SEQ ID NO: 10), and optionally an LCDR3 comprising QQGNTLPRT (SEQ ID NO: 11).

[0006] In another aspect, the disclosure provides an isolated TfR binding protein comprising (i) an antibody or antigen-binding fragment thereof, and (ii) a cargo linked thereto. In some embodiments, the cargo is a therapeutic compound (e.g., a protein such as an enzyme, e.g., a lysosomal enzyme). In particular embodiments, the enzyme is acid α-glucosidase (GAA).

[0007] Also provided is a pharmaceutical composition comprising the antibody, antigen-binding fragment, or TfR-binding protein; and a pharmaceutically acceptable excipient.

[0008] In another aspect, the disclosure provides nucleic acids and expression vectors encoding the present antibodies, antigen-binding fragments, and TfR-binding proteins, host cells containing such nucleic acids or expression vectors, and methods of producing the antibodies, antigen-binding fragments, and TfR-binding proteins herein using the host cells. In some embodiments, the method of production comprises culturing the host cells under conditions that allow expression of the antibody or antigen-binding fragment or TfR-binding protein, and isolating the antibody or antigen-binding fragment or TfR-binding protein from the cell culture.

[0009] In another aspect, the disclosure provides a method of making a therapeutic molecule capable of crossing the BBB in a human subject, the method comprising linking (e.g., chemically or recombinantly) the therapeutic portion of the molecule to an antibody or antigen-binding fragment.

[0010] In another aspect, the present disclosure provides a method of delivering a therapeutic molecule across the BBB in a subject in need thereof, comprising administering a therapeutic molecule to the subject, wherein the therapeutic molecule is linked to an antibody or antigen-binding fragment herein. Also provided are antibodies or antigen-binding fragments herein for use in delivering a therapeutic molecule across the BBB in a subject in need thereof. Further provided is use of an antibody or antigen-binding fragment herein for delivering a therapeutic molecule across the BBB in a subject in need thereof, or in the manufacture of a medicament for said purpose.

[0011] In another aspect, the disclosure provides a method of treating a subject in need thereof, the method comprising administering an isolated TfR binding protein herein comprising an enzyme (e.g., a lysosomal enzyme), wherein the subject is deficient in the enzyme or its activity. Also provided are isolated TfR binding proteins herein comprising an enzyme (e.g., a lysosomal enzyme) for use in treating a subject deficient in the enzyme or its activity. Also provided are uses of isolated TfR binding proteins herein comprising an enzyme (e.g., a lysosomal enzyme) for treating, or for the manufacture of a medicament for treating, a subject deficient in the enzyme or its activity. In some embodiments, the enzyme is GAA, and the subject is suffering from Pompe disease.

[0012] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. It should be understood, however, that this detailed description, while indicating embodiments and aspects of the present invention, is given by way of example only, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0013] [Figure 1A] Schematic diagram showing the amino acid residues of the human TfR (FIG. 1A) and cynomolgus monkey TfR (FIG. 1B) with atoms within 4 angstroms of 531v25 Fab atoms as determined by cryo-electron microscopy. [Figure 1B] Schematic diagram showing the amino acid residues of the human TfR (FIG. 1A) and cynomolgus monkey TfR (FIG. 1B) with atoms within 4 angstroms of 531v25 Fab atoms as determined by cryo-electron microscopy. [Figure 2A] 1 is a panel of schematic diagrams showing three exemplary formats of anti-TfR binding proteins. "mAb": monoclonal antibody. Fab: Fab fragment of an intact antibody. "Fab-FcOL": a monovalent antibody fragment having a light chain, an intact heavy chain, and an Fc polypeptide that dimerizes with the intact heavy chain to form a dimeric Fc domain. [Figure 2B]1 is a panel of schematic diagrams showing four exemplary formats of anti-TfR binding proteins containing a GAA cargo: "Fab-LC-GAA": GAA fused to the C-terminus of the light chain (LC) of an anti-TfR Fab; "Fab-FcOL-LC-GAA": GAA fused to the C-terminus of the LC of an anti-TfR Fab-FcOL; "Fab-FcOL-GAA": GAA fused to the C-terminus of the Fc polypeptide of an anti-TfR Fab-FcOL; and "mAb-GAA": GAA fused to the C-terminus of one of the two heavy chains (HC) of an anti-TfR mAb. [Figure 3A] Figure 1 shows an alignment of two exemplary humanized 531 heavy chains (531 v1 VH and 531 v2 VH) with their corresponding human germline sequences, IGHV3-23*05 and IGHJ6-01. HCDRs 1-3 are underlined. Mutations in HCDR1 are shown in bold. [Figure 3B] Figure 1 shows an alignment of three additional exemplary humanized 531 heavy chains (531 v3 VH, 531 v4 VH, and 531 v5 VH) with their corresponding human germline sequences IGHV1-46*01 and IGHJ6-01. HCDRs 1-3 are underlined. Mutations in HCDR1 are shown in bold. [Figure 3C] Alignment of four exemplary humanized 531 light chains (531 v1 VL, 531 v2 VL, 531 v3 VL, and 531 v4 VL) with their corresponding human germline sequences IGKV1-39*01 and IGKJ4-01. LCDRs 1-3 are underlined. Mutations in LCDR1 are shown in bold. [Figure 4A-B]Graphs showing glycogen levels (mean ± SD) in the brain (FIG. 4A), spinal cord (FIG. 4B), heart (FIG. 4C), diaphragm (FIG. 4D), gastrocnemius (FIG. 4E), quadriceps (FIG. 4F), and triceps (FIG. 4G) of experimental animals administered various 531v25-GAA constructs. Asterisks: genders combined. "f": females (only if significantly different). "m": males (only if significantly different). Dark (top) asterisk: p<0.05 vs. vehicle. Light (bottom) asterisk: p<0.05 vs. neoGAA. "GAA": recombinant GAA without carrier. [Figure 4C-D] Same as above. [Figure 4E-G] Same as above. [Figure 5] This is a listing of the amino acid sequences for the three subunits of the 531v25 Fab-FcOL-GAA molecule. The protein is of the human IgG1 isotype. In the light chain (Fab-FcOL-GAA light chain), LCDRs 1-3 are shown in bold, and the constant region is underlined. In the complete heavy chain (Fab-FcOL-GAA heavy chain_HOLE), HCDRs 1-3 are shown in bold; constant regions are underlined; NNAS (SEQ ID NO: 53) mutations are double underlined (linear positions of S301N / T302A / Y303S corresponding to S298N / T299A / Y300S EU numbering); hole mutations are shown in italics and bold (linear positions of Y352C / T369S / L371A / Y410V corresponding to Y349C / T366S / L368A / Y407V EU numbering); RF mutations are shown in bold and double underlined (linear positions of H438R / Y439F corresponding to H435R / Y436F EU numbering). In the Fc polypeptide / GAA fusion (Fab-FcOL-GAA FcOL_KNOB+GAA), the Fc polypeptide positions are underlined, the GGGG (SEQ ID NO: 42) linker is underlined in bold; the NNAS mutation is double underlined (linear position of S78N / T79A / Y80S corresponding to S298N / T299A / Y300S Eu numbering); and the knob mutation is shown in italics and bold (linear position of S134C / T146W corresponding to S354C / T366W Eu numbering). Linear positions refer to the actual positions in the sequences shown. [Figure 6] 1 is a series of graphs showing tissue-specific knockdown of MALAT1 mRNA in the brain, heart, gastrocnemius muscle, quadriceps muscle, spleen, and sciatic nerve of experimental animals administered anti-hTfR-MALAT1-ASO conjugate ("anti-hTfR-ASO") compared to free MALAT1-ASO ("ASO") or vehicle (saline). *=p<0.05, **=p<0.005, ***=p<0.0001 vs. vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure provides isolated binding proteins, such as antibodies and antigen-binding fragments thereof, that bind to human TfR (hTfR). These TfR-binding proteins bind to epitopes on the extracellular domain of hTfR and do not interfere with the interaction between hTfR and its natural ligand, transferrin. The TfR-binding proteins are excellent BBB transporters and have improved transcytosis efficiency. Furthermore, the TfR-binding proteins cross-react with cynomolgus monkey TfR (cTfR), thus enabling preclinical testing of proteins in non-human primates (NHPs).

[0015] The present disclosure also provides specific uses of the BBB transporters for transporting enzymes, e.g., lysosomal enzymes such as acid alpha-glucosidase (GAA), to the central nervous system (CNS). The GAA-loaded transporters can be used to restore CNS (e.g., brain and spinal cord) and muscle glycogen to normal levels in patients in need thereof (e.g., Pompe disease patients). The GAA-loaded transporters may exhibit superior glycogen clearance in the CNS compared to recombinant GAA used in conventional enzyme replacement therapy.

[0016] The BBB transporters can also be used to transport oligonucleotides, such as antisense oligonucleotides or siRNA, into the central nervous system (CNS). Such oligonucleotide-loaded transporters can be used, for example, to knock down specific mRNA targets in the CNS for therapeutic purposes.

[0017] I. TfR-binding proteins The present disclosure provides hTfR binding proteins, including anti-hTfR antibodies and antigen-binding fragments thereof (collectively also referred to herein as "BBB transporters"), as well as hTfR binding proteins comprising such antibodies or antigen-binding fragments and cargo to be transported across the BBB.

[0018] A.TfR binding characteristics The human TfR is a homodimer composed of two disulfide-linked subunits. An exemplary human TfR amino acid sequence can be found at UniProt accession number P02786 and NCBI accession number NP001121620.1, and has the following amino acid sequence: [ka]

[0019] Amino acid residues 1-61 are cytoplasmic. Amino acid residues 62-89 are transmembrane. Amino acid residues 90-760 are extracellular. The extracellular portion (extracellular domain) has three domains: a helical domain (residues 606-760), a protease-like domain (residues 121-183, 384-605), and an apical domain (residues 184-383) (Sjoestroem, D., Linnaeus University Dissertations, No. 406 / 2021; Lawrence et al., Science (1999) 286(5440):779-82).

[0020] The antibody or antigen-binding fragment binds to an epitope located in a region of the protease-like domain (residues 384-605) of hTfR that corresponds to the lateral region of the receptor. This represents a unique epitope distinct from known anti-TfR antibodies that bind to the apical domain of hTfR (see, e.g., antibodies 3 and 3N disclosed in EP 3088518 A1, which are shown in WO 2022 / 174114 to bind to the apical region of hTfR). The TfR-binding protein does not compete with transferrin for binding to TfR.

[0021] In some embodiments, the TfR binding proteins bind to at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of K261, K358, T491, S492, N493, K495, H515, V517, T518, Q520, Q524, D525, N527, S530, K531, E533, D560, and E582 of hTfR (SEQ ID NO: 1) (see Figure 1A). In certain embodiments, UCSF Chimera software (available on the University of California, San Francisco server) is used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues. The term "paratope" refers to residues of an antibody that are involved in recognizing and binding to an epitope of an antigen. In some embodiments, a paratope / epitope pair may be characterized, for example, by the distance (e.g., within 4 Å or 5 Å) between the antibody and antigen atoms in the bound antibody / antigen complex. In some embodiments, a paratope / epitope pair may be characterized by participation in hydrogen bonding interactions and / or salt bridging interactions between the antibody and antigen residues.

[0022] In some embodiments, the TfR binding protein binds to at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) or all of K261, K358, T491, S492, N493, F494, K495, H515, V517, T518, Q520, L522, Q524, D525, N527, S530, K531, E533, D560, and E582 of hTfR. In certain embodiments, PISA software (available at the European Bioinformatics Institute server) and / or UCSF ChimeraX (available at the University of California, San Francisco server) are used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues.

[0023] In some embodiments, the TfR binding protein binds to at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) or all of K261, K287, K358, T491, S492, N493, F494, K495, M510, H515, V517, T518, Q520, F521, L522, Y523, Q524, D525, N527, S530, K531, V532, E533, E559, D560, D562, and E582 of hTfR. In certain embodiments, UCSF Chimera software is used for visualization of the paratope / epitope structure and determination of distances (in Å) and interactions between paratope and epitope residues.

[0024] In some embodiments, the TfR binding proteins bind to an epitope that resides in whole or in part in the region spanning T491 to D562 of the hTfR, and thus bind to one or more residues in this region.

[0025] In some embodiments, the subject TfR binding proteins also bind to TfRs of non-human primates (NHPs), such as macaques (e.g., Macaca fascicularis, also known as cynomolgus monkeys). An exemplary amino acid sequence of cynomolgus monkey TfR (cTfR) can be found at NCBI Accession No. XP_045243212.1 and is shown below: [ka]

[0026] In some embodiments, the TfR binding proteins bind to at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of K261, K358, T491, S492, N493, K495, H515, V517, T518, R520, Q524, D525, N527, S530, K531, E533, D560, and E582 of TfR (see Figure 1B). These residues are identical between hTfR and cTfR, except for the Q520R substitution from hTfR to cTfR. In certain embodiments, UCSF Chimera software is used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues.

[0027] In some embodiments, the TfR binding proteins bind to at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of K261, K358, T491, S492, N493, F494, K495, H515, V517, T518, R520, L522, Q524, D525, N527, S530, K531, E533, D560, and E582 of cTfR (see Figure 1B). These residues are identical between hTfR and cTfR, except for the Q520R substitution from hTfR to cTfR. In certain embodiments, PISA and / or UCSF ChimeraX software is used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues.

[0028] In some embodiments, the TfR binding protein binds to at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) or all of K261, K287, K358, T491, S492, N493, F494, K495, M510, H515, V517, T518, R520, S521, L522, Y523, Q524, D525, N527, S530, K531, V532, E533, E559, D560, D562, and E582 of cTfR. These residues are identical between hTfR and cTfR, except for the Q520R and F521S substitutions from hTfR to cTfR. In certain embodiments, UCSF Chimera software is used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues.

[0029] Residues of hTfR and / or cTfR bound by the present TfR-binding proteins can be identified using, for example, UCSF Chimera, PISA, or UCSF ChimeraX software, or any combination thereof.

[0030] In some embodiments, the TfR binding proteins bind to an epitope that resides in whole or in part in the region spanning T491 to D562 of the cTfR, and thus bind to one or more residues in this region.

[0031] The extracellular domains of hTfR and cTfR (residues 90-760) are 95% identical and 97% homologous to each other. An alignment between the two extracellular domains is shown below. In the alignment, an "*" (asterisk) indicates a position with a single, completely conserved residue; a ":" (colon) indicates conservation between groups of strongly similar properties (scores >0.5 in the Gonnet PAM 250 matrix), and a "." (period) indicates conservation between groups of weakly similar properties (scores ≤0.5 in the Gonnet PAM 250 matrix). The region forming the protease-like domain (Sjoeström, supra) is underlined. [ka]

[0032] In some embodiments, the TfR-binding protein binds to residues conserved between the hTfR and cTfR extracellular domains. In further embodiments, the TfR-binding protein binds to residues conserved between the protease-like domains of the hTfR and cTfR extracellular domains. In further embodiments, the TfR-binding protein binds to one or more residues in a region spanning positions 384-605 of the hTfR (e.g., one or more residues in a region spanning positions 491-562), e.g., residues conserved between the hTfR and cTfR in this region. In some embodiments, the TfR-binding protein binds to at least one or all of the boxed residues in the above alignment.

[0033] The present anti-TfR antibodies and antigen-binding fragments thereof specifically bind to hTfR and cTfR. By "specifically," we mean that the antibodies and fragments bind to hTfR and cTfR with the affinities described herein or higher. To function as a carrier across the BBB, BBB transporters may have suitable affinity for hTfR. Furthermore, to facilitate preclinical testing of BBB transporters in NHP animal models, BBB transporters may have suitable affinity for cTfR, and the difference (ratio) between the affinity of BBB transporters for cTfR and their affinity for hTfR may be within about 1 log. Several techniques, such as surface plasmon resonance (SPR, e.g., using BIAcore™) or biolayer interferometry (BLI, e.g., using Octet™ from ForteBio), can be used to measure TfR binding affinity (K D Flow cytometry assays (e.g., FACS) using cells expressing membrane-bound hTfR or cTfR can also characterize the EC of BBB transporters. 50 or IC 50 These values ​​are indicative of binding to human and cynomolgus TfR in their native conformations.

[0034] In some embodiments, the BBB transporter has a K of about 1-50 nM (e.g., 1-30, 1-20, or 1-10 nM) for hTfR as determined by SPR (e.g., using BIAcore™). D and a K for cTfR of about 1 to 200 nM (e.g., 1 to 150 or 1 to 100 nM). DIn some embodiments, the SPR assay is performed using the extracellular domain of the TfR being evaluated. Such an assay is exemplified in Example 2B below. In some embodiments, the ratio of binding affinity of the BBB transporter for hTfR compared to cTfR is 1:1 to 1:20, 1:1 to 1:15, 1:1 to 1:10, 1:1 to 1:9, 1:2 to 1:9, 1:3 to 1:9, 1:4 to 1:9, 1:5 to 1:9, or 1:6 to 1:9 (e.g., about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20).

[0035] In some embodiments, the BBB transporter has a K of about 1-50 nM (e.g., 1-30, 1-20, or 1-10 nM) for hTfR as determined by BLI (e.g., using Octet®). D and a K for cTfR of about 1 to 200 nM (e.g., 1 to 150 or 1 to 100 nM). D In some embodiments, BLI is performed using the extracellular domain of the TfR being evaluated. Such an assay is further exemplified in Example 3A, below. In some embodiments, the ratio of binding affinity of the BBB transporter for hTfR compared to cTfR is 1:1 to 1:20, 1:1 to 1:15, 1:1 to 1:10, 1:1 to 1:9, 1:2 to 1:9, 1:3 to 1:9, 1:4 to 1:9, 1:5 to 1:9, or 1:6 to 1:9 (e.g., about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20).

[0036] In some embodiments, the BBB transporter has an EC of about 1-50 nM (e.g., 1-30, 1-20, or 1-10 nM) for hTfR as determined by flow cytometry (e.g., FACS) using mammalian cells modified to express the TfR being evaluated. 50and an EC for cTfR of about 1-200 nM (e.g., 1-150 or 1-100 nM). 50 In some embodiments, the mammalian cells can be mouse cells (e.g., mouse pre-B cells), hamster cells, or human cells. Such assays are further exemplified in Example 5A below. In some embodiments, the EC of BBB transporters for hTfR compared to cTfR is 50 The ratio is 1:1 to 1:20, 1:1 to 1:15, 1:1 to 1:10, 1:1 to 1:9, 1:2 to 1:9, 1:3 to 1:9, 1:4 to 1:9, 1:5 to 1:9, or 1:6 to 1:9 (e.g., about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20).

[0037] BBB transporters having any combination of the above functional properties are also contemplated.

[0038] Exemplary formats of TfR binding proteins are shown in Figure 2A (antibodies and fragments thereof) and Figure 2B (cargo-loaded TfR binding proteins). The formats are further described below.

[0039] B. Anti-TfR Antibodies and Antigen-Binding Fragments TfR-binding proteins herein include chimeric or humanized anti-TfR antibodies and antigen-binding fragments having a murine-derived antigen-binding domain, which can be used as transporters to transport cargo (payload) across the BBB.

[0040] The term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region or domain (V H ) and heavy chain constant region (C H Each light chain is composed of a light chain variable region or domain (V L) and the light chain constant region (CL). H and V L Domains can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs (HCDR herein refers to the CDR from the heavy chain; LCDR herein refers to the CDR from the light chain) and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0041] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al., 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) ("Kabat" system); Al-Lazikani et al., J Mol Biol. (1997) 273:927-48) ("Chothia" system); MacCallum et al., J Mol Biol. (1996) 262:732-45 ("contact" system); Lefranc et al., Dev Comp Immunol. (2003) 27(1):55-77 ("IMGT" system); Honegger and Plueckthun, J Mol Biol. (2001) 309(3):657-70 ("Aho" system); and Whitelegg and Rees, Protein Eng. (2000) 13(12):819-24 (the "AbM" system). The boundaries of a given CDR or FR may vary depending on the system used. For example, the Kabat system is based on sequence alignment, while the Chothia system is based on structural information. Numbering for both the Kabat and Chothia systems is based on the most common antibody region sequence length, and insertions are accommodated by an insertion letter, e.g., "30a." The two systems place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The contact system is based on the analysis of complex crystal structures and is similar in many respects to the Chothia system. In certain embodiments, the CDRs of the antibodies described herein may be defined by a system selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof. Unless otherwise specified, the CDRs herein are defined by the IMGT system.

[0042] In some embodiments, the BBB transporters herein have a structure different from that of a complete antibody, which has two full-length heavy chains and two full-length light chains. For example, a BBB transporter may be an antigen-binding fragment of a complete tetrameric antibody, yet retain the TfR-binding properties of the complete antibody. The term "antigen-binding fragment" or "antigen-binding portion" herein encompasses genetically engineered and / or otherwise modified forms of immunoglobulins that do not have the traditional full-length tetrameric structure. This term encompasses intrabodies, peptibodies, diabodies, triabodies, tetrabodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-chain antibody molecules (e.g., scFv or sFv), tandem di-scFv, and tandem tri-scFv. Exemplary anti-TfR antigen-binding fragments include the Fab fragment and Fab-FcOL fragment described herein.

[0043] In some embodiments, the anti-TfR antibody herein is a humanized antibody. A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs (e.g., mouse) and all or substantially all FR amino acid residues are derived from human FRs (i.e., acceptor). A humanized antibody may also comprise at least a portion of an antibody heavy and / or light chain constant region derived from a human antibody. Compared to the non-human parent antibody from which the humanized antibody is derived, a humanized antibody has reduced immunogenicity in humans. To retain the specificity and affinity of the parent antibody, some FR residues in the human acceptor may be substituted with the corresponding residues from the non-human parent antibody (backmutation).

[0044] 1. TfR binding domain The TfR-binding domain of a BBB transporter (including an anti-TfR antibody or an antigen-binding fragment thereof) is an antibody heavy chain variable domain (V) in which heavy chain CDRs (HCDRs) 1 to 3 and light chain CDRs (LCDRs) 1 and 2 are derived from a mouse parent anti-TfR antibody. H ) and antibody light chain variable domain (V L In certain embodiments, HCDR1-3 and LCDR1-3 are derived from a murine parent anti-TfR antibody.

[0045] In some embodiments, the BBB transporters herein comprise an HCDR1 comprising X1YTFTRYX2, where optionally, X1 can be G or D and X2 can be W or Y (SEQ ID NO: 50). For example, HCDR1 can be GYTFTRYY (SEQ ID NO: 26), or GYTFTRYW (SEQ ID NO: 27), or DYTFTRYW (SEQ ID NO: 5).

[0046] In some embodiments, the BBB transporters herein comprise an HCDR2 comprising IDPSVSEX3, and optionally, X3 can be T or C (SEQ ID NO: 51). For example, HCDR2 can comprise IDPSVSET (SEQ ID NO: 28) or IDPSVSEC (SEQ ID NO: 6).

[0047] In some embodiments, the BBB transporter herein comprises an HCDR3 comprising SQIRLPYYYAMDS (SEQ ID NO: 7).

[0048] In some embodiments, the BBB transporter herein comprises an LCDR1 comprising QDIX4SF, and optionally, X4 can be S or N (SEQ ID NO: 52). For example, LCDR1 can comprise QDISSF (SEQ ID NO: 29) or QDINSF (SEQ ID NO: 9).

[0049] In some embodiments, the BBB transporter herein comprises an LCDR2 comprising YTS (SEQ ID NO: 10).

[0050] In some embodiments, the BBB transporter herein comprises an LCDR3 comprising QQGNTLPRT (SEQ ID NO: 11).

[0051] In some embodiments, the BBB transporter comprises a) HCDRs 1-3 and b) LCDRs 1 and 2 or LCDRs 1-3, as described in the preceding paragraphs. That is, HCDRs 1-3 comprise SEQ ID NOs: 26, 28, and 7, respectively; SEQ ID NOs: 27, 28, and 7, respectively; or SEQ ID NOs: 5, 6, and 7, respectively; and / or i) LCDRs 1 and 2 comprise SEQ ID NOs: 29 and 10, respectively, or SEQ ID NOs: 9 and 10, respectively; or ii) LCDRs 1-3 comprise SEQ ID NOs: 29, 10, and 11, respectively; or SEQ ID NOs: 9, 10, and 11, respectively. In certain embodiments, the BBB transporter comprises (i) HCDRs 1-3 comprising SEQ ID NOs: 26, 28, and 7, respectively, and LCDRs 1 and 2 comprising SEQ ID NOs: 29 and 10, respectively, or (ii) HCDRs 1-3 comprising SEQ ID NOs: 5-7, respectively, and LCDRs 1 and 2 comprising SEQ ID NOs: 9 and 10, respectively. In certain embodiments, the BBB transporter comprises (i) HCDRs 1 to 3 comprising SEQ ID NOs: 26, 28, and 7, respectively, and LCDRs 1 to 3 comprising SEQ ID NOs: 29, 10, and 11, respectively, or (ii) HCDRs 1 to 3 comprising SEQ ID NOs: 5 to 7, respectively, and LCDRs 1 to 3 comprising SEQ ID NOs: 9 to 11.

[0052] In some embodiments, the BBB transporter comprises HCDR1-3, LCDR1 and LCDR2 as described in the paragraphs above, and further comprises a heavy chain framework (HFR) 1 whose last residue is serine (S), a light chain framework (LFR) 3 whose first residue is arginine (R), or both. For example, in certain embodiments, the BBB transporter comprises: HCDR1 comprising GYTFTRYY (SEQ ID NO: 26), GYTFTRYW (SEQ ID NO: 27), or DYTFTRYW (SEQ ID NO: 5); - HCDR2 comprising IDPSVSET (SEQ ID NO: 28) or IDPSVSEC (SEQ ID NO: 6); and - an HCDR3 comprising SQIRLPYYYAMDS (SEQ ID NO: 7); and LCDR1 comprising QDISSF (SEQ ID NO: 29) or QDINSF (SEQ ID NO: 9), - LCDR2 comprising YTS (SEQ ID NO: 10), and optionally, an LCDR3 comprising QQGNTLPRT (SEQ ID NO: 11) Including, Further included are HFR1s in which the last residue is S and / or LFR3s in which the first residue is R (eg, HFR1s in which the last residue is S and LFR3s in which the first residue is R). In certain embodiments, the BBB transporter is HCDR1 comprising GYTFTRYY (SEQ ID NO: 26), GYTFTRYW (SEQ ID NO: 27), or DYTFTRYW (SEQ ID NO: 5); - HCDR2 comprising IDPSVSET (SEQ ID NO: 28) or IDPSVSEC (SEQ ID NO: 6); and - an HCDR3 comprising SQIRLPYYYAMDS (SEQ ID NO: 7); and LCDR1 comprising QDISSF (SEQ ID NO: 29) or QDINSF (SEQ ID NO: 9), - LCDR2 comprising YTS (SEQ ID NO: 10), and LCDR3 containing QQGNTLPRT (SEQ ID NO: 11) Including, Further included are HFR1s in which the last residue is S and / or LFR3s in which the first residue is R (eg, HFR1s in which the last residue is S and LFR3s in which the first residue is R).

[0053] In some embodiments, HCDRs 1-3 comprise SEQ ID NOs: 26, 28, and 7, respectively; SEQ ID NOs: 27, 28, and 7, respectively; or SEQ ID NOs: 5, 6, and 7, respectively; LCDRS 1 and 2 comprise SEQ ID NOs: 29 and 10, respectively; or SEQ ID NOs: 9 and 10, respectively; HFR1 has a last residue of S and / or LFR3 has a first residue of R. In certain embodiments, a BBB transporter comprises HCDRs 1-3 comprising SEQ ID NOs: 26, 28, and 7, respectively; LCDRS 1 and 2 comprising SEQ ID NOs: 29 and 10, respectively; and HFR1 whose last residue is S and / or LFR3 whose first residue is R (e.g., HFR1 whose last residue is S and LFR3 whose first residue is R). In other embodiments, the BBB transporter comprises HCDRs 1-3 comprising SEQ ID NOs: 5-7, respectively; LCDRs 1 and 2 comprising SEQ ID NOs: 9 and 10, respectively; and HFR1 whose last residue is S and / or LFR3 whose first residue is R (e.g., HFR1 whose last residue is S and LFR3 whose first residue is R).

[0054] In some embodiments, a BBB transporter comprises HCDR1-3 and LCDR1-3 as described in the preceding paragraphs, and further comprises an HFR1 whose last residue is S, an LFR3 whose first residue is R, or both. For example, in certain embodiments, HCDR1-3 comprise SEQ ID NOs: 26, 28, and 7, respectively; SEQ ID NOs: 27, 28, and 7, respectively; or SEQ ID NOs: 5, 6, and 7, respectively; and LCDR1-3 comprise SEQ ID NOs: 29, 10, and 11, respectively; or SEQ ID NOs: 9-11, respectively; HFR1 has a last residue of S and / or LFR3 has a first residue of R. In certain embodiments, a BBB transporter comprises HCDR1-3 comprising SEQ ID NOs: 26, 28, and 7, respectively; LCDR1-3 comprising SEQ ID NOs: 29, 10, and 11, respectively; and an HFR1 whose last residue is S and / or an LFR3 whose first residue is R (e.g., an HFR1 whose last residue is S and an LFR3 whose first residue is R). In other embodiments, the BBB transporter comprises HCDR1-3 comprising SEQ ID NOs: 5-7, respectively; LCDR1-3 comprising SEQ ID NOs: 9-11, respectively; and HFR1 whose last residue is S and / or LFR3 whose first residue is R (e.g., HFR1 whose last residue is S and LFR3 whose first residue is R).

[0055] In some embodiments, the BBB transporter comprises HCDRs 1-3 of SEQ ID NOs: 26, 28, and 7, respectively, and LCDRs 1 and 2 of SEQ ID NOs: 29 and 10, respectively, wherein the HCDRs 1-3 and LCDRs 1 and 2 comprise a total of 1 to 5 (e.g., 1, 2, 3, 4, or 5, 1 to 2, 1 to 3, or 1 to 4) mutations across the five CDRs, wherein the mutations are at the following positions: Y2, R6, Y7, and optionally Y8 of SEQ ID NO: 26 (HCDR1); D2, S4, V5, and E7 of SEQ ID NO: 28 (HCDR2); R4, L5, P6, Y7, Y8, and Y9 of SEQ ID NO: 7 (HCDR3); F6 of SEQ ID NO: 29 (LCDR1), and Y1 of SEQ ID NO: 10 (LCDR2) does not occur in BBB transporters further include an HFR1 in which the last residue is S and / or an LFR3 in which the first residue is R (eg, an HFR1 in which the last residue is S and an LFR3 in which the first residue is R).

[0056] In some embodiments, the BBB transporter comprises HCDRs 1-3 of SEQ ID NOs: 26, 28, and 7, respectively, and LCDRs 1-3 of SEQ ID NOs: 29, 10, and 11, respectively, wherein the HCDRs 1-3 and LCDRs 1-3 comprise a total of 1-5 (e.g., 1, 2, 3, 4, or 5, 1-2, 1-3, or 1-4) mutations across the six CDRs, wherein the mutations are at the following positions: Y2, R6, Y7, and optionally Y8 of SEQ ID NO: 26 (HCDR1); D2, S4, V5, and E7 of SEQ ID NO: 28 (HCDR2); R4, L5, P6, Y7, Y8, and Y9 of SEQ ID NO: 7 (HCDR3); F6 of SEQ ID NO: 29 (LCDR1), and Y1 of SEQ ID NO: 10 (LCDR2) does not occur in BBB transporters further include an HFR1 in which the last residue is S and / or an LFR3 in which the first residue is R (eg, an HFR1 in which the last residue is S and an LFR3 in which the first residue is R).

[0057] In some embodiments, the BBB transporter, when bound to hTfR, V, as defined by IMGT numbering, includes: H : optionally, S26, which is within 4.0 Å of residues K261 and K358 of SEQ ID NO: 1; Y28, which is within 4.0 Å of residue K261 of SEQ ID NO:1; R36, which is within 4.0 Å of residues K495, K531, and E533 of SEQ ID NO:1; Y37, which is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; optionally, Y38, which is within 4.0 Å of residue D525 of SEQ ID NO: 1; D57, which is within 4.0 Å of residues N527 and K531 of SEQ ID NO:1; S59, which is within 4.0 Å of residues S530 and K531 of SEQ ID NO:1; V62, which is within 4.0 Å of residues N527 and S530 of SEQ ID NO:1; E64, which is within 4.0 Å of residue N527 of SEQ ID NO:1; R108, which is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; L109, which is within 4.0 Å of residues S492 and D560 of SEQ ID NO:1; P110, which is within 4.0 Å of residues S492 and N493 of SEQ ID NO:1; Y111, which is within 4.0 Å of residues S492, F494, H515, T518, and L522 of SEQ ID NO: 1; Y112, which is within 4.0 Å of residues T491, S492, H515, V517, and T518 of SEQ ID NO: 1; Y113, which is within 4.0 Å of residues Q520 and Q524 of SEQ ID NO:1; and VLs, as defined by IMGT numbering, include: F38, which is within 4.0 Å of residue Q520 of SEQ ID NO:1; Y56, which is within 4.0 Å of residues V517 and T518 of SEQ ID NO:1; R66, which is within 4.0 Å of residue E582 of SEQ ID NO:1 Includes: In certain embodiments, UCSF Chimera, PISA, or UCSF ChimeraX software, or any combination thereof, is used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues.

[0058] In some embodiments, the BBB transporter, when bound to hTfR, V, as defined by IMGT numbering, includes: H : optionally, S26, which can form a hydrogen bond with residue K261 of SEQ ID NO: 1; R36, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; Y37, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; optionally, Y38, which can form a hydrogen bond with residue D525 of SEQ ID NO: 1; D57, which can form a hydrogen bond with residue N527 of SEQ ID NO: 1 and a salt bridge with residue K531 of SEQ ID NO: 1; S59, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; R108, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1 and a salt bridge with residue D560 of SEQ ID NO: 1; Y111, which can form a hydrogen bond with residue S492 of SEQ ID NO:1 and with residue L522 of SEQ ID NO:1; and Y113, which can form a hydrogen bond with residue Q524 of SEQ ID NO: 1; and V, as defined by IMGT numbering, includes: L : Y56, which can form a hydrogen bond with residue V517 and with residue T518 of SEQ ID NO: 1; and R66, which can form a salt bridge with residue E582 of SEQ ID NO: 1 Includes:

[0059] In some embodiments, the BBB transporter comprises HCDRs 1-3 of SEQ ID NOs: 26, 28, and 7, respectively, LCDRs 1 and 2 of SEQ ID NOs: 29 and 10, respectively, and optionally an LCDR3 of SEQ ID NO: 11, and optionally an HFR1 whose last residue is S and / or an LFR3 whose first residue is R (e.g., an HFR1 whose last residue is S and an LFR3 whose first residue is R), wherein, when bound to hTfR, the BBB transporter: V, as defined by IMGT numbering, includes: H : a) S26, which is within 4.0 Å of residues K261 and K358 of SEQ ID NO:1; b) Y28, which is within 4.0 Å of residue K261 of SEQ ID NO:1; c) R36, which is within 4.0 Å of residues K495, K531, and E533 of SEQ ID NO:1; d) Y37, which is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; e) Y38, which is within 4.0 Å of residue D525 of SEQ ID NO:1; f) D57, which is within 4.0 Å of residues N527 and K531 of SEQ ID NO:1; g) S59, which is within 4.0 Å of residues S530 and K531 of SEQ ID NO:1; h) V62, which is within 4.0 Å of residues N527 and S530 of SEQ ID NO:1; i) E64, which is within 4.0 Å of residue N527 of SEQ ID NO:1; j) R108, which is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; k) L109, which is within 4.0 Å of residues S492 and D560 of SEQ ID NO:1; l) P110, which is within 4.0 Å of residues S492 and N493 of SEQ ID NO:1; m) Y111, which is within 4.0 Å of residues S492, F494, H515, T518, and L522 of SEQ ID NO: 1; n) Y112, which is within 4.0 Å of residues T491, S492, H515, V517, and T518 of SEQ ID NO: 1; and / or o) Y113, which is within 4.0 Å of residues Q520 and Q524 of SEQ ID NO: 1; and / or V, as defined by IMGT numbering, includes: L : p) F38, which is within 4.0 Å of residue Q520 of SEQ ID NO:1; q) Y56, which is within 4.0 Å of residues V517 and T518 of SEQ ID NO: 1; and / or r) R66, which is within 4.0 Å of residue E582 of SEQ ID NO:1; Or any combination of a) to r) (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18) In certain embodiments, UCSF Chimera, PISA, or UCSF ChimeraX software, or any combination thereof, is used to visualize the paratope / epitope structure and determine the distances (in Å) and interactions between paratope and epitope residues.

[0060] In some embodiments, the BBB transporter comprises HCDRs 1-3 of SEQ ID NOs: 26, 28, and 7, respectively, LCDRs 1 and 2 of SEQ ID NOs: 29 and 10, respectively, and optionally an LCDR3 of SEQ ID NO: 11, and optionally an HFR1 whose last residue is S and / or an LFR3 whose first residue is R (e.g., an HFR1 whose last residue is S and an LFR3 whose first residue is R), wherein, when bound to hTfR, the BBB transporter: V, as defined by IMGT numbering, includes: H : a) S26, which can form a hydrogen bond with residue K261 of SEQ ID NO: 1; b) R36, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; c) Y37, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; d) Y38, which can form a hydrogen bond with residue D525 of SEQ ID NO: 1; e) D57, which can form a hydrogen bond with residue N527 of SEQ ID NO: 1 and a salt bridge with residue K531 of SEQ ID NO: 1; f) S59, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; g) R108, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1 and a salt bridge with residue D560 of SEQ ID NO: 1; h) Y111, which is capable of forming a hydrogen bond with residue S492 of SEQ ID NO: 1 and a hydrogen bond with residue L522 of SEQ ID NO: 1; and / or i) Y113, which is capable of forming a hydrogen bond with residue Q524 of SEQ ID NO: 1; and / or V, as defined by IMGT numbering, includes: L : j) Y56, which can form a hydrogen bond with residue V517 and with residue T518 of SEQ ID NO: 1; and k) R66, which is capable of forming a salt bridge with residue E582 of SEQ ID NO: 1; Or any combination of a) to k) (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11) Includes:

[0061] In some embodiments, the BBB transporter is a V transporter comprising residues S26, Y28, R36, Y37, Y38, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113. H and V comprising residues F38, Y56, and R66 L the residue positions are defined according to IMGT numbering. In certain embodiments, the BBB transporter binds to an epitope of hTfR comprising residues K261, K358, T491, S492, N493, F494, K495, H515, V517, T518, Q520, L522, Q524, D525, N527, S530, K531, E533, and D560 and E582 of SEQ ID NO:1.

[0062] In some embodiments, the BBB transporter comprises residues Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113, and optionally further comprises S26 and / or Y38. H and V comprising residues F38, Y56, and R66 L the residue positions are according to IMGT numbering. In certain embodiments, the BBB transporter binds to an epitope of hTfR comprising residues K261, T491, S492, N493, F494, K495, H515, V517, T518, Q520, L522, Q524, D525, N527, S530, K531, E533, D560, and E582 of SEQ ID NO: 1, and optionally further comprising K358 and / or D525 of SEQ ID NO: 1. In certain embodiments, when the BBB transporter is bound to hTfR, S26 is within 4.0 Å of residue K261 of SEQ ID NO:1; S26 is within 4.0 Å of residue K358 of SEQ ID NO:1; Y28 is within 4.0 Å of residue K261 of SEQ ID NO:1; R36 is within 4.0 Å of residue K495 of SEQ ID NO:1; R36 is within 4.0 Å of residue K531 of SEQ ID NO:1; R36 is within 4.0 Å of residue E533 of SEQ ID NO:1; Y37 is within 4.0 Å of residue N493 of SEQ ID NO:1; Y37 is within 4.0 Å of residue D560 of SEQ ID NO:1; Y38 is within 4.0 Å of residue D525 of SEQ ID NO:1; D57 is within 4.0 Å of residue N527 of SEQ ID NO:1; D57 is within 4.0 Å of residue K531 of SEQ ID NO:1; S59 is within 4.0 Å of residue S530 of SEQ ID NO:1; S59 is within 4.0 Å of residue K531 of SEQ ID NO:1; V62 is within 4.0 Å of residue N527 of SEQ ID NO:1; V62 is within 4.0 Å of residue S530 of SEQ ID NO:1; E64 is within 4.0 Å of residue N527 of SEQ ID NO:1; R108 is within 4.0 Å of residue N493 of SEQ ID NO:1; R108 is within 4.0 Å of residue D560 of SEQ ID NO:1; L109 is within 4.0 Å of residue S492 of SEQ ID NO:1; L109 is within 4.0 Å of residue D560 of SEQ ID NO:1; P110 is within 4.0 Å of residue S492 of SEQ ID NO:1; P110 is within 4.0 Å of residue N493 of SEQ ID NO:1; Y111 is within 4.0 Å of residue S492 of SEQ ID NO:1; Y111 is within 4.0 Å of residue F494 of SEQ ID NO:1; Y111 is within 4.0 Å of residue H515 of SEQ ID NO:1; Y111 is within 4.0 Å of residue T518 of SEQ ID NO:1; Y111 is within 4.0 Å of residue L522 of SEQ ID NO:1; Y112 is within 4.0 Å of residue T491 of SEQ ID NO:1; Y112 is within 4.0 Å of residue S492 of SEQ ID NO:1; Y112 is within 4.0 Å of residue H515 of SEQ ID NO:1; Y112 is within 4.0 Å of residue V517 of SEQ ID NO:1; Y112 is within 4.0 Å of residue T518 of SEQ ID NO:1; Y113 is within 4.0 Å of residue Q520 of SEQ ID NO:1; Y113 is within 4.0 Å of residue Q524 of SEQ ID NO:1; F38 is within 4.0 Å of residue Q520 of SEQ ID NO:1; Y56 is within 4.0 Å of residue V517 of SEQ ID NO:1; Y56 is within 4.0 Å of residue T518 of SEQ ID NO: 1; R66 is within 4.0 Å of residue E582 of SEQ ID NO:1; or any combination thereof (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or all 38 thereof). In certain embodiments, UCSF Chimera, PISA, or UCSF ChimeraX software, or any combination thereof, is used to visualize the paratope / epitope structure and determine the distance (in Å) and interactions between the paratope and epitope residues. In addition to or instead of the 4.0 Å distance embodiment described above, in certain embodiments, when the BBB transporter is bound to hTfR, S26 can form a hydrogen bond with residue K261 of SEQ ID NO: 1; R36 can form a hydrogen bond with residue K531 of SEQ ID NO: 1; Y37 can form a hydrogen bond with residue N493 of SEQ ID NO: 1; Y38 can form a hydrogen bond with residue D525 of SEQ ID NO: 1; D57 can form a hydrogen bond with residue N527 of SEQ ID NO: 1; D57 can form a salt bridge with residue K531 of SEQ ID NO: 1; S59 can form a hydrogen bond with residue K531 of SEQ ID NO: 1; R108 can form a hydrogen bond with residue N493 of SEQ ID NO: 1; R108 can form a salt bridge with residue D560 of SEQ ID NO: 1; Y111 can form a hydrogen bond with residue S492 of SEQ ID NO: 1; Y111 can form a hydrogen bond with residue L522 of SEQ ID NO: 1; Y113 can form a hydrogen bond with residue Q524 of SEQ ID NO: 1; Y56 can form a hydrogen bond with residue V517 of SEQ ID NO: 1; Y56 can form a hydrogen bond with residue T518 of SEQ ID NO: 1; R66 can form a salt bridge with residue E582 of SEQ ID NO: 1; or any combination thereof (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 of them).

[0063] In some embodiments, the BBB transporter herein is V H Amino acid residues (i.e., residues in VH) R36, Y37, D57, S59, R108, Y111, and Y113; and V L In some embodiments, the paratope comprises amino acid residues (i.e., residues in the VL) Y56 and R66, numbering according to IMGT. H In some embodiments, the paratope further comprises amino acid residue S26.H In some embodiments, the paratope further comprises amino acid residue Y38 (or W38). H In some embodiments, the paratope further comprises amino acid residues S26 and Y38 (or W38). In some embodiments, the paratope further comprises at least one V selected from Y28, V62, E64, L109, P110, and Y112. H In some embodiments, the paratope further comprises an amino acid residue V H In some embodiments, the paratope further comprises amino acid residue Y28. H In some embodiments, the paratope further comprises amino acid residue V62. H In some embodiments, the paratope further comprises amino acid residue E64. H In some embodiments, the paratope further comprises amino acid residue L109. H In some embodiments, the paratope further comprises amino acid residue P110. H In some embodiments, the paratope further comprises amino acid residue Y112. H In some embodiments, the paratope further comprises amino acid residues S26, Y28, Y38 (or W38), V62, E64, L109, P110, and Y112. H In some embodiments, the paratope further comprises amino acid residues Y28, V62, E64, L109, P110, and Y112. L In some embodiments, the paratope further comprises amino acid residue F38. H Amino acid residues S26, Y28, Y38 (or W38), V62, E64, L109, P110, and Y112, and V L In some embodiments, the paratope further comprises amino acid residue F38. H Amino acid residues Y28, V62, E64, L109, P110 and Y112 and V L It further comprises amino acid residue F38.

[0064] In some embodiments, the BBB transporter is V Hat least nine (e.g., at least 10, at least 11, at least 12, at least 13, or at least 14) amino acid residues selected from the group consisting of S26, Y28, R36, Y37, Y38 (or W38), D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; and / or V L and a paratope comprising at least two amino acid residues selected from the group consisting of F38, Y56, and R66; numbering according to IMGT.

[0065] In some embodiments, the BBB transporter is V H In some embodiments, the BBB transporter comprises a paratope comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residue selected from the group consisting of amino acid residues S26, Y28, R36, Y37, Y38 (or W38), D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113, numbering according to IMGT. L In some embodiments, the BBB transporter comprises a paratope comprising at least one (e.g., one, two, or three) amino acid residues selected from the group consisting of amino acid residues F38, Y56, and R66, numbering according to IMGT. H at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residue selected from the group consisting of amino acid residues S26, Y28, R36, Y37, Y38 (or W38), D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; and (ii) V L The amino acid sequence of the present invention comprises a paratope comprising at least one (e.g., one, two, or three) amino acid residues selected from the group consisting of amino acid residues F38, Y56, and R66, where the numbering is according to IMGT. In some embodiments, the numbering is according to a different system (Kabat, Chothia, Aho, AbM, or a combination thereof), and the positions are adapted accordingly.

[0066] In some embodiments, the BBB transporter is V H In some embodiments, the BBB transporter comprises a paratope comprising amino acid residues Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; optionally, further comprising S26 and / or Y38 (or W38), numbering according to IMGT. L In some embodiments, the BBB transporter comprises a paratope comprising amino acid residues F38 and Y56, and optionally further comprising R66, numbering according to IMGT. H comprising amino acid residues Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; optionally, further comprising S26 and / or Y38 (or W38); and (ii) V L It comprises a paratope comprising amino acid residues F38 and Y56, and optionally further comprising R66. In some embodiments, the numbering is according to a different system (Kabat, Chothia, Aho, AbM, or a combination thereof), and the positions are adapted accordingly.

[0067] In some embodiments, the BBB transporter comprises an HCDR1 comprising Y28, R36, Y37, and Y38 (or W38); an HCDR2 comprising D57, S59, V62, and E64; an HCDR3 comprising R108, L109, P110, Y111, Y112, and Y113; an LCDR1 comprising F38; an LCDR2 comprising Y56; and an LFR3 starting with an arginine residue (R66). In some embodiments, the BBB transporter comprises an HCDR1 comprising Y28, R36, Y37, and Y38 (or W38); an HCDR2 comprising D57, S59, V62, and E64; and an HCDR3 comprising R108, L109, P110, Y111, Y112, and Y113. In some embodiments, the BBB transporter comprises an LCDR1 that comprises F38; an LCDR2 that comprises Y56; and an LFR3 that starts with an arginine residue (R66).

[0068] In some embodiments, the BBB transporter is V, including H optionally, HFR1 terminating in a serine residue (S26); HCDR1 comprising Y28, R36, Y37 and optionally Y38 (or W38), and comprising 1 to 5 additional amino acid residues of SEQ ID NO: 26; HCDR2 comprising D57, S59, V62, and E64 and comprising 1 to 4 additional amino acid residues of SEQ ID NO: 28; and an HCDR3 comprising R108, L109, P110, Y111, Y112, Y113 and 1 to 7 additional amino acid residues of SEQ ID NO: 7; and / or V, including L LCDR1 comprising F38 and 1 to 5 additional amino acid residues of SEQ ID NO: 29; LCDR2 comprising Y56 and one to two additional amino acid residues of SEQ ID NO: 10; LFR3 starting from an arginine residue (R66); and Optionally, an LCDR3 comprising 1 to 9 amino acid residues of SEQ ID NO: 11. Includes: In some embodiments, the BBB transporter is a V, including: H optionally, HFR1 terminating in a serine residue (S26); HCDR1 comprising Y28, R36, Y37 and optionally Y38 (or W38), and comprising 1 to 5 additional amino acid residues of SEQ ID NO: 26; HCDR2 comprising D57, S59, V62, and E64 and comprising 1 to 4 additional amino acid residues of SEQ ID NO: 28; and HCDR3 comprising R108, L109, P110, Y111, Y112, Y113 and 1 to 7 additional amino acid residues of SEQ ID NO: 7 Includes: In some embodiments, the BBB transporter is a V, including: L LCDR1 comprising F38 and 1 to 5 additional amino acid residues of SEQ ID NO: 29; LCDR2 comprising Y56 and one to two additional amino acid residues of SEQ ID NO: 10; LFR3 starting from an arginine residue (R66); and Optionally, an LCDR3 comprising 1 to 9 amino acid residues of SEQ ID NO: 11. Includes:

[0069] In some embodiments, the BBB transporter is V, including H optionally, HFR1 terminating in a serine residue (S26); HCDR1 comprising R36 and Y37 and further comprising 1 to 6 amino acid residues of SEQ ID NO: 26; HCDR2 comprising D57 and S59 and further comprising 1 to 6 amino acid residues of SEQ ID NO: 28; HCDR3 comprising R108, Y111, Y113 and further comprising 1 to 10 amino acid residues of SEQ ID NO: 7; and / or V, including L LCDR2 comprising Y56, which further comprises 1 to 2 amino acid residues of SEQ ID NO: 10; LFR3 starting from an arginine residue (R66); and Optionally, an LCDR3 comprising 1 to 9 amino acid residues of SEQ ID NO: 11. Includes: In certain embodiments, the number of amino acid residues in the CDRs of the BBB transporter are the same as those shown in the respective SEQ ID NOs: HCDR1: 8 residues, HCDR2: 8 residues, HCDR3: 13 residues, LCDR1: 6 residues, LCDR2: 3 residues, and LCDR3 (if present): 9 residues.

[0070] In some embodiments, a BBB transporter comprises, in addition to the set of paratope residues defined herein, an HCDR1, HCDR2, and HCDR3 having the sequences set forth in SEQ ID NOs: 26, 28, and 7, respectively. In some embodiments, a BBB transporter comprises, in addition to the set of paratope residues defined herein, at least one (e.g., one, two, or three) HCDRs selected from HCDR1, HCDR2, and HCDR3 having the sequences set forth in SEQ ID NOs: 26, 28, and 7, respectively. In some embodiments, a BBB transporter comprises, in addition to the set of paratope residues defined herein, an LCDR1 and an LCDR2 having the sequences set forth in SEQ ID NOs: 29 and 10, respectively; and optionally, further comprises an LCDR3 having the sequence set forth in SEQ ID NO: 11. In some embodiments, a BBB transporter comprises, in addition to the set of paratope residues defined herein, at least one (e.g., one, two, or three) LCDRs selected from LCDR1, LCDR2, and LCDR3 having the sequences set forth in SEQ ID NOs: 29, 10, and 11, respectively.

[0071] In some embodiments, a BBB transporter is provided that, in addition to the set of paratope residues defined herein, comprises HCDR1, HCDR2, and HCDR3 having the sequences set forth in SEQ ID NOs: 26, 28, and 7, respectively, and optionally further comprises LCDR1 and LCDR2 having the sequences set forth in SEQ ID NOs: 29 and 10, respectively; and optionally further comprises LCDR3 having the sequence set forth in SEQ ID NO: 11. In some embodiments, the BBB transporter, in addition to the set of paratope residues defined herein, comprises at least one (e.g., one, two, or three) HCDR selected from HCDR1, HCDR2, and HCDR3 having the sequences set forth in SEQ ID NOs: 26, 28, and 7, respectively, and optionally further comprises at least one (e.g., one, two, or three) LCDR selected from LCDR1, LCDR2, and LCDR3 having the sequences set forth in SEQ ID NOs: 29, 10, and 11, respectively. The CDR sequences disclosed in the above embodiments are annotated according to IMGT. In some embodiments, the numbering follows a different system (Kabat, Chothia, Aho, AbM, or a combination thereof), and the sequences of the CDRs are adapted accordingly. In some embodiments, the BBB transporter further comprises an HFR1 that terminates with a serine residue (i.e., a serine residue at the carboxy terminus of HFR1). In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, an LFR3 that starts with an arginine residue (i.e., an arginine residue at the amino-terminal start of LFR3). In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, an HFR1 that terminates with a serine residue and an LFR3 that starts with an arginine residue. In some embodiments, the HFR1 has a sequence that is at least 50% identical to the HFR1 sequence contained in SEQ ID NO: 21, optionally with the proviso that HFR1 terminates with a serine residue. In some embodiments, the LFR3 has a sequence that is at least 50% identical to the LFR3 sequence contained in SEQ ID NO: 25, optionally with the proviso that the LFR3 starts with an arginine residue. In some embodiments, the framework regions have a sequence as contained in SEQ ID NOs: 21 and 25.The framework sequences disclosed in the above embodiments are annotated according to IMGT. In some embodiments, the numbering follows a different system (Kabat, Chothia, Aho, AbM, or a combination thereof), and the sequences of the framework regions are adapted accordingly.

[0072] In some embodiments, the BBB transporter comprises a V having a sequence that is at least 50% (at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%) identical to the sequence of SEQ ID NO:21 in addition to the set of paratope residues defined herein. H and V having a sequence at least 50% (at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%) identical to the sequence of SEQ ID NO: 25. L Includes:

[0073] In some embodiments, the BBB transporter comprises a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO: 21 in addition to the set of paratope residues defined herein. H and V having a sequence that is at least 95% identical to the sequence of SEQ ID NO: 25. L In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, a V having a sequence that is at least 50% (at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%) identical to the sequence of SEQ ID NO:21. H In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO:21. HIn some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, a V having a sequence that is at least 50% (at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%) identical to the sequence of SEQ ID NO:25. L In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO:25. L Includes:

[0074] In some embodiments, the BBB transporter comprises a V having a sequence set forth in SEQ ID NO:21 in addition to the set of paratope residues defined herein. H and V having the sequence set forth in SEQ ID NO: 25 L In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, a V having the sequence set forth in SEQ ID NO:21. H In some embodiments, the BBB transporter comprises, in addition to the set of paratope residues defined herein, a V having the sequence set forth in SEQ ID NO:25. L Includes:

[0075] In some embodiments, the above-defined paratope residues of a BBB transporter, when bound to hTfR (represented by SEQ ID NO: 1), can form hydrogen bonds and / or salt bridges with epitope residues. In some embodiments, the following paratope residues, if present, when bound, can form hydrogen bonds with at least one epitope residue of hTfR: V H The paratope residues in are: a) S26, which can form a hydrogen bond with residue K261 of SEQ ID NO: 1; b) R36, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; c) Y37, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; d) Y38, which can form a hydrogen bond with residue D525 of SEQ ID NO: 1; e) D57, which can form a hydrogen bond with residue N527 of SEQ ID NO: 1; f) S59, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; g) R108, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; h) Y111, which can form hydrogen bonds with residues S492 and L522 of SEQ ID NO: 1; and i) Y113, which is capable of forming hydrogen bonds with residues Q524 and Q520 of SEQ ID NO: 1; V L The paratope residues in are: j) Y56, which can form a hydrogen bond with residue V517 and with residue T518 of SEQ ID NO:1.

[0076] In some embodiments, the following paratope residues, if present, when bound, are capable of forming a salt bridge with at least one epitope residue of hTfR: V H The paratope residues in a) R108, which is capable of forming a salt bridge with residue N560 of SEQ ID NO: 1; and b) D57, which is capable of forming a salt bridge with residue K531 of SEQ ID NO: 1; V L The paratope residues in are: j) R66, which is capable of forming a salt bridge with residue E582 of SEQ ID NO:1.

[0077] In some embodiments, the following paratope residues, when present, are capable of forming hydrogen bonds and / or salt bridges with at least one epitope residue of hTfR when bound, and the paratope residues in VH are: a) S26, which can form a hydrogen bond with residue K261 of SEQ ID NO: 1; b) R36, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; c) Y37, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; d) Y38, which can form a hydrogen bond with residue D525 of SEQ ID NO: 1; e) D57, which can form a hydrogen bond with residue N527 of SEQ ID NO: 1; f) D57, which can form a salt bridge with residue K531 of SEQ ID NO: 1; g) S59, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; h) R108, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; i) R108, which is capable of forming a salt bridge with residue N560 of SEQ ID NO: 1; j) Y111, which can form hydrogen bonds with residues S492 and L522 of SEQ ID NO: 1; and k) Y113, which is capable of forming hydrogen bonds with residues Q524 and Q520 of SEQ ID NO: 1; The paratope residues in VL are: l) Y56, which can form a hydrogen bond with residue V517 and with residue T518 of SEQ ID NO: 1; and m) R66, which is capable of forming a hydrogen bond with residue E582 of SEQ ID NO:1.

[0078] In some embodiments, the above-defined paratope residues of a BBB transporter may have additional properties when bound to hTfR (according to SEQ ID NO: 1). -V H The following paratope residues located within, if present, may have the following additional properties when bound to hTfR (represented in SEQ ID NO: 1): a) S26 is within 4.0 Å of residues K261 and K358 of SEQ ID NO:1; b) Y28 is within 4.0 Å of residue K261 of SEQ ID NO:1; c) R36 is within 4.0 Å of residues K495, K531, and E533 of SEQ ID NO:1; d) Y37 is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; e) Y38 is within 4.0 Å of residue D525 of SEQ ID NO:1; f) D57 is within 4.0 Å of residues N527 and K531 of SEQ ID NO:1; g) S59 is within 4.0 Å of residues S530 and K531 of SEQ ID NO:1; h) V62 is within 4.0 Å of residues N527 and S530 of SEQ ID NO: 1; i) E64 is within 4.0 Å of residue N527 of SEQ ID NO:1; j) R108 is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; k) L109 is within 4.0 Å of residues S492 and D560 of SEQ ID NO:1; l) P110 is within 4.0 Å of residues S492 and N493 of SEQ ID NO:1; m) Y111 is within 4.0 Å of residues S492, F494, H515, T518, and L522 of SEQ ID NO: 1; n) Y112 is within 4.0 Å of residues T491, S492, H515, V517, and T518 of SEQ ID NO: 1; o) Y113 is within 4.0 Å of residues Q520 and Q524 of SEQ ID NO: 1; and -V L The following paratope residues located within, if present, may have the following additional properties when bound to hTfR (represented in SEQ ID NO: 1): p) F38 is within 4.0 Å of residue Q520 of SEQ ID NO: 1; q) Y56 is within 4.0 Å of residues V517 and T518 of SEQ ID NO: 1; r) R66 is within 4.0 Å of residue E582 of SEQ ID NO:1.

[0079] The paratope residues disclosed in the above embodiments are annotated according to IMGT. In some embodiments, the numbering follows a different system (Kabat, Chothia, Aho, Abm, or a combination thereof), and the positions of the paratope residues are adapted accordingly.

[0080] The present disclosure provides, for example, the following numbered embodiments of the anti-TfR antibodies or antigen-binding fragments thereof of the invention:

[0081] 1. An anti-human transferrin receptor (hTfR) antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (V H ) amino acid residues R36, Y37, D57, S59, R108, Y111 and Y113; and Light chain variable region (V L ) amino acid residues Y56 and R66; (wherein numbering is according to IMGT).

[0082] 2. The paratope is -V H amino acid residues S26 and / or Y38 (or W38); 2. The antibody or antigen-binding fragment of embodiment 1, further comprising: (wherein numbering is according to IMGT).

[0083] 3. The paratope is -V H Amino acid residues Y28, V62, E64, L109, P110 and Y112; and -V L amino acid residue F38; 10. The antibody or antigen-binding fragment of any one of the preceding embodiments, further comprising:

[0084] 4. V, including H - a heavy chain CDR (HCDR) 1 comprising 1 to 5 additional amino acid residues of SEQ ID NO: 26, including Y28, R36, Y37, and optionally Y38 (or W38); HCDR2 comprising D57, S59, V62 and E64 and comprising 1 to 4 additional amino acid residues of SEQ ID NO: 28; and an HCDR3 comprising 1 to 7 additional amino acid residues of SEQ ID NO: 7, comprising R108, L109, P110, Y111, Y112 and Y113; and / or V, includingL a light chain CDR (LCDR)1 comprising F38 and 1 to 5 additional amino acid residues of SEQ ID NO: 29; - LCDR2 comprising Y56 and one to two additional amino acid residues of SEQ ID NO: 10; - light chain framework region (LFR) 3 starting from arginine residue (R66); and optionally, an LCDR3 comprising 1 to 9 amino acid residues of SEQ ID NO: 11; further including; Optionally, the antibody or antigen-binding fragment further comprises a heavy chain framework region (HFR) 1 terminating in a serine residue; 10. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein numbering is according to IMGT.

[0085] 5. An anti-human transferrin receptor (hTfR) antibody or an antigen-binding fragment thereof, - heavy chain CDRs (HCDR) 1, HCDR2 and HCDR3 having the sequences set forth in SEQ ID NOs: 26, 28 and 7, respectively; - light chain CDRs (LCDR)1 and LCDR2 having the sequences set forth in SEQ ID NOs: 29 and 10, respectively; and optionally, LCDR3 having the sequence set forth in SEQ ID NO: 11; - a heavy chain framework region (HFR) 1 ending in a serine residue; and -Light chain framework region (LFR) 3 starting with an arginine residue An antibody or antigen-binding fragment thereof comprising:

[0086] 6. - V having a sequence that is at least 50% identical to the sequence of SEQ ID NO: 21 H and - V having a sequence that is at least 50% identical to the sequence of SEQ ID NO: 25 L 2. The antibody or antigen-binding fragment of any one of the preceding embodiments, comprising:

[0087] 7. The following paratope residues, if present, when bound, are capable of forming hydrogen bonds with at least one epitope residue of hTfR: V H The paratope residues in are: a) S26, which can form a hydrogen bond with residue K261 of SEQ ID NO: 1; b) R36, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; c) Y37, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; d) Y38, which can form a hydrogen bond with residue D525 of SEQ ID NO: 1; e) D57, which can form a hydrogen bond with residue N527 of SEQ ID NO: 1; f) S59, which can form a hydrogen bond with residue K531 of SEQ ID NO: 1; g) R108, which can form a hydrogen bond with residue N493 of SEQ ID NO: 1; h) Y111, which can form hydrogen bonds with residues S492 and L522 of SEQ ID NO: 1; and i) Y113, which is capable of forming hydrogen bonds with residues Q524 and Q520 of SEQ ID NO: 1; V L The paratope residues in are: j) Y56, which is capable of forming a hydrogen bond with residue V517 and with residue T518 of SEQ ID NO: 1; 10. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein numbering is according to IMGT.

[0088] 8. The following paratope residues, when present, are capable of forming salt bridges with at least one epitope residue of hTfR when bound: where V H The paratope residues in a) R108 capable of forming a salt bridge with residue D560 of SEQ ID NO: 1; b) D57, which is capable of forming a salt bridge with residue K531 of SEQ ID NO: 1; V L The paratope residues in are: c) R66, which is capable of forming a hydrogen bond with residue E582 of SEQ ID NO: 1; 10. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein numbering is according to IMGT.

[0089] 9. The following paratope residues, if present, when bound, are within 4.0 Å of at least one epitope residue of hTfR: V H The paratope residues in are: a) S26, which is within 4.0 Å of residues K261 and K358 of SEQ ID NO:1; b) Y28, which is within 4.0 Å of residue K261 of SEQ ID NO:1; c) R36, which is within 4.0 Å of residues K495, K531, and E533 of SEQ ID NO:1; d) Y37, which is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; e) Y38, which is within 4.0 Å of residue D525 of SEQ ID NO:1; f) D57, which is within 4.0 Å of residues N527 and K531 of SEQ ID NO:1; g) S59, which is within 4.0 Å of residues S530 and K531 of SEQ ID NO:1; h) V62, which is within 4.0 Å of residues N527 and S530 of SEQ ID NO:1; i) E64, which is within 4.0 Å of residue N527 of SEQ ID NO:1; j) R108, which is within 4.0 Å of residues N493 and D560 of SEQ ID NO:1; k) L109, which is within 4.0 Å of residues S492 and D560 of SEQ ID NO:1; l) P110, which is within 4.0 Å of residues S492 and N493 of SEQ ID NO:1; m) Y111, which is within 4.0 Å of residues S492, F494, H515, T518, and L522 of SEQ ID NO: 1; n) Y112, which is within 4.0 Å of residues T491, S492, H515, V517, and T518 of SEQ ID NO: 1; o) Y113, which is within 4.0 Å of residues Q520 and Q524 of SEQ ID NO: 1; VL The paratope residues in are: p) F38, which is within 4.0 Å of residue Q520 of SEQ ID NO:1; q) Y56, which is within 4.0 Å of residues V517 and T518 of SEQ ID NO: 1; and r) R66, which is within 4.0 Å of residue E582 of SEQ ID NO: 1; 10. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein numbering is according to IMGT.

[0090] In some embodiments, the BBB transporter is the human germline sequence IGVH3-23 * 05 or IGHV1-46 * Exemplary humanized VH sequences generated using these human germline sequences include the HCDRs described herein inserted into human heavy chain framework sequences derived from IGHJ6_01 and IGHJ6_01. H The sequences are shown in Figures 3A and 3B.

[0091] In some embodiments, the BBB transporter comprises an LCDR described herein inserted into a human kappa light chain framework sequence derived from the human germline sequences IGKV1-39*01 and IGKJ4-01. Exemplary humanized VL1- and VL2-encoded sequences generated using these human germline sequences are shown in Table 1. L The sequence is shown in Figure 3C.

[0092] In some embodiments, the BBB transporter comprises a V comprising any one of SEQ ID NOs: 17-21. H or an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical thereto; and / or a V comprising any one of SEQ ID NOs: 22 to 25. L In a further embodiment, the BBB transporter comprises an amino acid sequence that is at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NOs: 21 and 25, SEQ ID NOs: 20 and 25, SEQ ID NOs: 19 and 25, SEQ ID NOs: 18 and 25, SEQ ID NOs: 17 and 25, SEQ ID NOs: 21 and 24, SEQ ID NOs: 20 and 24, SEQ ID NOs: 19 and 24, SEQ ID NOs: 18 and 24, SEQ ID NOs: 17 and 24, SEQ ID NOs: 21 and 23, SEQ ID NOs: 20 and 23, SEQ ID NOs: 19 and 23, SEQ ID NOs: 18 and 23, SEQ ID NOs: 17 and 23, SEQ ID NOs: 21 and 22, SEQ ID NOs: 20 and 22, SEQ ID NOs: 19 and 22, SEQ ID NOs: 18 and 22, SEQ ID NOs: 17 and 22, or V comprising SEQ ID NOs: 4 and 8, respectively H and V L , or an amino acid sequence that is at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to a pair of SEQ ID NOs listed above. In some embodiments, the mutant V H and V L The sequences comprise the HCDRs and LCDRs (e.g., as defined by IMGT) in the SEQ ID NO pairs listed above, with sequence variations occurring in the framework regions. H and V L retain the properties of their parent sequences with respect to TfR binding properties.

[0093] In a further embodiment, the BBB transporter is SEQ ID NOs: 21 and 25, SEQ ID NOs: 21 and 24, SEQ ID NOs: 21 and 23, SEQ ID NOs: 19 and 23, SEQ ID NOs: 18 and 23, SEQ ID NOs: 17 and 23, SEQ ID NOs: 21 and 22, SEQ ID NOs: 20 and 22, SEQ ID NOs: 18 and 22, SEQ ID NOs: 17 and 22, or V comprising SEQ ID NOs: 4 and 8, respectively H and V L , or an amino acid sequence that is at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to a pair of SEQ ID NOs listed above. In some embodiments, the mutant V H and V L The sequences comprise the HCDRs and LCDRs (e.g., as defined by IMGT) in the SEQ ID NO pairs listed above, with sequence variations occurring in the framework regions. H and V L retain the properties of their parent sequences with respect to TfR binding properties.

[0094] The percent identity of two amino acid sequences (or two nucleic acid sequences) can be obtained, for example, by BLAST® using default parameters (available at the US National Library of Medicine's National Center for Biotechnology Information website). The length of the reference sequence aligned for comparison is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90% of the reference sequence). H , V L For HC, or LC sequence identity or homology, the percent identity and homology are calculated based on the full-length query and reference V H , V L , HC, or LC sequences.

[0095] The present disclosure also contemplates BBB transporters structurally defined according to any of the above embodiments having any combination of the functional properties described herein for BBB transporters.

[0096] 2. BBB transporter formats In some embodiments, the BBB transporters herein are formatted as a full tetrameric antibody. The antibody can be of any immunoglobulin isotype, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The antibodies herein preferably comprise a human IgG (e.g., IgG1) constant region. In some embodiments, the IgG constant region can comprise mutations that improve the clinical potential of the antibody, such as mutations that reduce or eliminate antibody effector function (ADCC and / or CDC) (see, e.g., Wang et al., Protein Cell (2018) 9(1):63-73). For example, the monospecific or multispecific antibodies herein can comprise a human IgG1 constant region with the mutation L235E, a "LALA" mutation (L234A / L235A), or a "LALAGA" mutation (L234A / L235A / G237A) (EU numbering). In some embodiments, the heavy chain comprises one, two, or all three of the following mutations: S298N, T299A, and Y300S ("NNAS" mutations).

[0097] The IgG constant region may contain mutations that improve the serum half-life of the antibody, such as the M428L mutation, M252Y / S254T / T256E ("YTE" mutation), and mutations described in WO 2019 / 147973.

[0098] To promote proper protein assembly at the BBB during manufacturing, the antibody heavy chain (e.g., IgG1 heavy chain) may contain knob-in-hole mutations (e.g., Y349C, T366S, L368A, and Y407V for IgG1 hole mutations; and S354C and T366W for IgG1 knob mutations). In some embodiments, the hole heavy chain contains a H435R / Y436F ("RF") double mutation, which allows for easy removal of hole-hole homodimer and hole-half IgG by-products during manufacturing.

[0099] All such mutated human constant regions are still considered "human" constant regions herein. Unless otherwise indicated, all residue numbers in IgG constant regions are EU numbers.

[0100] In some embodiments, the anti-TfR antibody comprises a HC comprising SEQ ID NO: 32, or an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical thereto; and / or a LC comprising SEQ ID NO: 31, or an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical thereto. In some embodiments, the anti-TfR antibody comprises a HC comprising SEQ ID NO: 32 and a LC comprising SEQ ID NO: 31 (531v25 mAb). In some embodiments, the variant HC and LC sequences comprise the HCDRs and LCDRs (e.g., as defined by IMGT) in SEQ ID NOs: 32 and 31, with sequence variations occurring in the framework and / or constant regions. In some embodiments, the variant HC and LC retain the properties of their parent sequences with respect to TfR binding properties.

[0101] BBB transporters can also be antigen-binding fragments of complete antibodies. In some embodiments, the transporters are monovalent for TfR (i.e., each transporter has only one binding site for TfR) and / or comprise a Fab. For example, BBB transporters can be V H and C H1HC (e.g., IgG C H1 ) and V L and C L LC (e.g., κC L ) ). This transporter is also referred to herein as a "Fab transporter." In some embodiments, the Fab transporter comprises a HC comprising SEQ ID NO: 30, or an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical thereto; and / or a LC comprising SEQ ID NO: 31, or an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical thereto. In some embodiments, the Fab transporter comprises a HC comprising SEQ ID NO: 30 and a LC comprising SEQ ID NO: 31 (531v25Fab). In some embodiments, the Fab transporter comprises a V set forth in SEQ ID NO: 21. H and V as set forth in SEQ ID NO: 25 L In some embodiments, the variant HC and LC sequences comprise the HCDRs and LCDRs (e.g., as defined by IMGT) in SEQ ID NOs: 30 and 31 (or SEQ ID NOs: 21 and 25), with sequence variations occurring in the framework and / or constant regions. In some embodiments, the variant HC and LC retain the properties of their parent sequences with respect to TfR binding properties.

[0102] In some embodiments, the BBB transporter resembles a complete IgG antibody, except that it has only one TfR-binding domain. Such a BBB transporter is a heterotrimer comprising an LC, a complete antibody HC, and an Fc polypeptide that dimerizes with the constant region of the complete antibody HC to form an Fc domain. This transporter is also referred to herein as a "Fab-Fc" or "Fab-FcOL" transporter. In IgG, the Fc domain is a disulfide-linked dimeric structure formed by the CH2 and CH3 of the Hc pair. As used herein, an "Fc polypeptide" refers to a polypeptide that can dimerize via disulfide bonds to form an Fc domain, but does not include the V of the complete antibody HC. H In some embodiments, an Fc polypeptide contains CH2 and CH3 and at least a portion of the hinge region of a human IgG (e.g., human IgG1, IgG2, or IgG4) such that it can dimerize with a fully human IgG (e.g., human IgG1, IgG2, or IgG4) heavy chain to form a human IgG1, IgG2, or IgG4 Fc domain.

[0103] In some embodiments, the Fc polypeptide and the complete heavy chain may include one or more of the mutations described above that improve the clinical potential of the BBB transporter, such as mutations that reduce or eliminate effector function (e.g., "LALA" and "NNAS" mutations), mutations that improve the serum half-life of the BBB transporter (e.g., "YTE" mutations and those described in WO 2019 / 147973), mutations that promote proper protein assembly of the BBB transporter (e.g., knobs-in-holes mutations), and mutations that improve purification of the BBB transporter during manufacturing (e.g., "RF" mutations).

[0104] Exemplary TfR-binding antibody fragments include the following molecules: (i) V set forth in SEQ ID NO: 21 H HC comprising: V as set forth in SEQ ID NO: 25 Land a Fab-FcOL TfR-binding antibody fragment comprising an Fc polypeptide (optionally set forth in SEQ ID NO: 34); (ii) a Fab-FcOL TfR-binding antibody fragment comprising an HC comprising SEQ ID NO: 30, an LC comprising SEQ ID NO: 31, and an Fc polypeptide (optionally set forth in SEQ ID NO: 34); (iii) a Fab-FcOL TfR-binding antibody fragment comprising an HC comprising SEQ ID NO: 33, an LC comprising SEQ ID NO: 31, and an Fc polypeptide comprising SEQ ID NO: 34 (531v25Fab-FcOL); and (iv) A Fab-FcOL TfR-binding antibody fragment (531v25Fab-FcOL with engineered cysteines) comprising an HC comprising SEQ ID NO: 56, an LC comprising SEQ ID NO: 31, and an Fc polypeptide comprising SEQ ID NO: 34.

[0105] Further exemplary BBB transporters include the following molecules: (i) an antibody or antigen-binding fragment thereof comprising an HC comprising HCDRs 1-3 set forth in SEQ ID NOs: 26, 28, and 7, respectively, and an LC (and optionally an Fc polypeptide) comprising LCDRs 1-3 set forth in SEQ ID NOs: 29, 10, and 11, respectively; (ii) V set forth in SEQ ID NO: 21 H and V as set forth in SEQ ID NO: 25 L (iii) an antibody or antigen-binding fragment thereof comprising an HC comprising SEQ ID NO: 30 and an LC comprising SEQ ID NO: 31 (and optionally an Fc polypeptide); and (iv) an antibody or antigen-binding fragment thereof comprising an HC comprising SEQ ID NO: 30 and an LC (and optionally an Fc polypeptide) comprising SEQ ID NO: 31. These BBB transporters are referred to herein as "531v25 BBB transporters." A 531v25 BBB transporter having a Fab-FcOL format defined herein is referred to as a "531v25 Fab-FcOL BBB transporter."

[0106] C. Cargo The present BBB transporters (including the 531v25 BBB transporter) can be used to transport various cargoes across the BBB to the brain for diagnostic, prophylactic, and therapeutic purposes. The cargo can be chemically conjugated to the transporter, for example, via a lysine or cysteine ​​residue in the transporter. In some embodiments, the cargo can be chemically conjugated to an engineered residue, for example, an engineered cysteine ​​residue (THIOMAB™ technology) or an engineered lysine residue.

[0107] Alternatively, if the cargo is a peptide or polypeptide, it can be recombinantly fused to the transporter. For example, if the transporter is a complete antibody transporter (having two light chains and a full-length heavy chain), such as 531v25mAb, the cargo can be fused to the N- or C-terminus of one or both light chains, or to the N- or C-terminus of one or both heavy chains. In a further embodiment, the cargo can be fused to the C-terminus of one of the heavy chains (e.g., the knob heavy chain or the hole heavy chain).

[0108] In some embodiments, the transporter is a Fab transporter (such as 531v25Fab), and the cargo may be fused to the N- or C-terminus of the light chain or the N- or C-terminus of the heavy chain. In further embodiments, the cargo may be fused to the C-terminus of the heavy chain. In certain embodiments, the cargo may be fused to the C-terminus of the light chain.

[0109] In some embodiments, the transporter is a Fab-FcOL transporter (such as 531v25Fab-FcOL), and the cargo may be fused to the N- or C-terminus of the light chain, the N- or C-terminus of the complete heavy chain, or the N- or C-terminus of the Fc polypeptide. In further embodiments, the cargo may be fused to the C-terminus of the Fc polypeptide. In certain embodiments, the cargo may be fused to the C-terminus of the light chain.

[0110] The cargo may be linked to the anti-TfR antibody or antigen-binding fragment thereof via a peptide linker. In some embodiments, the peptide linker may comprise primarily the following amino acid residues: Gly, Ser, Ala, or Thr. The peptide linker may be long enough to link two molecules such that they assume the correct conformation relative to each other so that they retain their respective desired activities. In some embodiments, the linker is 1 to 50 (e.g., 1 to 30, 1 to 20, 1 to 10, or 1 to 5) amino acids in length. Useful linkers include, for example, glycine-serine polymers, including (GS)n, (GSGGS)n (SEQ ID NO: 44), (GGGGS)n (SEQ ID NO: 45), and (GGGS)n (SEQ ID NO: 46), where n is an integer of at least 1; glycine-alanine polymers; alanine-serine polymers; XTEN linkers; and other flexible linkers. In some embodiments, the linker is GGGG (SEQ ID NO: 42) and SGSGGGG (SEQ ID NO: 43). Further exemplary linkers for linking antibody fragments or single-chain variable fragments can include AAEPKSS (SEQ ID NO: 47), AAEPKSDKTHTCPPCP (SEQ ID NO: 48), GGGG (SEQ ID NO: 42), or GGGGDKTHTCPPCP (SEQ ID NO: 49).

[0111] Cargo transportable by the present BBB transporters (including the 531v25 BBB transporter) can be a diagnostic agent, for example an imaging agent for the brain.

[0112] The cargo transportable by the present BBB transporters (including the 531v25 BBB transporter) can be a therapeutic agent. Exemplary therapeutic agents are peptides / polypeptides and oligonucleotides.

[0113] In some embodiments, the therapeutic agent is an enzyme. In particular embodiments, the enzyme is a lysosomal enzyme. In further embodiments, the enzyme is acid alpha-glucosidase (GAA), such as recombinant human GAA. GAA is also known as alpha-1,4-glucosidase and acid maltase. It is an enzyme that helps break down glycogen in lysosomes. An example of a recombinant human GAA is alglucosidase alfa (Myozyme® and Lumizyme®). In some embodiments, the recombinant GAA comprises or consists of SEQ ID NO:35.

[0114] In some embodiments, the TfR binding protein for transporting GAA is a Fab-FcOL transporter comprising HC, LC, and Fc polypeptides and a human GAA sequence fused to the C-terminus of the (a) HC, (b) LC, or (c) Fc polypeptide, wherein the heavy chain and Fc polypeptide dimerize to form an Fc domain. In a further embodiment, the TfR binding protein for transporting GAA comprises a HC, LC, and Fc polypeptide, and a human GAA sequence fused to the C-terminus of the LC, wherein the heavy chain and Fc polypeptide dimerize to form an Fc domain. In a further embodiment, the TfR binding protein for transporting GAA is a 531v25 Fab-FcOL BBB transporter comprising HC, LC, and Fc polypeptides, wherein the human GAA sequence is fused to the C-terminus of the LC. In a further embodiment, the TfR binding protein is a Fab-FcOL (531v25Fab-FcOL-LC-GAA) comprising three polypeptides comprising SEQ ID NOs: 33, 36, and 34, respectively. In another embodiment, the TfR binding protein for transporting GAA comprises an HC, an LC, an Fc polypeptide, and a human GAA sequence fused to the C-terminus of the Fc polypeptide, wherein the heavy chain and the Fc polypeptide dimerize to form an Fc domain. In a further embodiment, the TfR binding protein for transporting GAA is a 531v25 Fab-FcOL BBB transporter comprising an HC, an LC, and an Fc polypeptide, wherein the human GAA sequence is fused to the C-terminus of the Fc polypeptide. In a further embodiment, the TfR binding protein is a Fab-FcOL (531v25Fab-FcOL-GAA) comprising three polypeptides comprising SEQ ID NOs: 33, 31, and 37, respectively.

[0115] In some embodiments, the TfR binding protein for transporting GAA comprises a Fab and a human GAA sequence fused to the C-terminus of the HC or LC of the Fab. In further embodiments, the TfR binding protein for transporting GAA comprises a Fab and a human GAA sequence fused to the C-terminus of the LC of the Fab. In a further embodiment, the TfR binding protein for transporting GAA is a 531v25 BBB transporter comprising the HC and LC in a Fab format, with the human GAA sequence fused to the C-terminus of the LC. In a further embodiment, the TfR binding protein comprises two polypeptides comprising SEQ ID NOs: 30 and 36, respectively (531v25Fab-GAA).

[0116] In some embodiments, the TfR binding protein for transporting GAA comprises two HCs, two LCs, and a human GAA sequence fused to the C-terminus of one of the two HCs. In a further embodiment, the TfR binding protein for transporting GAA is the 531v25 BBB transporter, comprising two HCs, two LCs, and a human GAA sequence fused to the C-terminus of one of the two HCs. In a further embodiment, the two LCs each comprise SEQ ID NO: 31, one of the HCs comprises SEQ ID NO: 33, and the other HC comprises SEQ ID NO: 37 (531v25mAb-GAA).

[0117] In some embodiments, the therapeutic agent is an oligonucleotide. In certain embodiments, the oligonucleotide is an antisense oligonucleotide or an siRNA, for example, targeted to a CNS gene.

[0118] In some embodiments, the TfR binding protein for transporting oligonucleotides is a Fab-FcOL transporter comprising HC, LC, and Fc polypeptides, where the heavy chain and Fc polypeptide dimerize to form an Fc domain, and the oligonucleotide is conjugated to a residue (e.g., lysine or cysteine) in one of the polypeptides. In certain embodiments, the oligonucleotide is conjugated to an engineered residue (e.g., an engineered cysteine ​​residue introduced by THIOMAB™ technology or an engineered lysine residue) for site-specific conjugation. In further embodiments, the TfR binding protein for transporting oligonucleotides is a 531v25 Fab-FcOL BBB transporter comprising HC, LC, and Fc polypeptides, where, for example, the oligonucleotide is conjugated to one of the transporter polypeptides as described above. In further embodiments, the TfR binding protein is a Fab-FcOL comprising three polypeptides comprising SEQ ID NOs: 31, 34, and 56, respectively.

[0119] In some embodiments, the TfR binding protein for transporting an oligonucleotide comprises a Fab. In further embodiments, the TfR binding protein for transporting an oligonucleotide is a 531v25 BBB transporter comprising an HC and LC in Fab format, and the oligonucleotide is conjugated to a residue (e.g., lysine or cysteine) of the HC or LC. In certain embodiments, the oligonucleotide is conjugated to an engineered residue (e.g., an engineered cysteine ​​residue introduced by THIOMAB™ technology or an engineered lysine residue) for site-specific conjugation.

[0120] In some embodiments, the TfR-binding protein for transporting an oligonucleotide comprises two HCs, two LCs, and an oligonucleotide conjugated to a residue (e.g., lysine or cysteine) of one of the HCs and LCs. In certain embodiments, the oligonucleotide is conjugated to an engineered residue (e.g., an engineered cysteine ​​residue introduced by THIOMAB™ technology or an engineered lysine residue) for site-specific conjugation. In further embodiments, the TfR-binding protein for transporting an oligonucleotide is a 531v25 BBB transporter comprising two HCs, two LCs, and an oligonucleotide conjugated to one of the HCs and LCs as described above. In certain embodiments, the two LCs each comprise SEQ ID NO: 31, one of the HCs comprises SEQ ID NO: 33, and the other HC comprises SEQ ID NO: 56. In certain embodiments, the two LCs each comprise SEQ ID NO: 31, and the two HCs each comprise SEQ ID NO: 56.

[0121] It should be understood that the attachment of cargo to a BBB transporter herein does not affect the TfR binding properties of the transporter. Cargo-loaded BBB transporters have binding properties (e.g., TfR binding affinity) as described in Section A (supra).

[0122] II. Preparation of TfR-binding proteins The TfR binding proteins described herein can be recombinantly produced using isolated nucleic acid molecules, such as expression constructs, encoding each chain of the protein. A biological molecule (e.g., a nucleic acid or polypeptide molecule) referred to herein as "isolated" or "purified" is one that (1) is separated from the biological molecule (e.g., genomic DNA or cellular RNA nucleic acid, or polypeptide of their origin; and / or (2) is not naturally occurring. The coding sequence for each polypeptide chain may be cloned into a single vector or into separate vectors.

[0123] Methods for producing proteins such as antibodies are well known. The present binding proteins, such as antibodies, can be produced, for example, in mammalian host cells using appropriate expression constructs. Mammalian cell lines available as hosts for expression include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and several other cell lines. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells, and yeast cell lines. Cell lines can be selected based on their expression levels. The binding proteins can be isolated and purified from the host cell culture using well-known methods such as centrifugation, ultracentrifugation, Protein A, Protein G, Protein A / G, or Protein L purification, and / or ion exchange chromatography.

[0124] III. Pharmaceutical Compositions and Uses The present disclosure also provides pharmaceutical compositions comprising the TfR binding proteins herein. Pharmaceutical compositions may include one or more pharmaceutically acceptable excipients, carriers, or diluents. As used herein, "pharmaceutically acceptable" with respect to "carriers," "excipients," or "diluents" includes suitable solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. In some embodiments, the pharmaceutical composition is a sterile aqueous solution and may include a buffer; a surfactant; a polyol; an antioxidant; and / or a chelating agent.

[0125] Pharmaceutical compositions comprising the BBB transporters herein loaded with an enzyme (e.g., a lysosomal enzyme) are useful for treating human patients having or at risk of developing an enzyme deficiency (e.g., a lysosomal enzyme deficiency). In some embodiments, the enzyme or enzyme activity is deficient (e.g., insufficient or completely absent) in the patient. In some embodiments, the GAA transporters herein can be used to treat patients in need of higher GAA activity levels, for example, patients with a congenital GAA deficiency. In some embodiments, the GAA BBB transporters can be used to restore GAA activity. For example, the GAA BBB transporters can be used to treat Pompe disease in human patients in need thereof. Pompe disease (also known as acid α-glucosidase deficiency, acid maltase deficiency, glycogen storage disease type II, GSD type II, and glycogen storage disease type II) is an inherited disorder of glycogen metabolism caused by the absence or significant deficiency of the lysosomal enzyme GAA. The GAA transporters herein can be used to treat late-onset Pompe disease (LOPD) and / or infantile-onset Pompe disease (IOPD).

[0126] Pharmaceutical compositions comprising the BBB transporters herein loaded with an oligonucleotide (e.g., ASO or siRNA) are useful, for example, for treating human patients who would benefit from delivery of the oligonucleotide to the CNS. In some embodiments, the BBB transporter can be used to transport the oligonucleotide to specific non-CNS target tissues rich in TfR expression, such as in the peripheral nervous system (e.g., sciatic nerve), skeletal muscle, internal organs (e.g., heart or spleen), or another tissue. When the oligonucleotide is an ASO or siRNA, the oligonucleotide-loaded BBB transporter can be useful for treating human patients who would benefit from knockdown of the target of the ASO or siRNA.

[0127] As used herein, the terms "treat," "treatment," and "treating" refer to a deliberate intervention in a physiological disease state that results in a lessening of the severity of a disease or condition; a decrease in the duration of a disease or condition; an improvement or elimination of one or more symptoms associated with a disease or condition; or the provision of a beneficial effect to a subject with a disease or condition. Treatment does not require a cure of the underlying disease or condition.

[0128] The pharmaceutical composition can be provided to a patient at a dosage strength and frequency determined as necessary by a medical professional. A therapeutically effective amount is an amount sufficient to ameliorate one or more symptoms associated with the disease or affliction being treated. A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a binding protein herein prevents a subject from developing a disease or promotes the regression or stabilization of a disease, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention or delay of functional impairment or disability resulting from the disease affliction.

[0129] The present disclosure also provides use of the present antibody or antigen-binding fragment for the manufacture of a medicament comprising a therapeutic molecule, wherein the medicament is capable of crossing the BBB. In some embodiments, the present disclosure provides use of the present enzyme-loaded TfR binding protein for the manufacture of a medicament for treating a subject with an enzyme deficiency (e.g., a GAA-loaded TfR binding protein for treating Pompe disease). In some embodiments, the present disclosure provides use of an oligonucleotide-loaded TfR binding protein for the manufacture of a medicament for treating a subject in need thereof, wherein the oligonucleotide can be an ASO or siRNA.

[0130] The present disclosure also provides uses of the present antibodies or antigen-binding fragments for diagnostic processes (e.g., in vitro or ex vivo). For example, the antibodies and antigen-binding fragments can be used to detect and / or measure levels of TfR in biological samples (e.g., tumor biopsies, tissue samples, or blood samples) from patients. Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistochemistry. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the antibodies or antigen-binding fragments described herein.

[0131] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations thereof, such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or otherwise clear from the context.

[0132] According to this disclosure, a backward reference in a dependent claim is intended as a shorthand for a direct and explicit disclosure of any and all combinations of the claims indicated by the backward reference. Furthermore, the headings herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0133] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Example]

[0134] Example 1: Generation and characterization of murine 531 mAb A. m531 Design Protocol: 531-1L2_ Mouse The purpose of this study was to generate and identify antibodies that bind to TfR. To this end, mice were immunized with human tissue expressing human TfR. Hybridomas were then selected, and the sequence of the antibody, designated 531-1L2_mouse, was obtained by RT-PCR and sequencing. The V of 531-1L2_mouse mAb H and V L The sequences are set forth in SEQ ID NOs: 4 and 8, respectively. The HCDRs are set forth in SEQ ID NOs: 5 to 7, and the LCDRs are set forth in SEQ ID NOs: 9 to 11.

[0135] B. Measurement of affinity of 531-1L2 mice by SPR The binding of 531-1L2_mouse Ab to human, cynomolgus monkey, and mouse TfR was assessed using SPR at pH 7.4. Experiments were performed on a Bruker MASS-2 instrument using HBS-EP+ (Cytiva Life Sciences, #BR100826) as the running buffer and sample diluent. An HCA sensor chip (Bruker, #1862614) was immobilized with an anti-his antibody (His Capture Kit, Cytiva Life Sciences, #28995056) according to the supplier's instructions. Approximately 10,000 RU of anti-his was obtained. His-tagged human transferrin receptor protein—human TfR extracellular domain of SEQ ID NO:39, cynomolgus monkey TfR extracellular domain of SEQ ID NO:40, or mouse TfR extracellular domain of SEQ ID NO:41 (5 μg / ml)—was captured at 10 μL / min for 1 minute. A concentration series of 531-1L2_mouse Ab was then injected over the human transferrin receptor surface at 30 μL / min for 4 min, and dissociation was monitored for 5 min. The surface was regenerated with a single 1-min pulse of 10 mM glycine-HCl pH 1.5. Bruker Sierra Analyzer software was used for analysis: sensorgrams were double-referenced with a reference surface (subtraction of bulk and weak nonspecific binding) and a blank (drift removal), and curves were fitted with a 1:1 binding model.

[0136] This experiment confirms that the 531-1L2_mouse antibody binds to the TfR extracellular domain. The binding affinity to TfR is shown in Table 1 below.

[0137] [Table 1]

[0138] C. Competition between 531-1L2 mouse antibody and transferrin by SPR The purpose of this study was to determine whether the 531-1L2_mouse antibody affects the binding of transferrin to its receptor.

[0139] Experiments were performed on a Biacore™ T200 instrument using HBS-EP+ (Cytiva Life Sciences, #BR100826) as the running buffer and sample diluent. Approximately 500 RU of 531-1L2 mouse antibody was covalently immobilized onto a C1 sensor chip (Cytiva Life Sciences, #BR100540) using an amine coupling kit (Cytiva Life Sciences, #BR100050). A 3-minute injection of 50 nM hTfR (R&D Systems, #2474-TR; SEQ ID NO: 1, with a G142S SNP substitution associated with Caucasian populations) onto the 531-1L2 mouse surface was immediately followed by a 3-minute injection of 100 nM human transferrin (Sigma, #T4132; represented as SEQ ID NO: 54, where the peptide signal is cleaved) at 10 μL / min. Approximately 150 RU of hTfR bound to the 531-1L2_mouse antibody and approximately 100 RU of human transferrin bound to the hTfR were measured. A 3-minute injection of 50 nM hTfR onto the 531-1L2_mouse surface was immediately followed by a 3-minute injection of 100 nM 531-1L2_mouse antibody at 10 μL / min. Approximately 150 RU of hTfFR bound to the 531-1L2_mouse antibody was measured, and no binding of the 531-1L2_mouse antibody was detectable (competitive control). The surface was regenerated with a 30-second pulse of 10 mM glycine-HCl (pH 2.5) at 10 μL / min.

[0140] The inventors observed that there was no competition between the 531-1L2_mouse antibody and transferrin for binding to its receptor.

[0141] Example 2: Generation of humanized Fab antibodies The goal of this study was to generate humanized 531 variants (Fab format) with optimal binding properties to hTfR and cTfR (i.e., approximately 1 log or less between the affinity for cTfR and the affinity for hTfR).

[0142] A. Generation and Expression of Humanized Fab Variants The technique applied to engineer the humanized Fab variants of the 531-1L2_murine antibody was CDR grafting, in which the CDRs of a non-human antibody are grafted onto a human framework.

[0143] First, the V of 531-1L2 mouse antibody (SEQ ID NOs: 4 and 8) H and V L From the CDR positions identified within the sequences, residues important for CDR loop conformation, antibody structure, and affinity and specificity for human and cynomolgus TfR (e.g., Vernier residues, contact residues, and anchors) were identified. Several human heavy chain germline (IGHV3-23*05 set forth in SEQ ID NO: 12 and IGHV1-46*01 set forth in SEQ ID NO: 13) and human light chain germline (IGKV1-39*01 set forth in SEQ ID NO: 15) sequences were selected as acceptors for CDR grafting, as they provided frameworks that were closest to the murine framework and were thought to support the murine CDR structures.

[0144] The sequences of the human IMGT CDRs were then replaced with residues from the mouse IMGT CDRs. One or more of the critical residues identified above were backmutated (reverted to the mouse sequence), and residues within the CDRs were modified as needed to avoid chemical instability. Five humanized V H Sequences (SEQ ID NOs: 17-21) and four humanized V L The sequences (SEQ ID NOs: 22-25) were generated and combined to generate humanized Fab variants.

[0145] Humanized anti-TfR variants in a Fab-His tag format were expressed by transient transfection of HEK293 cells. The Fab proteins were purified using a two-step process (IMAC and SEC) to achieve a purity of at least 95%. Prior to affinity determination by SPR, the His tag was removed by cleavage with TEV protease.

[0146] B. Affinity Measurement of Humanized Fab Variants for Various Species TfRs by SPR The binding of the humanized Fab variants to human, cynomolgus monkey, and mouse TfR was assessed using SPR at pH 7.4. Experiments were performed on a Bruker 2 instrument using HBS-EP+ (Cytiva Life Sciences, #BR100826) as the running buffer and sample diluent. HCA sensor chips (Bruker, #1862614) were immobilized with anti-His antibodies (His Capture Kit, Cytiva Life Sciences, #28995056) according to the manufacturer's instructions. Approximately 10,000 RU of anti-His was obtained. His-tagged hTfR ectodomain (SEQ ID NO:39), cTfR ectodomain (SEQ ID NO:40), or mouse TfR ectodomain (SEQ ID NO:41) (5 μg / ml) was captured at 10 μL / min for 1 minute. A concentration series of the humanized Fab variants was then injected over the hTfR surface at 30 μL / min for 4 min, and dissociation was monitored for 5 min. The surface was regenerated with a single 1-min pulse of 10 mM glycine-HCl at pH 1.5. Bruker Sierra Analyzer software was used for analysis: sensorgrams were double-referenced with a reference surface (subtraction of bulk and weak nonspecific binding) and a blank (drift removal), and curves were fitted with a 1:1 binding model.

[0147] Candidate mutants were selected based on their affinity to human and cynomolgus monkey TfR and the cynomolgus monkey / human ratio. This selection process yielded 10 mutants (see Table 2; SEQ: sequence number; NB: no binding). None of the mutants bound to mouse TfR (mTfR). We then analyzed the V-likelihood of these 10 mutants and their respective CDRs and CDR combinations. H and V L The sequence was determined, and one mutant (531v25) was selected for further experiments (see Example 3).

[0148] [Table 2]

[0149] The HC and LC of 531v25 Fab are set forth in SEQ ID NOs: 30 and 31, respectively. H and V L The sequences are set forth in SEQ ID NOs: 21 and 25, respectively. HCDRs 1 to 3 are set forth in SEQ ID NOs: 26, 28 and 7, respectively, and LCDRs 1 to 3 are set forth in SEQ ID NOs: 29, 10 and 11, respectively.

[0150] Example 3: TfR binding properties of 531v25-derived constructs A. Affinity measurement The purpose of this study was to compare the TfR binding affinity of various 531v25-derived constructs: (1) 531v25Fab ​​as in Example 2, and (2) 531v25Fab-FcOL (LC is set forth in SEQ ID NO: 31, HC is set forth in SEQ ID NO: 33, and Fc polypeptide is set forth in SEQ ID NO: 34).

[0151] The affinity of the constructs for human and cynomolgus TfR (and cynomolgus / human ratio) was assessed using SPR and biolayer interferometry (BLI) (see Table 3).

[0152] [Table 3]

[0153] Affinity was assessed by SPR as described in Example 2B, except that 531v25Fab ​​or 531v25Fab-FcOL was used instead of the humanized Fab variants.

[0154] Affinity by BLI was assessed using an Octet® HTX instrument under the following conditions: equilibrium dissociation constant (K DThe binding activity of the ligands was determined with Octet® in HBSP buffer (10 mM HEPES, 150 mM NaCl, 0.05% polysorbate 20, pH 7.4). A streptavidin probe was used to immobilize a fixed concentration of ligand: hTfR or cTfR (both with site-specific biotinylation on the N-terminal Avitag); in this experiment, the hTfR consisted of an AviTag (SEQ ID NO: 55) fused to the N-terminus of the extracellular domain of human TfR (residues 90-760 of SEQ ID NO: 1), and the cTfR consisted of an AviTag fused to the N-terminus of the extracellular domain of cTfR (amino acid residues 90-760 of SEQ ID NO: 2). The ligands were tested for binding and dissociation to the analyte (531v25-Fab-FcOL) in a two-fold serial dilution series from 250 nM to 3.91 nM. A 1:1 binding model with global fitting was applied after double subtraction for buffer and probe-analyte interactions with an association window of 0–90 s and a dissociation window of 0–60 s.

[0155] SPR data confirm that the 531v25Fab-FcOL construct retained the affinity characteristics of 531v25Fab, including the cynomolgus / human affinity ratio. Furthermore, BLI yielded similar affinity determinations, and therefore similar affinity ratios, for 531v25Fab-FcOL for human and cynomolgus TfR.

[0156] Competition for transferrin by B.531v25Fab-FcOL was not observed by BLI. We sought to confirm the absence of binding competition with transferrin (for TfR) upon saturation with either holo-TF (holo-transferrin) or 531v25Fab-FcOL using BLI. The 531v25Fab-FcOL molecule was as detailed in Example 3A.

[0157] Equilibrium dissociation constant (K D) was determined with Octet® in HBSP buffer (10 mM HEPES, 150 mM NaCl, 0.05% polysorbate 20, pH 7.4). A streptavidin probe was used to immobilize a fixed concentration of ligand hTfR with site-specific biotinylation on an N-terminal AviTag fused to the N-terminus of the hTfR extracellular domain. The immobilized hTfR was then saturated with 500 nM of either human holo-transferrin (#2914-HT, R&D systems) or 531v25Fab-FcOL. Potentially competing ligands were then tested for binding and dissociation to the blocked analyte (531v25Fab-FcOL) in a two-fold serial dilution series from 500 nM to 31.25 nM. A 1:1 binding model with global fitting was applied after double subtraction for buffer and probe-analyte interactions with a 0-90 s association and 0-60 s dissociation window. Ligands were tested for binding and dissociation to the analyte (531v25Fab-FcOL) in a two-fold serial dilution series from 250 nM to 3.91 nM. A 1:1 binding model with global fitting was applied after double subtraction for buffer and probe-analyte interactions.

[0158] The data show that transferrin binding was not affected by pre-binding of 531v25Fab-FcOL. Saturation of holo-transferrin with hTfR had minimal effect on 531v25Fab-FcOL binding to hTfR. These data also show that there is no competition between 531v25Fab-FcO and transferrin for binding to the transferrin receptor (see Table 4).

[0159] [Table 4]

[0160] C. Biodistribution of monovalent 531v25-Fab-FcOL after a single dose in hTfR-KI mice hTfR-KI mice were administered either 531v25-Fab-FcOL (as described above) or a human IgG1 control Ab (Southern Biotech, catalog no. 0151K-14) at a dose of 70 nmol / kg via intravenous injection. Mice were lethally anesthetized 1, 3, and 24 hours after administration. Blood was collected in EDTA for plasma isolation, followed by perfusion (8 mL / min for 5 minutes) with Dulbecco's phosphate-buffered saline containing heparin and Ca / Mg. Tissues were homogenized using a Precellys® 2 mL Hard Tissue Homogenization Ceramic Bead Kit in PBS containing 1% NP-40 Surfact Amps detergent (ThermoFisher) and Pierce protease inhibitor (A32955). Finally, the anti-TfR construct concentration in the tissue (per wet tissue weight) was quantified by human IgG MSD (MSD Human / NHP Isotyping Panel #K150JLD-1, Meso Scale Discovery) according to the manufacturer's protocol.

[0161] The monovalent 531v25-Fab-FcOL had increased brain and spinal cord biodistribution (see Table 5) and faster plasma clearance (data not shown) compared to the control IgG, confirming that the 531v25 binding domain can reach the brain and CNS via the transferrin receptor.

[0162] [Table 5]

[0163] Example 4: Epitope and paratope mapping of 531v25 Fab We sought to determine the localization of the human and cynomolgus TfR epitopes recognized by 531v25 Fab using cryo-electron microscopy (cryo-EM).

[0164] In this study, purified complexes between hTfR (SEQ ID NO: 39) or cTfR (SEQ ID NO: 40) and 531v25 Fab were further purified using a Superdex200 3.3 / 300 column pre-equilibrated with PBS buffer. UltraAufoil® 0.6 / 1 on a 300-well gold mesh grid was glow-discharged at 22 mA for 45 seconds, and 3 μL of each sample, at concentrations of 1 and 0.64 mg / ml, respectively, was added to the grid and quenched in liquid ethane. Data from the grid was collected on a Glacios™ microscope (Thermo Fisher) equipped with a Flacon 4 camera at 200 keV. Images in EER format were recorded using an EPU at a nominal magnification of 240,000x with a pixel size of 0.58 Å and a defocus range of -0.8 to -2.2. The dose on the camera during an exposure time of 4.72 seconds was 60 e- / Å2 and the number of fractions was 162 with 54 final frames.

[0165] A total of 7,000 and 4,630 images were taken for each grid, respectively. Data analysis was performed using CryoSPARC v3. The values ​​at which the FSC curves fell below 0.143 were used to estimate the resolution of the final reconstructions at 2.5 Å and 2.73 Å for human and cynomolgus TfR, respectively. The two cryo-EM maps were sharpened using Phenix and then used to fit the atomic coordinates of the TfR and 531v25 Fab. The atomic coordinates underwent several rounds of manual (in Coot) and real-space refinement (in Phenix). The amino acid residues of hTfR and cTfR with atoms within 4 Å of 531v25 Fab atoms are depicted in Figures 1A and 1B, respectively. These distances were measured initially using UCSF Chimera software, followed by PISA and UCSF ChimeraX software. Amino acid residues of hTfR and cTfR with atoms within 5 angstroms distance from 531v25 Fab atoms (as determined using UCSF Chimera software) are represented in the alignment below.

[0166] As shown in Figures 1A and 1B, 531v25 Fab binds to K261, K358, T491, S492, N493, K495, H515, V517, T518, Q520 (R520 for cTfR), Q524, D525, N527, S530, K531, E533, D560, and E582 of hTfR and cTfR within a resolution distance of 4 Å as determined by UCSF Chimera software. The 531v25 Fab binds to K261, K358, T491, S492, N493, F494, K495, H515, V517, T518, Q520 (R520 for cTfR), L522, Q524, D525, N527, S530, K531, E533, D560, and E582 of hTfR and cTfR within a resolution distance of 4 Å as determined by PISA and UCSF ChimeraX software.

[0167] As shown in the alignment below, 531v25 Fab binds to K261, K287, K358, T491, S492, N493, F494, K495, M510, H515, V517, T518, Q520 (R520 for cTfR), F521 (S521 for cTfR), L522, Y523, Q524, D525, N527, S530, K531, V532, E533, E559, D560, D562 and E582 (boxed below) of hTfR and cTfR within a resolution distance of 5 Å. [ka]

[0168] Interestingly, we observed that 531v25 Fab binds to the protease-like domain (side) of TfR, which corresponds to a portion of TfR that is less variable between humans and cynomolgus monkeys. Two amino acid differences between human and cynomolgus monkey TfR have been identified (at positions 520 and 521), which have been shown by cryo-EM to not affect the interaction between 531v25Fab ​​and TfR.

[0169] In parallel with the determination of the epitope residues, the 531v25 paratope residues and the interactions between these paratope and epitope residues were determined using PISA and UCSF ChimeraX software.

[0170] The following V H 13 residues and V L Three residues of were found to be within 4.0 Å of the epitope residues (human TfR): V H Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112 and Y113 and V L F38, Y56, and R66 in (all residue positions are defined according to IMGT numbering). Twelve of these residues are located in the CDRs: Y28, R36, and Y37 are located in HCDR1; D57, S59, V62, and E64 are located in HCDR2; R108, L109, P110, Y111, Y112, and Y113 are located in HCDR3; F38 is located in LCDR1; and Y56 is located in LCDR2. One of the residues, R66, is located in LFR3.

[0171] Due to the flexibility of the human TfR, V H Two additional residues (S26 located in HFR1 and Y38 located in HCDR1) were found to be within 4.0 Å of the epitope residues depending on the conformation of the hTfR monomer. S26 and Y38 can be within 4.0 Å of the epitope residues in one hTfR monomer conformation and slightly more than 4.0 Å away from the epitope residues in another hTfR monomer conformation. Therefore, these two residues are thought to have weaker interactions with human TfR.

[0172] The interface residues within 4.0 Å were determined and are listed below: V of 531v25 H -Y28(531v25 V H ) is within 4.0 Å of residue K261 of human TfR (SEQ ID NO: 1); -R36 is within 4.0 Å of residues K495, K531, and E533; -Y37 is within 4.0 Å of residues N493 and D560; -D57 is within 4.0 Å of residues N527 and K531; -S59 is within 4.0 Å of residues S530 and K531; -V62 is within 4.0 Å of residues N527 and S530; -E64 is within 4.0 Å of residue N527; -R108 is within 4.0 Å of residues N493 and D560; -L109 is within 4.0 Å of residues S492 and D560; -P110 is within 4.0 Å of residues S492 and N493; -Y111 is within 4.0 Å of residues S492, F494, H515, T518, and L522; -Y112 is within 4.0 Å of residues T491, S492, H515, V517, and T518; -Y113 is within 4.0 Å of residues Q520 and Q524.

[0173] V of 531v25 L : -F38(531v25 V L ) is within 4.0 Å of residue Q520 of human TfR (SEQ ID NO: 1); -Y56 is within 4.0 Å of residues V517 and T518; -R66 is within 4.0 Å of residue E582.

[0174] V H The interface residues within 4.0 Å for the two additional residues are: -S26(531v25 V H ) is within 4.0 Å of residues K261 and K358 of human TfR (SEQ ID NO: 1); -Y38 is within 4.0 Å of residue D525.

[0175] Among the interface residues within 4.0 Å defined above, the paratope residues involved in hydrogen bonds and salt bridges with the epitope residues have been determined (hydrogen bonds and salt bridges are considered to be strong interactions between an antibody and an antigen): V H R36, Y37, D57, S59, R108, Y111, and Y113 and V L Y56 and R66 in V (residue positions are defined according to IMGT numbering). H Residues S26 and Y38 in were also shown to form hydrogen bonds with epitope residues when within 4.0 Å. The following hydrogen bonds and salt bridges were determined: V of 531v25 H -R36(531v25 V H ) can form a hydrogen bond with residue K531 of human TfR (SEQ ID NO: 1); -Y37 can form a hydrogen bond with residue N493; -D57 can form a hydrogen bond with residue N527 and a salt bridge with residue K531; -S59 is capable of forming a hydrogen bond with residue K531 of SEQ ID NO: 1; - R108 can form a hydrogen bond with residue N493 and a salt bridge with residue D560; -Y111 can form a hydrogen bond with residue S492 and a hydrogen bond with residue L522; -Y113 can form a hydrogen bond with residue Q524.

[0176] V of 531v25 L -Y56(531v25 V L ) can form a hydrogen bond with residue V517 and residue T518 of human TfR (SEQ ID NO: 1); -R66 can form a salt bridge with residue E582.

[0177] If it is within 4.0 Å of an epitope residue, V H The hydrogen bonds of the two additional residues in are as follows: -S26(531v25 V H ) can form a hydrogen bond with residue K261 of human TfR (SEQ ID NO: 1); -Y38 can form a hydrogen bond with residue D525.

[0178] Example 5: Generation and characterization of the 531v25-GAA molecule Four anti-TfR-GAA molecules with fusions of GAA with different parts of the anti-TfR binding entity: - anti-TfR 531v25mAb-GAA: LC of SEQ ID NO: 31, HC of SEQ ID NO: 33 and HC-GAA fusion of SEQ ID NO: 38; - anti-TfR 531v25-Fab-LC-GAA: LC of SEQ ID NO: 36 and HC of SEQ ID NO: 30; - anti-TfR 531v25-Fab-FcOL-GAA: LC of SEQ ID NO: 31, HC of SEQ ID NO: 33 and Fc-GAA fusion of SEQ ID NO: 37; and - Anti-TfR 531v25-Fab-FcOL-LC-GAA: LC of SEQ ID NO: 36, HC of SEQ ID NO: 33 and Fc polypeptide of SEQ ID NO: 34.

[0179] A. Affinity Measurements and GAA Activity Next, we sought to compare the GAA activity and binding properties of these four anti-TfR-GAA molecules (see Table 6 for activity and ELISA data, and Table 7 for SPR and BLI data). Recombinant GAA (SEQ ID NO: 35) was used as a control.

[0180] Specific (GAA) activity was measured using a fluorometric enzyme assay with a synthetic substrate (4-methylumbelliferyl α-D-glucopyranoside, M9766, Sigma). Briefly, standard curves (3.9–250 ng / mL) of GAA and anti-TfR-GAA constructs were prepared in dilution buffer (0.1% BSA, 0.2 M sodium acetate, 0.4 M KCl, pH 3.9). 15 μL of each standard sample (GAA or anti-TfR-GAA construct) was mixed with 50 μL of 5 mM 4-methylumbelliferyl α-D-glucopyranoside and incubated at 37°C for 1 h. The reaction was stopped by adding 135 μL of 1 M glycine-NaOH buffer (pH 12.5). Fluorescence of the reaction solution was measured (excitation at 360 nm and emission at 450 nm). The amount of product was calculated by fitting a 4-methylumbelliferone (M-1381, Sigma) standard curve (0.039–5 nmol / well) by linear regression. Specific activity (µmol product / min / mg GAA) was calculated by dividing the amount of product by the reaction time and the amount of GAA.

[0181] The enzymatic activity of all molecules tested ranged from 70 to 120% of GAA activity. This assay also demonstrated similar activity of purified anti-TfR-GAA protein compared to GAA (Table 6).

[0182] The binding kinetics of four anti-TfR-GAA molecules to hTfR- and cTfR-expressing 300.19 cells were determined by flow cytometry. 300.19 is a lymphoma-derived murine pre-B cell line stably transfected by nuclear perforation with plasmids expressing either hTfR or cTfR (SEQ ID NO: 1 and SEQ ID NO: 2, respectively).

[0183] 300.19 TFRC-expressing cells were plated at 5 × 10 5Plates were coated with 100 μL / well of TfR-GAA molecules, incubated for 45 minutes at 4°C, and washed three times with PBS 1% BSA. 100 μL / well of goat anti-human IgG conjugated with Alexa488 (Jackson ImmunoResearch, #109-545-098) was added, incubated for 45 minutes at 4°C, and washed three times with PBS 1% BSA. Antibody binding was assessed after centrifugation and resuspension of the cells by adding 200 μL / well of PBS 1% BSA and reading using a Guava® easyCyte™ 8HT flow cytometry system. EC 50 Values ​​were estimated using BIOST@T-BINDING and are reported in Table 6.

[0184] [Table 6]

[0185] The affinity of 531v25Fab-FcOL-LC-GAA and 531v25Fab-FcOL-GAA (SPR) was evaluated as in Example 3A and reported in Table 7. The affinity of 531v25-mAb-GAAx2 molecule (GAA sequence fused to both HCs) was also measured by SPR.

[0186] These data indicate that the affinity characteristics and cynomolgus / human ratio of the two (monovalent) 531v25 Fab-FcOL molecules are preserved in the fusion construct with GAA. The FACS data also confirmed that the 531v25 antibody can bind to hTfR and cTfR in their native conformations. Data obtained with the 531v25-mAb-GAAx2 molecule demonstrate both affinity and avidity for TfR binding as a result of the two TfR binding sites.

[0187] [Table 7]

[0188] We also sought to evaluate the affinity of 531v25Fab-FcOL-GAA by BLI and compare it to 531v25-Fab-FcOL (i.e., without GAA). The affinity of 531v25Fab-FcOL-GAA by BLI was evaluated as in Example 3A, and the data are reported in Table 8. These data indicate that the affinity profile of the 531v25 molecule, as well as the cynomolgus / human ratio, are preserved in the fusion construct with GAA.

[0189] [Table 8]

[0190] B. No competition for transferrin and human TfR binding from 531v25-Fab-FcOL-GAA was observed as determined by BLI. We sought to confirm by BLI that saturation with either holo-TF (holo-transferrin) or 531v25Fab-FcOL-GAA did not result in binding competition with transferrin for TfR, as assayed in Example 3B, and the data are reported in Table 9.

[0191] The data show that transferrin binding was not affected by pre-binding of 531v25Fab-FcOL-GAA. Saturation of holotransferrin to the human TfR had minimal effect on the binding of 531v25Fab-FcOL-GAA to the hTfR. These data indicate that there is no competition between 531v25Fab-FcOL-GAA and transferrin for binding to the transferrin receptor.

[0192] [Table 9]

[0193] Example 6: Evaluation of the 531v25-GAA fusion molecule in mice Next, we compared four anti-TfR-GAA molecules in a mouse model of Pompe disease in which the endogenous GAA gene was knocked out and the extracellular domain of the human TfR gene was knocked in. After administration of the anti-TfR-GAA molecules in mice, glycogen levels were assessed in various organs (brain, spinal cord, heart, and four muscles) (Figures 4A-G and Table 10). The anti-TfR-GAA molecules evaluated—531v25mAb-GAA, 531v25Fab-LC-GAA, 531v25Fab-FcOL-GAA, and 531v25Fab-FcOL-LC-GAA—were as detailed above. Vehicle and GAA were used as controls.

[0194] Pompe mice (GAA-KO 6neo / 6neo, GAAtm1 Rabn) (Raben et al., J. Biol. Chem. (1998) 273:19086-92) were crossed to become double homozygous for both alleles with humanized TfR knock-in (KI) mice (hTfR1-KI mice). hTfR1-KI mice [C57BL / 6-TfR-tm2618(TfR)Arte (Taconic)] express a chimeric TfR constructed with a human extracellular domain fused to mouse transmembrane and cytoplasmic domains (exons 4-19 of the mouse TfR were replaced by the corresponding human sequences in C57BL / 6 NTAc ES cells without disrupting the mouse 3' untranslated region). Expression is driven from the endogenous mouse TfR1 promoter to comparable levels in all tissues compared to TfR1 in wild-type mice, as determined by qPCR and whole-tissue immunoblot.

[0195] Nine- to 10-month-old hTfR-KI-Pompe mice were administered 180 nmol / kg test substance once weekly for four doses. This is the molar equivalent of 20 mg / kg alglucosidase alfa (GAA). Each test group contained seven mice per treatment (3M, 4F). The antihistamine diphenhydramine was administered to all animals starting with the second dose to mitigate hypersensitivity responses to human protein administration. Animals were euthanized 7 days after the last dose, perfused with PBS, and target tissues were harvested. Tissues were homogenized at 4°C using a bead homogenizer in a 1:10 or 1:50 volume / weight excess of water / tissue. Glycogen content was biochemically quantified using a commercially available colorimetric / fluorimetric assay kit (BioVision, Milpitas, CA) according to the manufacturer's instructions. A non-hydrolase-treated sample was used for each sample to correct for glucose background. All values ​​were back-calculated to mg glycogen / gr initial tissue.

[0196] Alglucosidase alfa (GAA), which does not cross the blood-brain barrier, did not alter glycogen levels in the brain or spinal cord of hTfR-KI-Pompe mice compared to vehicle controls. In contrast, treatment with all anti-TfR-GAA constructs resulted in significant glycogen clearance in the CNS (Figures 4A and 4B). Percent reductions are calculated relative to vehicle or GAA treatment, as shown in Table 10 below.

[0197] The data show that both 531v25Fab-LC-GAA and 531v25Fab-FcOL-GAA were most effective in the brain, with 84% and 74% glycogen reduction, respectively. Regarding the myocardium, all anti-TfR-targeted GAA constructs were more effective at lowering tissue glycogen, with Fab-LC-GAA and Fab-FcOL-GAA showing superior efficacy (Figure 4C and Table 10). In all skeletal muscles, anti-TfR-GAA constructs with Fc domains appeared more potent than Fab-LC-GAA, and mAb-GAA was comparable to or better than Fab-FcOL-GAA (Figure 4D-G and Table 10).

[0198] [Table 10]

[0199] Example 7: Pharmacokinetics (PK) of anti-TfR-GAA molecules in human TfR-knockin mice Next, we sought to determine the behavior of various anti-TfR-GAA molecules in mice. The following molecules were used in this study: 531v25mAb-GAA, 531v25Fab-GAA, 531v25Fab-FcOL-GAA, and 531v25Fab-FcOL-LC-GAA.

[0200] Experiments were conducted in hTfR-KI mice (see Example 6). All mice were naive, male and female, 4-6 months of age at the start of the study. For administration, anti-TfR-GAA molecules were prepared in 10 mM histidine (pH 6), 150 mM NaCl formulation buffer and administered as a single intravenous dose of 70 nmol / kg in a dose volume of 10 mL / kg into the tail vein. For each compound, a total of three animals per sampling time were evaluated over the 7-day study period using a terminal sampling procedure (0.25, 2, 5, 24, 48, 72, and 168 hours). Blood, quadriceps, and brain samples were collected according to the sampling time. Blood samples were centrifuged at 1500 g for 10 minutes at 4°C, and the isolated plasma was stored at -80°C until analysis. Before harvesting, brains were flushed in situ with saline to avoid blood contamination, and then homogenized in lysis buffer (1% NP-40). Brains and quadriceps muscles were frozen at -80°C immediately after harvesting.

[0201] After tissue sampling, 5 volumes of lysis buffer were added to tubes containing ceramic beads for the brain hemispheres and metal beads for the quadriceps muscles. Homogenization was achieved using Precellys®: for the brain, two 20-second cycles at 5500 rpm with a 10-second pause, and for the quadriceps muscles, two 20-second cycles at 7500 rpm with a 10-second pause. After centrifugation at 1400 rpm for 30 seconds at 4°C, the tubes were placed on a roller for 1 hour at 4°C, aliquoted into 100 μL volumes in low-binding tubes, and stored at -80°C until analysis. The concentration of each anti-TfR-GAA molecule at each time point was determined by immunoassay using an MSD platform (QuickPlex SQ120). The assay was based on the GAA recognition properties of a rabbit anti-GAA antibody coated onto a microtiter plate (standard MSD 96-well sector plate) (GAA capture) and the use of a goat anti-mouse kappa ruthenium tracer for detection by electrochemiluminescence (Fabκ LC detection). Samples (standards, quality controls, and test samples) were diluted 10-fold in PBS-Tween 0.1% BSA buffer and dispensed into a 96-well microtiter plate. All analyses were performed in duplicate, and the quantification range was 0.0078–1000 ng / mL.

[0202] The PK parameters of the anti-TfR-GAA molecules in hTfR-KI mice are summarized in Tables 11, 12, and 13.

[0203] 531v25Fab-GAA showed the highest clearance compared to the other constructs. There was rapid elimination of all constructs from plasma, with 531v25Fab-GAA having the shortest half-life (t 1 / 2 ) (Table 11). In the brain, the highest exposure was observed for the two 531v25Fab-FcOL constructs with a brain / plasma AUC=4%. The shortest elimination half-life from the brain was observed for the 531v25Fab-GAA construct (Table 12).

[0204] [Table 11]

[0205] Finally, the highest exposure in quadriceps muscle was observed for the two 531v25Fab-FcOL constructs (Table 12). The shortest elimination half-life was observed for 531v25Fab-GAA (Table 13).

[0206] [Table 12]

[0207] [Table 13]

[0208] Taken together, these results indicate that both the 531v25Fab-FcOL-LC-GAA and 531v25Fab-FcOL-GAA molecules exhibit the most favorable PK behavior compared to 531v25Fab-GAA and 531v25mAb-GAA, with the highest plasma exposure, lowest clearance, and highest distribution in the brain and quadriceps muscle.

[0209] Example 8: MALAT1 mRNA knockdown using anti-hTfR 531v25-Fab-FcOL-anti-MALAT-1 ASO oligonucleotide conjugate in hTfR-KI mice To assess whether additional moieties could be efficiently targeted to brain and skeletal muscle, we evaluated ASO-mediated knockdown of MALAT1, a nuclear RNA expressed in most tissues. For this study, the following reagents were prepared:

[0210] 531v25 Fab-FcOL (consisting of SEQ ID NOS: 31, 34, and 56) was transiently expressed in Expi293 cells and then purified by Protein A capture and size-exclusion chromatography final purification steps. SEQ ID NOS: 56 is similar to SEQ ID NOS: 33, but contains an A129C (IMGT numbering; A114C according to Kabat numbering) mutation for site-specific conjugation of MALAT1 ASO (THIOMAB™ technology). To generate the antibody-ASO conjugate (anti-hTfR-MALAT1-ASO conjugate), 531v25 Fab-FcOL was reduced with a 50-fold molar excess of TCEP at room temperature for 3 hours, followed by desalting on a HiPrep 26 / 10 desalting column. The sample was reoxidized overnight and then mixed with a 3 molar excess of mouse MALAT1-ASO (GCATTCTAATAGCAGC; SEQ ID NOS: 57) with an SMCC linker. The MALAT1 sequence is shown below with modifications: 5'-(SMCC)(NHC6)GbsCbsAbsdTsdTs(5MdC)sdTsdAsdAsdTsdAsdGs(5MdC)sAbsGbsCb-3' Nb: LNA residues (including LNA-5MeC and LNA T / LNA-5MeU) dN: DNA residue (5MdC): 5-methyl DNA C s: phosphorothioate backbone modification (NHC6): aminohexyl linker (SMCC): Succinimidyl-trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate

[0211] The progress of the conjugation was monitored on SDS-PAGE gels until completion, and the conjugated complex was then purified from excess unreacted ASO using size exclusion chromatography.

[0212] To generate the ASO alone, the maleimide in the SMCC linker was inactivated with 10 mM cysteine ​​for 30 min at room temperature.

[0213] To evaluate tissue-specific knockdown of MALAT1 mRNA, hTfR-KI mice were administered four doses (twice weekly with a 3-4 day interval) of 400 nmol / kg inactivated SMCC-MALAT1 ASO (N = 5), or 400 nmol / kg molar equivalent ASO in the form of anti-hTfR-MALAT1-ASO conjugate (DAR1) (N = 3), or saline vehicle (N = 5). Three days (72 h) after the fourth dose, mice were anesthetized with ketamine / xylazine and transcardially perfused with heparinized DBP containing Ca / Mg. Tissues (brain, heart, gastrocnemius muscle, quadriceps muscle, spleen, and sciatic nerve) were harvested and weighed for quantitative PCR of MALAT1, using β-actin as a housekeeping gene. RNA was isolated from frozen tissue samples. Tissues were homogenized using a bead mill homogenizer in TRIzol / chloroform in a 2 mL tube containing 2.8 mm ceramic beads. RNA was then isolated according to the Qiagen RNeasy 96 QIAcube HT kit instructions. cDNA was generated using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor. Finally, qPCR was performed in a QuantStudio™ 7 Flex using PrimeTime® Gene Expression Master Mix (IDT) and the following TaqMan® qPCR primer sets: β-actin forward 5′-GTACGACCAGAGGCATACAG-3′ (SEQ ID NO: 58); reverse 5′-ACCGTGAAAAGATGACCCAG-3′ (SEQ ID NO: 59) Probe / 5HEX / ACCTTCAAC / ZEN / ACCCCAGCCATGTA / 3IABkFQ / (ACCTTCAAC: SEQ ID NO: 60; ACCCAGCCATGTA: SEQ ID NO: 61); mouse MALAT1 from ThermoFisher TaqMan® MGB Probes: MALAT1 Mm01227912_s1 FAM-MGB mouse.

[0214] Our data show that the anti-hTfR-MALAT1-ASO conjugate significantly knocks down MALAT1 mRNA in the brain, heart, gastrocnemius, and quadriceps muscles, as well as in the spleen and sciatic nerve, compared with mice treated with equimolar intravenously administered free MALAT1-ASO or vehicle. Significance was determined using one-way ANOVA with Dunnett's multiple comparisons (GraphPad v9.5.0). * =p<0.05, ** =p<0.005, *** p<0.0001 vs. vehicle) (Figure 6).

[0215] These findings demonstrate that 531v25 Fab-FcOL can be used to target oligonucleotides such as MALAT1-ASO for gene knockdown in brain, sciatic nerve, and skeletal muscle tissue in vivo. These data provide evidence that various moieties (including enzymes and oligonucleotides) can be efficiently targeted to brain and muscle cells with functional effects such as recruitment of enzyme function (GAA) or reduction of gene expression levels (MALAT1) when conjugated to our 531v25-anti-TfR moiety.

[0216] array The sequences described in this disclosure are summarized in the table below (SEQ: Sequence Number).

[0217] [Table 14]

[0218] [Table 15]

[0219] [Table 16]

[0220] [Table 17]

[0221] Table 18

[0222] Table 19

[0223] Table 20

[0224] Table 21

[0225] Table 22

[0226] Table 23

Claims

1. An anti-human transferrin receptor (TfR) antibody or an antigen-binding fragment thereof, a heavy chain CDR (HCDR) 1 comprising GYTFTRYY (SEQ ID NO: 26), or GYTFTRYW (SEQ ID NO: 27), or DYTFTRYW (SEQ ID NO: 5); HCDR2 comprising IDPSVSET (SEQ ID NO: 28) or IDPSVSEC (SEQ ID NO: 6), and HCDR3 comprising SQIRLPYYYAMDS (SEQ ID NO: 7); and light chain CDR (LCDR) 1 comprising QDISSF (SEQ ID NO: 29) or QDINSF (SEQ ID NO: 9); LCDR2 comprising YTS (SEQ ID NO: 10), and Optionally, an LCDR3 comprising QQGNTLPRT (SEQ ID NO: 11) An antibody or antigen-binding fragment thereof comprising:

2. The HCDR1 to HCDR3 are SEQ ID NOs: 26, 28, and 7; SEQ ID NOs: 27, 28 and 7, or comprising SEQ ID NOs: 5, 6 and 7, respectively; and / or The LCDR1 to LCDR2 are SEQ ID NOs: 29 and 10, or comprising SEQ ID NOs: 9 and 10, respectively; and / or The antibody or antigen-binding fragment of claim 1 , wherein the LCDR3 comprises SEQ ID NO:

11.

3. (i) HCDR1-3 comprising SEQ ID NOs: 26, 28, and 7, respectively, and LCDR1-2 comprising SEQ ID NOs: 29 and 10, respectively; (ii) HCDR1-3 comprising SEQ ID NOs: 5-7, respectively, and LCDR1-2 comprising SEQ ID NOs: 9 and 10, respectively; (iii) HCDR1-3 comprising SEQ ID NOs: 26, 28, and 7, respectively, and LCDR1-3 comprising SEQ ID NOs: 29, 10, and 11, respectively; or (iv) HCDR1-3 comprising SEQ ID NOs: 5-7, respectively, and LCDR1-3 comprising SEQ ID NOs: 9-11, respectively.

2. The antibody or antigen-binding fragment of claim 1, comprising:

4. A heavy chain variable domain (V) comprising any one of SEQ ID NOs: 17 to 21. Ｈ ), and / or A light chain variable domain (V) comprising any one of SEQ ID NOs: 22 to 25 Ｌ ) 2. The antibody or antigen-binding fragment of claim 1, comprising:

5. SEQ ID NOs: 21 and 25, SEQ ID NOs: 21 and 24, SEQ ID NOs: 21 and 23, SEQ ID NOs: 19 and 23, SEQ ID NOs: 18 and 23, SEQ ID NOs: 17 and 23, SEQ ID NOs: 21 and 22, SEQ ID NOs: 20 and 22, SEQ ID NOs: 18 and 22, SEQ ID NOs: 17 and 22, or V comprising SEQ ID NOs: 4 and 8, respectively Ｈ and V Ｌ 2. The antibody or antigen-binding fragment of claim 1, comprising:

6. The antibody mutations that reduce antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), optionally including S298N, T299A, and Y300S; mutations that improve the serum half-life of the antibody, optionally including M252Y, S254T, and T256E; Optionally, knob-in-hole mutations include Y349C, T366S, L368A, and Y407V for hole mutations; and S354C and T366W for IgG1 knob mutations; and Purification-facilitating mutations, optionally including H435R and Y436F (EU numbering) Human IgG optionally containing one or more mutations selected from １ , IgG ２ , or IgG ４ The antibody according to any one of claims 1 to 5, which is of an isotype subtype.

7. The antibody of claim 6, wherein the antibody comprises a heavy chain and a light chain comprising SEQ ID NOs: 32 and 31, respectively.

8. The antigen-binding fragment of any one of claims 1 to 5, wherein the antigen-binding fragment is monovalent and / or comprises a Fab.

9. 9. The antigen-binding fragment of claim 8, comprising a heavy chain and a light chain comprising SEQ ID NOs: 30 and 31, respectively.

10. the antigen-binding fragment is a heterotrimer comprising a heavy chain, a light chain, and an Fc polypeptide, wherein the heavy chain and the Fc polypeptide dimerize to form an Fc domain, and optionally, the heavy chain and the Fc polypeptide are selected from the group consisting of human IgG １ , G ２ or IgG ４ The antigen-binding fragment of claim 8 , which is of an isotype subtype.

11. the heavy chain and the Fc polypeptide Mutations that reduce ADCC and / or CDC, optionally including S298N, T299A, and Y300S; mutations that improve the serum half-life of the antibody, optionally including M252Y, S254T, and T256E; Optionally, knob-in-hole mutations include Y349C, T366S, L368A, and Y407V for hole mutations; and S354C and T366W for IgG1 knob mutations; and Purification-facilitating mutations, optionally including H435R and Y436F (EU numbering) The antigen-binding fragment of claim 10, comprising one or more mutations selected from:

12. The antigen-binding fragment of claim 10, wherein the heavy chain comprises SEQ ID NO: 33, the light chain comprises SEQ ID NO: 31, and the Fc polypeptide comprises SEQ ID NO:

34.

13. (i) an antibody or antigen-binding fragment according to any one of claims 1 to 12, and (ii) a cargo linked to said antibody or antigen-binding fragment, optionally said cargo being a therapeutic compound, and further optionally said therapeutic compound being a protein; A TfR binding protein comprising:

14. The cargo optionally via a peptide linker, of the antibody or antigen-binding fragment, a heavy chain, optionally at the C-terminus of said heavy chain, If present, the Fc polypeptide, optionally at the N-terminus or C-terminus of said Fc polypeptide; or a light chain, optionally at the C-terminus of said light chain are connected, 14. The TfR binding protein of claim 13, further optionally, wherein the peptide linker comprises SEQ ID NO: 42 or 43.

15. 15. The TfR binding protein of claim 13 or 14, wherein the cargo is an enzyme, optionally the enzyme is a lysosomal enzyme, optionally the lysosomal enzyme is acid alpha-glucosidase (GAA), optionally further comprising SEQ ID NO:

35.

16. heavy chain (HC), Light chain (LC), Fc polypeptides, and a human GAA sequence fused to the C-terminus of (a) the HC, (b) the LC, or (c) the Fc polypeptide; 16. The TfR binding protein of claim 15, wherein the heavy chain and the Fc polypeptide dimerize to form an Fc domain.

17. SEQ ID NOs: 33, 36 and 34, or 17. The TfR binding protein of claim 16, comprising three polypeptides comprising SEQ ID NOs: 33, 31 and 37, respectively.

18. 17. The TfR binding protein of claim 16, comprising a Fab and a human GAA sequence fused to the C-terminus of the HC or LC of the Fab.

19. 20. The TfR binding protein of claim 18, comprising two polypeptides comprising SEQ ID NOs: 30 and 36, respectively.

20. It comprises two HCs, two LCs, and a human GAA sequence fused to the C-terminus of one of the two HCs; Optionally, the two LCs each comprise SEQ ID NO: 31, the first HC comprises SEQ ID NO: 33, and the second HC comprises SEQ ID NO:

37. The TfR binding protein of claim 16.

21. 21. A pharmaceutical composition comprising an antibody or antigen-binding fragment of any one of claims 1 to 12, or a TfR binding protein of any one of claims 13 to 20; and a pharmaceutically acceptable excipient.

22. One or more nucleic acid molecules or expression vectors encoding the antibody or antigen-binding fragment of any one of claims 1 to 12, or the TfR-binding protein of any one of claims 13 to 22.

23. 13. A method of making a therapeutic molecule capable of crossing the blood-brain barrier in a human subject, the method comprising linking a therapeutic portion of the molecule to an antibody or antigen-binding fragment of any one of claims 1 to 12.

24. 13. A method of delivering a therapeutic molecule across the blood-brain barrier in a subject in need thereof, comprising administering said therapeutic molecule to said subject, wherein said therapeutic molecule is linked to an antibody or antigen-binding fragment of any one of claims 1 to 12.

25. 13. The antibody or antigen-binding fragment of any one of claims 1 to 12 for use in delivering a therapeutic molecule across the blood-brain barrier in a subject in need thereof.

26. 13. Use of the antibody or antigen-binding fragment of any one of claims 1 to 12 in the manufacture of a medicament for delivering a therapeutic molecule across the blood-brain barrier in a subject in need thereof, wherein the antibody or antigen-binding fragment is linked to the therapeutic molecule.

27. 27. The method of claim 24, the antibody or antigen-binding fragment for use of claim 25, or the use of claim 26, wherein the therapeutic molecule is an enzyme, optionally a lysosomal enzyme, and the subject is deficient in the enzyme or its activity.

28. 16. A method of treating an enzyme deficiency in a subject in need thereof, comprising administering to said subject the TfR binding protein of claim 15.

29. 16. The TfR binding protein of claim 15 for use in treating a subject having a deficiency in the enzyme or its activity.

30. 16. Use of the TfR binding protein of claim 15 in the manufacture of a medicament for treating a subject having a deficiency in the enzyme or its activity.

31. 31. The method, antibody or antigen-binding fragment for use, TfR-binding protein for use, or use of any one of claims 27 to 30, wherein the enzyme is GAA and the subject is suffering from Pompe disease.

32. 13. A method of treating Pompe disease in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment is linked to GAA.

33. 13. The antibody or antigen-binding fragment of any one of claims 1 to 12, for use in treating Pompe disease in a subject in need thereof, wherein the antibody or antigen-binding fragment is linked to GAA.

34. 13. Use of the antibody or antigen-binding fragment of any one of claims 1 to 12 in the manufacture of a medicament for treating Pompe disease in a subject in need thereof, wherein the antibody or antigen-binding fragment is linked to GAA.

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