Transferrin receptor binding proteins

Abstract

The present disclosure provides antibodies and antigen binding fragments that target transferrin receptors. The use of these antibodies and antigen-binding fragments as vectors to deliver therapeutic molecules across the blood-brain barrier is also provided.

Description

Cross - reference to related applications

[0001] This application claims the priority of European Patent Application 22306784.4 filed on December 5, 2022. The disclosure of the priority application is incorporated herein by reference in its entirety. Sequence Listing

[0002] This application contains a sequence listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The electronic copy of the sequence listing created on December 3, 2023 is named 122548.TW005.xml and has a size of 92,235 bytes. Background Art

[0003] Transferrin receptor 1 (TfR), also known as CD71, is a widely expressed transmembrane glycoprotein that is involved in the cellular uptake of iron. TfR imports iron through receptor - mediated endocytosis of transferrin, an iron - binding protein. Since TfR is highly expressed by brain capillary endothelial cells that form the blood - brain barrier (BBB) and transports iron across the BBB via transcytosis, TfR has been investigated as a potential target for molecular shuttles that are 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 significant challenges in using TfR as a target for molecular shuttles. First, the presence of high blood levels of transferrin may require these shuttles to compete with transferrin for binding to TfR. Other challenges include specificity for brain tissue, potential lysosomal degradation, and significant transport into the brain parenchyma (Pulgar, Front Neurosci. (2019) 12:doi10.3389 / fnins.2018.01019). There is still a need for molecular shuttles that target TfR with sufficient brain specificity and efficient uptake. Summary of the Invention

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

[0006] In another aspect, the present disclosure provides an isolated TfR-binding protein comprising: (i) an antibody or antigen-binding fragment thereof as described herein 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 certain embodiments, the enzyme is acid alpha-glucosidase (GAA).

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

[0008] In another aspect, the present disclosure provides nucleic acids and expression vectors encoding the antibodies, antigen-binding fragments, and TfR-binding proteins of the invention, host cells containing such nucleic acids or expression vectors, and methods of using the host cells to produce the antibodies, antigen-binding fragments, and TfR-binding proteins described herein. In some embodiments, the production method comprises culturing the host cells under conditions that permit the 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 present disclosure provides a method of manufacturing a therapeutic molecule capable of crossing the blood-brain barrier (BBB) of a human subject, the method comprising (chemically or recombinantly) linking a 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 of a subject in need thereof, the method comprising administering to the subject a therapeutic molecule, wherein the therapeutic molecule is linked to an antibody or antigen-binding fragment herein. Also provided is an antibody or antigen-binding fragment herein for delivering a therapeutic molecule across the BBB of a subject in need thereof. Also provided is the use of an antibody or antigen-binding fragment herein for delivering a therapeutic molecule across the BBB of a subject in need thereof, or for use in the manufacture of a medicament for such purpose.

[0011] In another aspect, the present 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 lacks the enzyme or its activity. Also provided is an isolated TfR-binding protein herein comprising an enzyme (e.g., a lysosomal enzyme) for treating a subject lacking the enzyme or its activity. Also provided is the use of an isolated TfR-binding protein herein comprising an enzyme (e.g., a lysosomal enzyme) for treating a subject lacking the enzyme or its activity or for use in the manufacture of a medicament for treating a subject lacking the enzyme or its activity. In some embodiments, the enzyme is GAA and the subject has Pompe disease.

[0012] Other features, objects, and advantages of the invention will be apparent from the following detailed description. However, it should be understood that while the embodiments and aspects of the invention are indicated, the detailed description is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A and Figure 1B are diagrams showing the amino acid residues of human TfR ( Figure 1A ) and cynomolgus monkey TfR ( Figure 1B ) having atoms within 4 angstroms of the 531v25 Fab atoms as determined by cryo-electron microscopy.

[0014] Figure 2A is a set of schematic diagrams illustrating three exemplary forms of an anti-TfR binding protein. "mAb": monoclonal antibody. Fab: Fab fragment of a complete antibody. "Fab-fcOL": a monovalent antibody fragment having a light chain, a complete heavy chain, and an Fc polypeptide that dimerizes with the complete heavy chain to form a dimeric Fc domain.

[0015] Figure 2BA set of schematic diagrams showing four exemplary forms of anti-TfR binding proteins containing GAA cargo. "Fab-LC-GAA": GAA fused to the C-terminus of the light chain (LC) of anti-TfR Fab. "Fab-FcOL-LC-GAA": GAA fused to the C-terminus of the LC of anti-TfRFab-FcOL. "Fab-FcOL-GAA": GAA fused to the C-terminus of the Fc polypeptide of anti-TfR Fab-FcOL. "mAb-GAA": GAA fused to the C-terminus of one of the two heavy chains (HC) of anti-TfR mAb.

[0016] Figure 3A Are alignments of two exemplary humanized 531 heavy chains (531v1 V H and 531v2 V H ) with their corresponding human germline sequences IGHV3-23*05 and IGHJ6-01. HCDR1-3 are underlined. The changes in HCDR1 are in bold.

[0017] Figure 3B Are alignments of three additional exemplary humanized 531 heavy chains (531v3 V H 、531v4 V H and 531v5 V H ) with their corresponding human germline sequences IGHV1-46*01 and IGHJ6-01. HCDR1-3 are underlined. The changes in HCDR1 are in bold.

[0018] Figure 3C Are alignments of four exemplary humanized 531 light chains (531v1 VL, 531v2 VL, 531v3 VL and 531v4 VL) with their corresponding human germline sequences IGKV1-39*01 and IGKJ4-01. LCDR1-3 are underlined. The changes in LCDR1 are in bold.

[0019] Figure 4A - Figure 4G Shows the brain ( Figure 4A )、spinal cord ( Figure 4B )、heart ( Figure 4C )、diaphragm ( Figure 4D )、gastrocnemius ( Figure 4E )、quadriceps ( Figure 4F ) and triceps brachii ( Figure 4GGraph of glycogen levels (mean ± SD) in (). Asterisks: all males and females. "f": females (only significant). "m": males (only significant). Dark (top) asterisks: p < 0.05 compared to vehicle. Light (bottom) asterisks: p < 0.05 compared to neoGAA. "GAA": recombinant GAA without a vector.

[0020] Figure 5 Is a list of the amino acid sequences of the three subunits of the 531v25 Fab-FcOL-GAA molecule. The protein is of the human IgG1 isotype subtype. In the light chain (Fab-FcOL-GAA light chain), LCDR1-3 are in bold and the constant region is underlined. In the full heavy chain (Fab-FcOL-GAA heavy chain_jiu), HCDR1-3 are in bold; the constant region is underlined; the NNAS (SEQ ID NO: 53) mutation is double underlined (linear positions S301N / T302A / Y303S, corresponding to S298N / T299A / Y300S Eu numbering); the jiu mutation is italicized and bold (linear positions Y352C / T369S / L371A / Y410V, corresponding to Y349C / T366S / L368A / Y407V Eu numbering); and the RF mutation is bold and double underlined (linear positions H438R / Y439F, corresponding to H435R / Y436F Eu numbering). In the Fc polypeptide / GAA fusion (Fab-FcOL-GAA FcOL_chu + GAA), the Fc polypeptide part is underlined and the GGGG (SEQ ID NO: 42) linker is double underlined; the NNAS mutation is double underlined (linear positions S78N / T79A / Y80S, corresponding to S298N / T299A / Y300S Eu numbering); and the chu mutation is italicized and bold (linear positions S134C / T146W, corresponding to S354C / T366W Eu numbering). Linear positions refer to the actual positions in the sequences shown.

[0021] Figure 6 Is a set of graphs showing tissue-specific knockdown of MALAT1 mRNA in the brain, heart, gastrocnemius, quadriceps, spleen, and sciatic nerve of experimental animals receiving the 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, all compared to vehicle. Detailed Description

[0022] The present disclosure provides isolated binding proteins that bind to human TfR (hTfR), such as antibodies and antigen-binding fragments thereof. These TfR-binding proteins bind to epitopes on the extracellular region of hTfR and do not interfere with the interaction between hTfR and transferrin (its natural ligand). The TfR-binding proteins are excellent BBB transporters and have improved endocytic transport efficiency. In addition, the TfR-binding proteins of the present invention cross-react with cynomolgus monkey TfR (cTfR), thus allowing preclinical studies of proteins in non-human primates (NHPs).

[0023] The present disclosure also provides a specific use of the BBB transporter of the present invention to transport an enzyme (such as a lysosomal enzyme, such as acid α-glucosidase (GAA)) into the central nervous system (CNS). The GAA-loaded transporter of the present invention can be used to restore the CNS (such as the brain and spinal cord) and muscle glycogen of patients in need (such as patients with Pompe disease) to normal levels. As compared with the recombinant GAA used in conventional enzyme replacement therapy, the GAA-loaded transporter of the present invention will show excellent glycogen clearance in the CNS.

[0024] The BBB transporter of the present invention 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. I. TfR binding protein

[0025] The present disclosure provides hTfR-binding proteins (also collectively referred to herein as "BBB transporters") comprising anti-hTfR antibodies and antigen-binding fragments thereof, and hTfR-binding proteins comprising such antibodies or antigen-binding fragments and a cargo to be transported across the BBB. A. TfR Binding Characteristics

[0026] Human TfR is a homodimer composed of two subunits linked by disulfide bonds. Exemplary human TfR amino acid sequences can be found at UniProt accession number P02786 and NCBI accession number NP001121620.1 and have the following amino acid sequence: 1MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AVDEEENADN 51NTKANVTKPK RCSGSICYGT IAVIVFFLIG FMIGYLGYCK GVEPKTECER 101LAGTESPVRE EPGEDFPAAR RLYWDDLKRK LSEKLDSTDF TGTIKLLNEN 151SYVPREAGSQ KDENLALYVE NQFREFKLSK VWRDQHFVKIQVKDSAQNSV 201IIVDKNGRLV YLVENPGGYV AYSKAATVTG KLVHANFGTK KDFEDLYTPV 251NGSIVIVRAG KITFAEKVAN AESLNAIGVL IYMDQTKFPI VNAELSFFGH 301AHLGTGDPYT PGFPSFNHTQ FPPSRSSGLP NIPVQTISRA AAEKLFGNME 351GDCPSDWKTD STCRMVTSES KNVKLTVSNV LKEIKILNIF GVIKGFVEPD 401HYVVVGAQRD AWGPGAAKSG VGTALLLKLA QMFSDMVLKD GFQPSRSIIF 451ASWSAGDFGS VGATEWLEGY LSSLHLKAFT YINLDKAVLG TSNFKVSASP 501LLYTLIEKTM QNVKHPVTGQ FLYQDSNWAS KVEKLTLDNA AFPFLAYSGI 551PAVSFCFCED TDYPYLGTTM DTYKELIERIPELNKVARAA AEVAGQFVIK 601LTHDVELNLD YERYNSQLLS FVRDLNQYRA DIKEMGLSLQ WLYSARGDFF 651RATSRLTTDF GNAEKTDRFV MKKLNDRVMR VEYHFLSPYV SPKESPFRHV 701FWGSGSHTLP ALLENLKLRK QNNGAFNETL FRNQLALATW TIQGAANALS 751GDVWDIDNEF(SEQ ID NO:1)

[0027] 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)( D., Linnaeus University Dissertations, No. 406 / 2021; Lawrence et al., Science (1999) 286(5440):779 - 82).

[0028] The antibody or antigen - binding fragment of the present invention binds to an epitope in a region corresponding to the lateral region of the receptor in the protease - like domain (residues 384 - 605) of hTfR. This represents a unique epitope different from known anti - TfR antibodies that bind to the apical domain of hTfR (see, for example, antibodies 3 and 3N disclosed in EP3088518A1, which have been shown in WO 2022 / 174114 to bind to the apical portion of hTfR). The TfR - binding protein of the present invention does not compete with transferrin for binding to TfR.

[0029] In some embodiments, the TfR - binding protein of the present invention binds 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 and interactions between paratope residues and epitope residues in angstroms. The term "paratope" refers to the antibody residues involved in recognizing and binding to an antigenic epitope. In some embodiments, a paratope / epitope pair can be characterized, for example, by the distance between the antibody and antigen atoms in the bound antibody / antigen complex (such as within 4 or 5 angstroms). In some embodiments, a paratope / epitope pair can be characterized by the hydrogen - bond interactions and / or salt - bridge interactions involved between antibody and antigen residues.

[0030] In some embodiments, the TfR-binding protein of the invention 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 on the European Bioinformatics Institute server) and / or UCSF ChimeraX (available on the University of California, San Francisco server) are used to visualize the paratope / epitope structure and determine the distances and interactions between paratope residues and epitope residues in terms of

[0031] In some embodiments, the TfR-binding protein of the invention 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 to visualize the paratope / epitope structure and determine the distances and interactions between paratope residues and epitope residues in terms of

[0032] In some embodiments, the TfR-binding protein of the invention binds to an epitope that is located, in whole or in part, in the region spanning T491 to D562 of hTfR and thus binds to one or more residues in this region.

[0033] In some embodiments, the TfR-binding protein of the invention also binds to the TfR of non-human primates (NHPs) such as macaques (e.g., cynomolgus macaques (Macaca fascicularis), also known as crab-eating macaques). An exemplary amino acid sequence of cynomolgus macaque TfR (cTfR) can be found in NCBI accession number XP_045243212.1 and is shown below:

[0034] In some embodiments, the TfR-binding protein of the invention binds 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 cTfR (see Figure 1B ). Except for the Q520R substitution from hTfR to cTfR, these residues are identical between hTfR and cTfR. In certain embodiments, UCSF Chimera software is used to visualize the paratope / epitope structure and determine the distances and interactions, measured in , between paratope residues and epitope residues.

[0035] In some embodiments, the TfR-binding protein of the invention binds 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 ). Except for the Q520R substitution from hTfR to cTfR, these residues are identical between hTfR and cTfR. In certain embodiments, PISA and / or UCSF ChimeraX software is used to visualize the paratope / epitope structure and determine the distances and interactions, measured in , between paratope residues and epitope residues.

[0036] In some embodiments, the TfR-binding protein of the present invention 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. Except for the Q520R and F521S substitutions from hTfR to cTfR, these residues are identical between hTfR and cTfR. In certain embodiments, UCSF Chimera software is used to visualize the paratope / epitope structure and determine the distances and interactions, measured in between the paratope residues and the epitope residues.

[0037] Residues of hTfR and / or cTfR bound by the TfR-binding protein of the present invention can be identified using, for example, UCSF Chimera, PISA, or UCSF ChimeraX software or any combination thereof.

[0038] In some embodiments, the cTfR-binding protein of the present invention binds to an epitope located in whole or in part in the region spanning T491 to D562 of cTfR and thus binds to one or more residues in this region.

[0039] The extracellular domains (residues 90 - 760) of hTfR and cTfR are 95% identical and 97% homologous to each other. The alignment between the two extracellular domains is shown below. In the alignment, "*" (asterisk) indicates positions with a single fully conserved residue; ":" (colon) indicates conservation between groups with strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix), and "." (period) indicates conservation between groups with weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix). The regions forming the protease-like domain ( same as above) are underlined.

[0040] In some embodiments, the TfR-binding protein of the invention binds to residues conserved between the extracellular domains of hTfR and cTfR. In additional embodiments, the TfR-binding protein of the invention binds to residues conserved between the protease-like domains of the extracellular domains of hTfR and cTfR. In additional embodiments, the TfR-binding protein of the invention binds to one or more residues in the region spanning positions 384 - 605 of hTfR (e.g., one or more residues in the region spanning positions 491 - 562), e.g., residues conserved between hTfR and cTfR in this region. In some embodiments, the TfR-binding protein of the invention binds to at least one or all of the boxed residues in the alignment above.

[0041] The anti-TfR antibodies and antigen-binding fragments of the invention specifically bind hTfR and cTfR. "Specifically" means that the antibodies and fragments bind to hTfR and cTfR with an affinity as described herein or higher. To act as a vector across the BBB, the BBB transporter can have a suitable affinity for hTfR. In addition, to facilitate preclinical studies of the BBB transporter in NHP animal models, the BBB transporter can have a suitable affinity for cTfR, and the difference (ratio) between the affinity of the BBB transporter for cTfR and its affinity for hTfR can be within about 1 log. Several techniques can be used to characterize the TfR-binding affinity (K D ), such as surface plasmon resonance (SPR, using e.g., BIAcore TM ) or biolayer interferometry (BLI, using e.g., Octet from ForteBio TM ). Flow cytometry assays using cells expressing membrane-bound hTfR or cTfR (e.g., FACS) can also be used to determine the EC 50 or IC 50 values of the BBB transporter; these values indicate binding to human and cynomolgus monkey TfR in their native conformation.

[0042] In some embodiments, the BBB transporter has a K D of about 1 - 50 nM (e.g., 1 - 30, 1 - 20, or 1 - 10 nM) for hTfR and a K D of about 1 - 200 nM (e.g., 1 - 150 or 1 - 100 nM) for cTfR, as determined by SPR (e.g., using BIAcore TM)。In some embodiments, surface plasmon resonance (SPR) assays are performed with the extracellular domain of the TfR to be evaluated. Such assays are described in Example 2B below. In some embodiments, the ratio of the binding affinity of the BBB transporter for hTfR to cTfR is between 1:1 and 1:20, between 1:1 and 1:15, between 1:1 and 1:10, between 1:1 and 1:9, between 1:2 and 1:9, between 1:3 and 1:9, between 1:4 and 1:9, between 1:5 and 1:9, or between 1:6 and 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).

[0043] 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 D , and a K of about 1 - 200 nM (e.g., 1 - 150 or 1 - 100 nM) for cTfR D , as determined by Biolayer Interferometry (BLI) (e.g., using Octet ). In some embodiments, BLI is performed with the extracellular domain of the TfR to be evaluated. Such assays are further described in Example 3A below. In some embodiments, the ratio of the binding affinity of the BBB transporter for hTfR to cTfR is between 1:1 and 1:20, between 1:1 and 1:15, between 1:1 and 1:10, between 1:1 and 1:9, between 1:2 and 1:9, between 1:3 and 1:9, between 1:4 and 1:9, between 1:5 and 1:9, or between 1:6 and 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).

[0044] 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 50 , and an EC of about 1 - 200 nM (e.g., 1 - 150 or 1 - 100 nM) for cTfR 50, as determined using mammalian cells engineered to express the TfR to be evaluated by flow cytometry (e.g., FACS). In some embodiments, the mammalian cells can be murine cells (e.g., murine pre-B cells), hamster cells, or human cells. Such assays are further illustrated in Example 5A below. In some embodiments, the BBB transporter has an EC 50 ratio for hTfR and cTfR between 1:1 and 1:20, between 1:1 and 1:15, between 1:1 and 1:10, between 1:1 and 1:9, between 1:2 and 1:9, between 1:3 and 1:9, between 1:4 and 1:9, between 1:5 and 1:9, or between 1:6 and 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).

[0045] Also contemplated are BBB transporters having any combination of the above functional properties.

[0046] Exemplary forms of the TfR-binding proteins are shown in Figure 2A (antibodies and their fragments) and Figure 2B (cargo-loaded TfR-binding proteins). These forms will be further described below. B. Anti-TfR antibodies and antigen-binding fragments

[0047] The TfR-binding proteins herein include chimeric or humanized anti-TfR antibodies and antigen-binding fragments having antigen-binding domains of murine origin. These antibodies and antigen-binding fragments can be used as transporters to carry a cargo (payload) across the BBB.

[0048] The term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region or domain (V H ) and a heavy chain constant region (C H ). Each light chain consists of a light chain variable region or domain (V L ) and a light chain constant region (CL). V H and V L domains can be further subdivided into regions of high variability, called "complementary determining regions" (CDRs), interspersed with more conserved regions, called "framework regions" (FRs). Each V H and V LComposed of three CDRs (HCDR herein refers to the CDR from the heavy chain; and LCDR herein refers to the CDR from the light chain) and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0049] The precise amino acid sequence boundaries of a given CDR or FR can be defined by several well-known systems, including those described by the following: 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 Plückthun, J Mol Biol. (2001) 309(3):657-70 ("Aho" system); and Whitelegg and Rees, Protein Eng. (2000) 13(12):819-24 ("AbM" system). The boundaries of a given CDR or FR can 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. The numbering of both the Kabat and Chothia systems is based on the most common antibody region sequence lengths, where insertions are provided by inserting letters (e.g., "30a"). These two systems place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The contact system is based on the analysis of complex crystal structures and is similar to the Chothia system in many respects. In certain embodiments, the CDRs of the antibodies described herein can 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.

[0050] In some embodiments, the BBB transporter proteins herein have a configuration different from that of a full - length antibody having two full - length heavy chains and two full - length light chains. For example, the BBB transporter protein is an antigen - binding fragment of a full - length tetrameric antibody and still retains the TfR - binding properties of the full - length antibody. The term "antigen - binding fragment" or "antigen - binding portion" herein encompasses immunoglobulin in genetically engineered and / or otherwise modified forms that do not have the conventional full - length tetrameric structure. The term encompasses intracellular antibodies, peptibodies, diabodies, triabodies, tetra - bodies, 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 fragments and Fab - FcOL fragments described herein.

[0051] In some embodiments, the anti - TfR antibodies herein are humanized antibodies. A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non - human CDRs (e.g., murine) and all or substantially all of the FR amino acid residues are derived from human FRs (i.e., the acceptor). A humanized antibody may also include at least a portion of the heavy - chain and / or light - chain constant regions derived from human antibodies. Compared to the non - human parental antibody from which the humanized antibody is derived, the humanized antibody has reduced immunogenicity in humans. To retain the specificity and affinity of the parental antibody, some FR residues in the human acceptor may be replaced with the corresponding residues from the non - human parental antibody (back - mutation). 1. TfR - binding domain

[0052] The TfR - binding domain of the BBB transporter protein (including the anti - TfR antibody or its antigen - binding fragment) may comprise a heavy - chain variable domain (V H ) and a light - chain variable domain (V L ), wherein the heavy - chain CDRs (HCDRs) 1 - 3 and the light - chain CDRs (LCDRs) 1 and 2 are derived from a murine parental anti - TfR antibody. In certain embodiments, HCDRs 1 - 3 and LCDRs 1 - 3 are derived from a murine parental anti - TfR antibody.

[0053] In some embodiments, the BBB transporter protein herein comprises an HCDR1 containing X1YTFTRYX2, optionally wherein 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).

[0054] In some embodiments, the BBB transporter herein comprises an HCDR2 containing IDPSVSEX3, optionally where X3 can be T or C (SEQ ID NO: 51). For example, the HCDR2 can comprise IDPSVSET (SEQ ID NO: 28) or IDPSVSEC (SEQ ID NO: 6).

[0055] In some embodiments, the BBB transporter herein comprises an HCDR3 containing SQIRLPYYYAMDS (SEQ ID NO: 7).

[0056] In some embodiments, the BBB transporter herein comprises an LCDR1 containing QDIX4SF, optionally where X4 can be S or N (SEQ ID NO: 52). For example, the LCDR1 can comprise QDISSF (SEQ ID NO: 29) or QDINSF (SEQ ID NO: 9).

[0057] In some embodiments, the BBB transporter herein comprises an LCDR2 containing YTS (SEQ ID NO: 10).

[0058] In some embodiments, the BBB transporter herein comprises an LCDR3 containing QQGNTLPRT (SEQ ID NO: 11).

[0059] In some embodiments, the BBB transporter comprises a) HCDR1-3 and b) LCDR1 and 2 or LCDR1-3, as described in the above paragraphs. That is, HCDR1-3 comprises SEQ ID NO:26, 28, and 7, respectively; comprises SEQ ID NO:27, 28, and 7, respectively; or comprises SEQ ID NO:5, 6, and 7, respectively; and / or i) LCDR 1 and 2 comprise SEQ ID NO:29 and 10, respectively, or comprise SEQ ID NO:9 and 10, respectively; or ii) LCDR1-3 comprises SEQ ID NO:29, 10, and 11, respectively; or comprises SEQ ID NO:9, 10, and 11, respectively. In certain embodiments, the BBB transporter comprises: (i) HCDR1-3 comprising SEQ ID NO:26, 28, and 7, respectively, and LCDR1 and 2 comprising SEQ ID NO:29 and 10, respectively, or (ii) HCDR1-3 comprising SEQ ID NO:5-7, respectively, and LCDR1 and 2 comprising SEQ ID NO:9 and 10, respectively. In certain embodiments, the BBB transporter comprises: (i) HCDR1-3 comprising SEQ ID NO:26, 28, and 7, respectively, and LCDR1-3 comprising SEQ ID NO:29, 10, and 11, respectively, or (ii) HCDR1-3 comprising SEQ ID NO:5-7, respectively, and LCDR1-3 comprising SEQ ID NO:9-11, respectively.

[0060] In some embodiments, the BBB transporter comprises HCDR1-3, LCDR1, and LCDR2 as described in the above paragraphs, 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 containing GYTFTRYY (SEQ ID NO:26), GYTFTRYW (SEQ ID NO:27), or DYTFTRYW (SEQ ID NO:5); - HCDR2 containing IDPSVSET (SEQ ID NO:28) or IDPSVSEC (SEQ ID NO:6); and - HCDR3 containing SQIRLPYYYAMDS (SEQ ID NO:7); and - LCDR1 containing QDISSF (SEQ ID NO:29) or QDINSF (SEQ ID NO:9), - LCDR2 containing YTS (SEQ ID NO:10), and - Optionally, an LCDR3 containing QQGNTLPRT (SEQ ID NO:11), and further comprising an HFR1 with the last residue being S and / or an LFR3 with the first residue being R (e.g., an HFR1 with the last residue being S and an LFR3 with the first residue being R). In certain embodiments, the BBB transporter comprises: - An HCDR1 containing GYTFTRYY (SEQ ID NO:26), GYTFTRYW (SEQ ID NO:27), or DYTFTRYW (SEQ ID NO:5); - An HCDR2 containing IDPSVSET (SEQ ID NO:28) or IDPSVSEC (SEQ ID NO:6); and an HCDR3 containing SQIRLPYYYAMDS (SEQ ID NO:7); and - An LCDR1 containing QDISSF (SEQ ID NO:29) or QDINSF (SEQ ID NO:9), - An LCDR2 containing YTS (SEQ ID NO:10), and - An LCDR3 containing QQGNTLPRT (SEQ ID NO:11), and further comprising an HFR1 with the last residue being S and / or an LFR3 with the first residue being R (e.g., an HFR1 with the last residue being S and an LFR3 with the first residue being R).

[0061] In some embodiments, HCDR1-3 respectively comprise SEQ ID NO:26, 28, and 7, respectively comprise SEQ ID NO:27, 28, and 7, or respectively comprise SEQ ID NO:5, 6, and 7; LCDR1 and 2 respectively comprise SEQ ID NO:29 and 10, or respectively comprise SEQ ID NO:9 and 10; and HFR1 has a last residue S and / or LFR3 has a first residue R. In certain embodiments, the BBB transporter comprises: HCDR1-3 respectively comprising SEQ ID NO:26, 28, and 7; LCDR1 and 2 respectively comprising SEQ ID NO:29 and 10; and HFR1 having a last residue S and / or LFR3 having a first residue R (e.g., HFR1 having a last residue S and LFR3 having a first residue R). In other embodiments, the BBB transporter comprises: HCDR1-3 respectively comprising SEQ ID NO:5-7; LCDR1 and 2 respectively comprising SEQ ID NO:9 and 10; and HFR1 having a last residue S and / or LFR3 having a first residue R (e.g., HFR1 having a last residue S and LFR3 having a first residue R).

[0062] In some embodiments, the BBB transporter comprises HCDR1-3 and LCDR1-3 as described in the above paragraph, and further comprises HFR1 having a last residue S, LFR3 having a first residue R, or both. For example, in certain embodiments, HCDR1-3 respectively comprise SEQ ID NO:26, 28, and 7, respectively comprise SEQ ID NO:27, 28, and 7, or respectively comprise SEQ ID NO:5, 6, and 7; LCDR1-3 respectively comprise SEQ ID NO:29, 10, and 11, or respectively comprise SEQ ID NO:9-11; and HFR1 has a last residue S and / or LFR3 has a first residue R. In certain embodiments, the BBB transporter comprises: HCDR1-3 respectively comprising SEQ ID NO:26, 28, and 7; LCDR1-3 respectively comprising SEQ ID NO:29, 10, and 11; and HFR1 having a last residue S and / or LFR3 having a first residue R (e.g., HFR1 having a last residue S and LFR3 having a first residue R). In other embodiments, the BBB transporter comprises: HCDR1-3 respectively comprising SEQ ID NO:5-7; LCDR1-3 respectively comprising SEQ ID NO:9-11; and HFR1 having a last residue S and / or LFR3 having a first residue R (e.g., HFR1 having a last residue S and LFR3 having a first residue R).

[0063] In some embodiments, the BBB transporter comprises: HCDR1-3 that are SEQ ID NO:26, 28, and 7, respectively, and LCDR1 and 2 that are SEQ ID NO:29 and 10, respectively, wherein said HCDR1-3 and LCDR1 and 2 together comprise one to five (e.g., 1, 2, 3, 4, or 5, one to two, one to three, or one to four) mutations in the five CDRs, and wherein the mutations do not occur 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), wherein the BBB transporter further comprises HFR1 with a last residue of S and / or LFR3 with a first residue of R (e.g., HFR1 with a last residue of S and LFR3 with a first residue of R).

[0064] In some embodiments, the BBB transporter comprises: HCDR1-3 that are SEQ ID NO:26, 28, and 7, respectively, and LCDR1-3 that are SEQ ID NO:29, 10, and 11, respectively, wherein said HCDR1-3 and LCDR1-3 together comprise one to five (e.g., 1, 2, 3, 4, or 5, one to two, one to three, or one to four) mutations in the six CDRs, and wherein the mutations do not occur 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), wherein the BBB transporter further comprises HFR1 with a last residue of S and / or LFR3 with a first residue of R (e.g., HFR1 with a last residue of S and LFR3 with a first residue of R).

[0065] In some embodiments, when bound to hTfR, the BBB transporter comprises: VH , as defined by the IMGT numbering, which includes: Optionally, S26 within 4.0 of residues K261 and K358 of SEQ ID NO:1 ; Y28 within 4.0 of residue K261 of SEQ ID NO:1 ; R36 within 4.0 of residues K495, K531 and E533 of SEQ ID NO:1 ; Y37 within 4.0 of residues N493 and D560 of SEQ ID NO:1 ; Optionally, Y38 within 4.0 of residue D525 of SEQ ID NO:1 ; D57 within 4.0 of residues N527 and K531 of SEQ ID NO:1 ; S59 within 4.0 of residues S530 and K531 of SEQ ID NO:1 ; V62 within 4.0 of residues N527 and S530 of SEQ ID NO:1 ; E64 within 4.0 of residue N527 of SEQ ID NO:1 ; R108 within 4.0 of residues N493 and D560 of SEQ ID NO:1 ; L109 within 4.0 of residues S492 and D560 of SEQ ID NO:1 ; P110 within 4.0 of residues S492 and N493 of SEQ ID NO:1 ; Y111 within 4.0 of residues S492, F494, H515, T518 and L522 of SEQ ID NO:1 ; Y112 within 4.0 of residues T491, S492, H515, V517 and T518 of SEQ ID NO:1 ; and Y113 within 4.0 of residues Q520 and Q524 of SEQ ID NO:1 ; and VL, as defined by the IMGT numbering, which includes: F38 within 4.0 of residue Q520 of SEQ ID NO:1 ; Y56 within 4.0 of residues V517 and T518 of SEQ ID NO:1 ; and R66 within 4.0 of residue E582 of SEQ ID NO:1 . In certain embodiments, UCSF Chimera, PISA, or UCSF ChimeraX software, or any combination thereof, is used to visualize the paratope / epitope structure and to determine the distances and interactions, in angstroms, between paratope residues and epitope residues.

[0066] In some embodiments, when bound to hTfR, the BBB transporter comprises: V H , as defined by IMGT numbering, which comprises: Optionally, S26, which may form a hydrogen bond with residue K261 of SEQ ID NO:1; R36, which may form a hydrogen bond with residue K531 of SEQ ID NO:1; Y37, which may form a hydrogen bond with residue N493 of SEQ ID NO:1; Optionally, Y38, which may form a hydrogen bond with residue D525 of SEQ ID NO:1; D57, which may 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 may form a hydrogen bond with residue K531 of SEQ ID NO:1; R108, which may 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 may form a hydrogen bond with residue S492 of SEQ ID NO:1 and a hydrogen bond with residue L522 of SEQ ID NO:1; and Y113, which may form a hydrogen bond with residue Q524 of SEQ ID NO:1; and V L , as defined by IMGT numbering, which comprises: Y56, which may form a hydrogen bond with residue V517 of SEQ ID NO:1 and a hydrogen bond with residue T518 of SEQ ID NO:1; and R66, which may form a salt bridge with residue E582 of SEQ ID NO:1.

[0067] In some embodiments, the BBB transporter comprises: HCDR1-3 of SEQ ID NO: 26, 28, and 7 respectively, LCDR1 and 2 of SEQ ID NO: 29 and 10 respectively, optionally LCDR3 of SEQ ID NO: 11, and optionally HFR1 with the last residue being S and / or LFR3 with the first residue being R (e.g., HFR1 with the last residue being S and LFR3 with the first residue being R), wherein when binding to hTfR, the BBB transporter comprises V H , as defined by the IMGT numbering, which comprises: a) S26 within 4.0 of residues K261 and K358 of SEQ ID NO: 1 ; b) Y28 within 4.0 of residue K261 of SEQ ID NO: 1 ; c) R36 within 4.0 of residues K495, K531, and E533 of SEQ ID NO: 1 ; d) Y37 within 4.0 of residues N493 and D560 of SEQ ID NO: 1 ; e) Y38 within 4.0 of residue D525 of SEQ ID NO: 1 ; f) D57 within 4.0 of residues N527 and K531 of SEQ ID NO: 1 ; g) S59 within 4.0 of residues S530 and K531 of SEQ ID NO: 1 ; h) V62 within 4.0 of residues N527 and S530 of SEQ ID NO: 1 ; i) E64 within 4.0 of residue N527 of SEQ ID NO: 1 ; j) R108 within 4.0 of residues N493 and D560 of SEQ ID NO: 1 ; k) L109 within 4.0 of residues S492 and D560 of SEQ ID NO: 1 ; l) P110 within 4.0 of residues S492 and N493 of SEQ ID NO: 1 ; m) Y111 within 4.0 of residues S492, F494, H515, T518, and L522 of SEQ ID NO:1 ; n) Y112 within 4.0 of residues T491, S492, H515, V517, and T518 of SEQ ID NO:1; and / or ; o) Y113 within 4.0 of residues Q520 and Q524 of SEQ ID NO:1; and / or ; V L , as defined by IMGT numbering, which includes: p) F38 within 4.0 of residue Q520 of SEQ ID NO:1 ; q) Y56 within 4.0 of residues V517 and T518 of SEQ ID NO:1; and / or ; r) R66 within 4.0 of residue E582 of SEQ ID NO:1 ; or any combination of a)-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 complementarity-determining region (CDR) / epitope structure and to determine the distances and interactions between CDR residues and epitope residues in angstroms.

[0068] In some embodiments, the BBB transporter comprises: HCDR1-3 of SEQ ID NO:26, 28, and 7, respectively, LCDR1 and 2 of SEQ ID NO:29 and 10, respectively, optionally LCDR3 of SEQ ID NO:11, and optionally HFR1 with a last residue of S and / or LFR3 with a first residue of R (e.g., HFR1 with a last residue of S and LFR3 with a first residue of R), wherein when bound to hTfR, the BBB transporter comprises V H , as defined by IMGT numbering, which includes: a) S26 that can form a hydrogen bond with residue K261 of SEQ ID NO:1 b) R36 that can form a hydrogen bond with residue K531 of SEQ ID NO:1 c) Y37 that can form a hydrogen bond with residue N493 of SEQ ID NO:1 d) Y38 that can form a hydrogen bond with residue D525 of SEQ ID NO:1; e) D57 that 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 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; g) R108 that 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 that can form 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 that can form a hydrogen bond with residue Q524 of SEQ ID NO:1; and / or V L , as defined by the IMGT numbering, which comprises: j) Y56 that can form a hydrogen bond with residue V517 of SEQ ID NO:1 and a hydrogen bond with residue T518 of SEQ ID NO:1; and k) R66 that can form a salt bridge with residue E582 of SEQ ID NO:1; or any combination of a)-k) (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11).

[0069] In some embodiments, the BBB transporter comprises a V that contains residues S26, Y28, R36, Y37, Y38, D57, S59, V62, E64, R108, L109, P110, Y111, Y112 and Y113 H ; and a V that contains residues F38, Y56 and R66 L ; wherein the residue positions are defined according to the IMGT numbering. In certain embodiments, the BBB transporter binds to an epitope of hTfR that comprises 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.

[0070] In some embodiments, the BBB transporter comprises: V containing residues Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113, optionally further containing S26 and / or Y38 H ; and V containing residues F38, Y56, and R66 L ; wherein the residue positions are according to the 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 a particular embodiment, when the BBB transporter binds to hTfR, S26 is within 4.0 of residue K261 of SEQ ID NO:1 ; S26 is within 4.0 of residue K358 of SEQ ID NO:1 ; Y28 is within 4.0 of residue K261 of SEQ ID NO:1 ; R36 is within 4.0 of residue K495 of SEQ ID NO:1 ; R36 is within 4.0 of residue K531 of SEQ ID NO:1 ; R36 is within 4.0 of residue E533 of SEQ ID NO:1 ; Y37 is within 4.0 of residue N493 of SEQ ID NO:1 ; Y37 is within 4.0 of residue D560 of SEQ ID NO:1 ; Y38 is within 4.0 of residue D525 of SEQ ID NO:1 ; D57 is within 4.0 of residue N527 of SEQ ID NO:1 ; D57 is within 4.0 of residue K531 of SEQ ID NO:1 ; S59 is within 4.0 of residue S530 of SEQ ID NO:1 inside; S59 is within 4.0 of residue K531 of SEQ ID NO:1 inside; V62 is within 4.0 of residue N527 of SEQ ID NO:1 inside; V62 is within 4.0 of residue S530 of SEQ ID NO:1 inside; E64 is within 4.0 of residue N527 of SEQ ID NO:1 inside; R108 is within 4.0 of residue N493 of SEQ ID NO:1 inside; R108 is within 4.0 of residue D560 of SEQ ID NO:1 inside; L109 is within 4.0 of residue S492 of SEQ ID NO:1 inside; L109 is within 4.0 of residue D560 of SEQ ID NO:1 inside; P110 is within 4.0 of residue S492 of SEQ ID NO:1 inside; P110 is within 4.0 of residue N493 of SEQ ID NO:1 inside; Y111 is within 4.0 of residue S492 of SEQ ID NO:1 inside; Y111 is within 4.0 of residue F494 of SEQ ID NO:1 inside; Y111 is within 4.0 of residue H515 of SEQ ID NO:1 inside; Y111 is within 4.0 of residue T518 of SEQ ID NO:1 inside; Y111 is within 4.0 of residue L522 of SEQ ID NO:1 inside; Y112 is within 4.0 of residue T491 of SEQ ID NO:1 inside; Y112 is within 4.0 of residue S492 of SEQ ID NO:1 inside; Y112 is within 4.0 of residue H515 of SEQ ID NO:1 inside; within 4.0 of residue V517 of SEQ ID NO:1 for Y112 ; within 4.0 of residue T518 of SEQ ID NO:1 for Y112 ; within 4.0 of residue Q520 of SEQ ID NO:1 for Y113 ; within 4.0 of residue Q524 of SEQ ID NO:1 for Y113 ; within 4.0 of residue Q520 of SEQ ID NO:1 for F38 ; within 4.0 of residue V517 of SEQ ID NO:1 for Y56 ; within 4.0 of residue T518 of SEQ ID NO:1 for Y56 ; within 4.0 of residue E582 of SEQ ID NO:1 for R66 ; 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). 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 and interactions between paratope residues and epitope residues in terms of addition to or alternatively to the above 4.0 distance embodiments, in certain embodiments, when the BBB transporter binds 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; 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).

[0071] In some embodiments, the BBB transporter herein comprises complementarity-determining regions that comprise V H amino acid residues (i.e., residues in VH) R36, Y37, D57, S59, R108, Y111, and Y113; and V L amino acid residues (i.e., residues in VL) Y56 and R66; wherein the numbering is according to IMGT. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue S26. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue Y38 (or W38). In some embodiments, the complementarity-determining regions further comprise V H amino acid residues S26 and Y38 (or W38). In some embodiments, the complementarity-determining regions further comprise at least one V selected from the following H amino acid residues: Y28, V62, E64, L109, P110, and Y112. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue Y28. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue V62. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue E64. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue L109. In some embodiments, the complementarity-determining regions further comprise V H amino acid residue P110. In some embodiments, the complementarity-determining regions further comprise VH The amino acid residue Y112. In some embodiments, the complementary site further comprises V H The amino acid residues S26, Y28, Y38 (or W38), V62, E64, L109, P110, and Y112. In some embodiments, the complementary site further comprises V H The amino acid residues Y28, V62, E64, L109, P110, and Y112. In some embodiments, the complementary site further comprises V L The amino acid residue F38. In some embodiments, the complementary site further comprises V H The amino acid residues S26, Y28, Y38 (or W38), V62, E64, L109, P110, and Y112 and V L The amino acid residue F38. In some embodiments, the complementary site further comprises V H The amino acid residues Y28, V62, E64, L109, P110, and Y112 and V L The amino acid residue F38.

[0072] In some embodiments, the BBB transporter comprises a complementary site that comprises: at least 9 (e.g., at least 10, at least 11, at least 12, at least 13, or at least 14) amino acid residues selected from S26, Y28, R36, Y37, Y38 (or W38), D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113 in V H ; and / or at least two amino acid residues selected from F38, Y56, and R66 in V L ; wherein the numbering is according to IMGT.

[0073] In some embodiments, the BBB transporter comprises a complementary site that comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues selected from V H The amino acid residues S26, Y28, R36, Y37, Y38 (or W38), D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113, wherein the numbering is according to IMGT. In some embodiments, the BBB transporter comprises a complementary site that comprises at least one (e.g., 1, 2, or 3) amino acid residues selected from V LAmino acid residues F38, Y56, and R66, where the numbering is according to IMGT. In some embodiments, the BBB transporter comprises a complementarity-determining region (CDR) that comprises: (i) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) selected from V H Amino acid residues S26, Y28, R36, Y37, Y38 (or W38), D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; and (ii) at least one (e.g., 1, 2, or 3) selected from V L 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 adjusted accordingly.

[0074] In some embodiments, the BBB transporter comprises a complementarity-determining region (CDR) that comprises V H Amino acid residues Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; and optionally also comprises S26 and / or Y38 (or W38), where the numbering is according to IMGT. In some embodiments, the BBB transporter comprises a complementarity-determining region (CDR) that comprises V L Amino acid residues F38 and Y56, and optionally also comprises R66, where the numbering is according to IMGT. In some embodiments, the BBB transporter comprises a complementarity-determining region (CDR) that comprises V H Amino acid residues Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112, and Y113; and optionally also comprises S26 and / or Y38 (or W38); and (ii) V L Amino acid residues F38 and Y56, and optionally also comprises 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 adjusted accordingly.

[0075] In some embodiments, the BBB transporter comprises: HCDR1 containing Y28, R36, Y37, and Y38 (or W38); HCDR2 containing D57, S59, V62, and E64; HCDR3 containing R108, L109, P110, Y111, Y112, Y113; LCDR1 containing F38; LCDR2 containing Y56; and LFR3 starting with an arginine residue (R66). In some embodiments, the BBB transporter comprises: HCDR1 containing Y28, R36, Y37, and Y38 (or W38); HCDR2 containing D57, S59, V62, and E64; and HCDR3 containing R108, L109, P110, Y111, Y112, Y113. In some embodiments, the BBB transporter comprises: LCDR1 containing F38; LCDR2 containing Y56; and LFR3 starting with an arginine residue (R66).

[0076] In some embodiments, the BBB transporter comprises: V H , which comprises Optionally, HFR1 (S26) ending with a serine residue; HCDR1, which contains Y28, R36, Y37, and optionally Y38 (or W38) and contains 1 - 5 additional amino acid residues of SEQ ID NO:26; HCDR2, which contains D57, S59, V62, and E64 and contains 1 - 4 additional amino acid residues of SEQ ID NO:28; and HCDR3, which contains R108, L109, P110, Y111, Y112, Y113 and contains 1 - 7 additional amino acid residues of SEQ ID NO:7; and / or V L , which comprises LCDR1, which contains F38 and contains 1 - 5 additional amino acid residues of SEQ ID NO:29; LCDR2, which contains Y56 and contains 1 - 2 additional amino acid residues of SEQ ID NO:10; LFR3 starting with an arginine residue (R66); and Optionally, LCDR3 containing 1 - 9 amino acid residues of SEQ ID NO:11. In some embodiments, the BBB transporter comprises V H , said V H comprises Optionally, HFR1 (S26) ending with a serine residue; HCDR1, which comprises Y28, R36, Y37 and optionally Y38 (or W38) and comprises 1 to 5 additional amino acid residues of SEQ ID NO:26; HCDR2, which comprises D57, S59, V62 and E64 and comprises 1 to 4 additional amino acid residues of SEQ ID NO:28; and HCDR3, which comprises R108, L109, P110, Y111, Y112, Y113 and comprises 1 to 7 additional amino acid residues of SEQ ID NO:7. In some embodiments, the BBB transporter comprises V L , said V L comprises LCDR1, which comprises F38 and comprises 1 to 5 additional amino acid residues of SEQ ID NO:29; LCDR2, which comprises Y56 and comprises 1 to 2 additional amino acid residues of SEQ ID NO:10; LFR3 starting with an arginine residue (R66); and optionally, LCDR3 comprising 1 to 9 amino acid residues of SEQ ID NO:11.

[0077] In some embodiments, the BBB transporter comprises: V H , which comprises optionally, HFR1 ending with a serine residue (S26); HCDR1, which comprises R36 and Y37 and additionally comprises 1 to 6 amino acid residues of SEQ ID NO:26; HCDR2, which comprises D57 and S59 and additionally comprises 1 to 6 amino acid residues of SEQ ID NO:28; HCDR3, which comprises R108, Y111, Y113 and additionally comprises 1 to 10 amino acid residues of SEQ ID NO:7; and / or V L , which comprises LCDR2, which comprises Y56 and additionally comprises 1 to 2 amino acid residues of SEQ ID NO:10; LFR3 starting with an arginine residue (R66); and optionally, LCDR3 comprising 1 to 9 amino acid residues of SEQ ID NO:11. In certain embodiments, the number of amino acid residues in the CDRs of the BBB transporter is the same as that shown in the corresponding SEQ ID NO, i.e., HCDR1: 8 residues, HCDR2: 8 residues, HCDR3: 13 residues, LCDR1: 6 residues, LCDR2: 3 residues, and LCDR3 (when present): 9 residues.

[0078] In some embodiments, in addition to the set of complementarity determining residues specified herein, the BBB transporter comprises HCDR1, HCDR2, and HCDR3 having the sequences according to SEQ ID NO: 26, 28, and 7, respectively. In some embodiments, in addition to the set of complementarity determining residues specified herein, the BBB transporter comprises at least one (e.g., 1, 2, or 3) HCDR selected from HCDR1, HCDR2, and HCDR3 having the sequences according to SEQ ID NO: 26, 28, and 7, respectively. In some embodiments, in addition to the set of complementarity determining residues specified herein, the BBB transporter comprises LCDR1 and LCDR2 having the sequences according to SEQ ID NO: 29 and 10, respectively; optionally further comprising LCDR3 having the sequence according to SEQ ID NO: 11. In some embodiments, in addition to the set of complementarity determining residues specified herein, the BBB transporter comprises at least one (e.g., 1, 2, or 3) LCDR selected from LCDR1, LCDR2, and LCDR3 having the sequences according to SEQ ID NO: 29, 10, and 11, respectively.

[0079] In some embodiments, a BBB transporter is provided that, in addition to a set of complementarity-determining residues specified herein, comprises HCDR1, HCDR2, and HCDR3 having the sequences according to SEQ ID NO: 26, 28, and 7, respectively, optionally further comprising LCDR1 and LCDR2 having the sequences according to SEQ ID NO: 29 and 10, respectively; and optionally LCDR3 having the sequence according to SEQ ID NO: 11. In some embodiments, in addition to a set of complementarity-determining residues specified herein, the BBB transporter comprises at least one (e.g., 1, 2, or 3) HCDR selected from HCDR1, HCDR2, and HCDR3 having the sequences according to SEQ ID NO: 26, 28, and 7, respectively, optionally further comprising at least one (e.g., 1, 2, or 3) LCDR selected from LCDR1, LCDR2, and LCDR3 having the sequences according to SEQ ID NO: 29, 10, and 11, respectively. The CDR sequences disclosed in the above embodiments have been annotated according to IMGT. In some embodiments, the numbering is according to different systems (Kabat, Chothia, Aho, AbM, or a combination thereof), and the sequences of the CDRs are adjusted accordingly. In some embodiments, the BBB transporter further comprises HFR1 ending with a serine residue (i.e., the serine residue at the carboxyl terminus of HFR1). In some embodiments, in addition to a set of complementarity-determining residues specified herein, the BBB transporter comprises LFR3 starting with an arginine residue (i.e., the arginine residue at the amino-terminal start of LFR3). In some embodiments, in addition to a set of complementarity-determining residues specified herein, the BBB transporter comprises HFR1 ending with a serine residue and LFR3 starting with an arginine residue. In some embodiments, HFR1 has a sequence that is at least 50% identical to the HFR1 sequence contained in SEQ ID NO: 21, optionally provided that HFR1 ends with a serine residue. In some embodiments, LFR3 has a sequence that is at least 50% identical to the LFR3 sequence contained in SEQ ID NO: 25, optionally provided that LFR3 starts with an arginine residue. In some embodiments, the framework regions have the sequences as contained in SEQ ID NO: 21 and 25. The framework sequences disclosed in the above embodiments have been annotated according to IMGT. In some embodiments, the numbering is according to different systems (Kabat, Chothia, Aho, AbM, or a combination thereof), and the sequences of the framework regions are adjusted accordingly.

[0080] In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further 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 H ; and 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 .

[0081] In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further comprises a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO: 21 H ; and a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO: 25 L . In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further 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 H . In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further comprises a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO: 21 H . In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further 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: 25 L . In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further comprises a V having a sequence that is at least 95% identical to the sequence of SEQ ID NO: 25 L .

[0082] In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further comprises a V having a sequence according to SEQ ID NO: 21 H ; and a V having a sequence according to SEQ ID NO: 25 L . In some embodiments, in addition to a set of complementary amino acid residues specified herein, the BBB transporter further comprises a V having a sequence according to SEQ ID NO: 21 H. In some embodiments, in addition to a set of complementary site residues specified herein, the BBB transporter further comprises a V having the sequence according to SEQ ID NO:25 L .

[0083] . In some embodiments, when binding to hTfR (according to SEQ ID NO:1), the above-specified complementary site residues of the BBB transporter can form hydrogen bonds and / or salt bridges with epitope residues. In some embodiments, the following complementary site residues (when present) can form hydrogen bonds with at least one epitope residue of hTfR upon binding, wherein the complementary site residues within V H are: a) S26 that can form a hydrogen bond with residue K261 of SEQ ID NO:1; b) R36 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; c) Y37 that can form a hydrogen bond with residue N493 of SEQ ID NO:1; d) Y38 that can form a hydrogen bond with residue D525 of SEQ ID NO:1. e) D57 that can form a hydrogen bond with residue N527 of SEQ ID NO:1; f) S59 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; g) R108 that can form a hydrogen bond with residue N493 of SEQ ID NO:1; h) Y111 that can form hydrogen bonds with residue S492 and residue L522 of SEQ ID NO:1; and i) Y113 that can form hydrogen bonds with residue Q524 and Q520 of SEQ ID NO:1; and wherein the complementary site residues within V L are: j) Y56 that can form a hydrogen bond with residue V517 of SEQ ID NO:1 and a hydrogen bond with residue T518 of SEQ ID NO:1.

[0084] . In some embodiments, the following complementary site residues (when present) can form salt bridges with at least one epitope residue of hTfR upon binding, wherein the complementary site residues within V H are a) R108 that can form a salt bridge with residue N560 of SEQ ID NO:1; and b) D57 that can form a salt bridge with residue K531 of SEQ ID NO:1; and wherein the complementary site residues within V L are: j) R66 that can form a salt bridge with residue E582 of SEQ ID NO:1.

[0085] In some embodiments, the following complementary site residues (when present) can form hydrogen bonds and / or salt bridges with at least one epitope residue of hTfR upon binding, wherein the complementary site residues within VH are: a) S26 that can form a hydrogen bond with residue K261 of SEQ ID NO:1; b) R36 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; c) Y37 that can form a hydrogen bond with residue N493 of SEQ ID NO:1; d) Y38 that can form a hydrogen bond with residue D525 of SEQ ID NO:1. e) D57 that can form a hydrogen bond with residue N527 of SEQ ID NO:1; f) D57 that can form a salt bridge with residue K531 of SEQ ID NO:1; g) S59 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; h) R108 that can form a hydrogen bond with residue N493 of SEQ ID NO:1; i) R108 that can form a salt bridge with residue N560 of SEQ ID NO:1; j) Y111 that can form hydrogen bonds with residue S492 and residue L522 of SEQ ID NO:1; and k) Y113 that can form hydrogen bonds with residue Q524 and Q520 of SEQ ID NO:1; and wherein the complementary site residues within VL are: l) Y56 that can form a hydrogen bond with residue V517 of SEQ ID NO:1 and a hydrogen bond with residue T518 of SEQ ID NO:1; and m) R66 that can form a hydrogen bond with residue E582 of SEQ ID NO:1.

[0086] In some embodiments, when binding to hTfR (according to SEQ ID NO:1), the above-specified complementary site residues of the BBB transporter may have additional properties: - The following complementary site residues located within V H within (when present) may have the following additional properties when binding to hTfR (according to SEQ ID NO:1): a) S26 is within 4.0 of residues K261 and K358 of SEQ ID NO:1 within; b) Y28 is within 4.0 of residue K261 of SEQ ID NO:1 ; c) R36 is within 4.0 of residues K495, K531 and E533 of SEQ ID NO:1 ; d) Y37 is within 4.0 of residues N493 and D560 of SEQ ID NO:1 ; e) Y38 is within 4.0 of residue D525 of SEQ ID NO:1 ; f) D57 is within 4.0 of residues N527 and K531 of SEQ ID NO:1 ; g) S59 is within 4.0 of residues S530 and K531 of SEQ ID NO:1 ; h) V62 is within 4.0 of residues N527 and S530 of SEQ ID NO:1 ; i) E64 is within 4.0 of residue N527 of SEQ ID NO:1 ; j) R108 is within 4.0 of residues N493 and D560 of SEQ ID NO:1 ; k) L109 is within 4.0 of residues S492 and D560 of SEQ ID NO:1 ; l) P110 is within 4.0 of residues S492 and N493 of SEQ ID NO:1 ; m) Y111 is within 4.0 of residues S492, F494, H515, T518 and L522 of SEQ ID NO:1 ; n) Y112 is within 4.0 of residues T491, S492, H515, V517 and T518 of SEQ ID NO:1 ; and o) Y113 is within 4.0 of residues Q520 and Q524 of SEQ ID NO:1 ; and - The following complementary site residues (when present) within V L may have the following additional characteristics when binding to hTfR (according to SEQ ID NO:1): p) F38 is within 4.0 of residue Q520 of SEQ ID NO:1 ; q) Y56 is within 4.0 of residues V517 and T518 of SEQ ID NO:1 ; and r) R66 is within 4.0 of residue E582 of SEQ ID NO:1 .

[0087] The complementarity-determining residues disclosed in the above embodiments have been annotated 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 of the complementarity-determining residues are adjusted accordingly.

[0088] The present disclosure provides embodiments numbered as follows for, for example, an anti-TfR antibody of the invention or an antigen-binding fragment thereof: 1. An anti-human transferrin receptor (hTfR) antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a complementarity-determining region that comprises: 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 the numbering is according to IMGT. 2. The antibody or antigen-binding fragment according to embodiment 1, wherein the complementarity-determining region further comprises: - V H amino acid residues S26 and / or Y38 (or W38); wherein the numbering is according to IMGT. 3. The antibody or antigen-binding fragment according to any one of the foregoing embodiments, wherein the complementarity-determining region further comprises: - V H amino acid residues Y28, V62, E64, L109, P110, and Y112; and - V L amino acid residue F38; wherein the numbering is according to IMGT. 4. The antibody or antigen-binding fragment according to any one of the foregoing embodiments, which comprises: V H , which comprises - heavy chain CDR (HCDR) 1, which comprises Y28, R36, Y37, and optionally Y38 (or W38) and comprises 1 - 5 additional amino acid residues of SEQ ID NO:26; -HCDR2, which comprises D57, S59, V62 and E64 and comprises 1 to 4 additional amino acid residues of SEQ ID NO:28; and -HCDR3, which comprises R108, L109, P110, Y111, Y112 and Y113 and comprises 1 to 7 additional amino acid residues of SEQ ID NO:7; and / or V L , which comprises -light chain CDR (LCDR) 1, which comprises F38 and comprises 1 to 5 additional amino acid residues of SEQ ID NO:29; -LCDR2, which comprises Y56 and comprises 1 to 2 additional amino acid residues of SEQ ID NO:10; -light chain framework region (LFR) 3 starting with an arginine residue (R66); and -optionally, LCDR3, which comprises 1 to 9 amino acid residues of SEQ ID NO:11; Optionally, wherein the antibody or antigen-binding fragment further comprises a heavy chain framework region (HFR) 1 ending with a serine residue, wherein the numbering is according to IMGT. 5. An anti-human transferrin receptor (hTfR) antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: -heavy chain CDRs (HCDRs) 1, HCDR2 and HCDR3 having sequences according to SEQ ID NO:26, 28 and 7, respectively; -light chain CDRs (LCDRs) 1 and LCDR2 having sequences according to SEQ ID NO:29 and 10, respectively; and optionally LCDR3 having a sequence according to SEQ ID NO:11; -heavy chain framework region (HFR) 1 ending with a serine residue; and -light chain framework region (LFR) 3 starting with an arginine residue. 6. The antibody or antigen-binding fragment according to any one of the preceding embodiments, which comprises: -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 . 7. The antibody or antigen-binding fragment according to any one of the preceding embodiments, wherein the following complementarity-determining residues, when present, can form hydrogen bonds with at least one epitope residue of hTfR upon binding, Among them, the complementary site residues within V H are: a) S26 that can form a hydrogen bond with residue K261 of SEQ ID NO:1; b) R36 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; c) Y37 that can form a hydrogen bond with residue N493 of SEQ ID NO:1; d) Y38 that can form a hydrogen bond with residue D525 of SEQ ID NO:1. e) D57 that can form a hydrogen bond with residue N527 of SEQ ID NO:1; f) S59 that can form a hydrogen bond with residue K531 of SEQ ID NO:1; g) R108 that can form a hydrogen bond with residue N493 of SEQ ID NO:1; h) Y111 that can form hydrogen bonds with residue S492 and residue L522 of SEQ ID NO:1; and i) Y113 that can form hydrogen bonds with residue Q524 and Q520 of SEQ ID NO:1; and Among them, the complementary site residues within V L are: j) Y56 that can form a hydrogen bond with residue V517 of SEQ ID NO:1 and a hydrogen bond with residue T518 of SEQ ID NO:1, where the numbering is according to IMGT. 8. The antibody or antigen-binding fragment according to any one of the foregoing embodiments, wherein when the following complementary site residues are present, they can form a salt bridge with at least one epitope residue of hTfR upon binding, Among them, the complementary site residues within V H are a) R108 that can form a salt bridge with residue D560 of SEQ ID NO:1; and b) D57 that can form a salt bridge with residue K531 of SEQ ID NO:1; and Among them, the complementary site residues within V L are: c) R66 that can form a hydrogen bond with residue E582 of SEQ ID NO:1, where the numbering is according to IMGT. 9. The antibody or antigen-binding fragment according to any one of the foregoing embodiments, wherein when the following complementary site residues are present, within 4.0 of at least one epitope residue of hTfR upon binding, within, Among them, within V H the complementary site residues are: a) S26 within 4.0 of residues K261 and K358 of SEQ ID NO:1 ; b) Y28 within 4.0 of residue K261 of SEQ ID NO:1 ; c) R36 within 4.0 of residues K495, K531 and E533 of SEQ ID NO:1 ; d) Y37 within 4.0 of residues N493 and D560 of SEQ ID NO:1 ; e) Y38 within 4.0 of residue D525 of SEQ ID NO:1 ; f) D57 within 4.0 of residues N527 and K531 of SEQ ID NO:1 ; g) S59 within 4.0 of residues S530 and K531 of SEQ ID NO:1 ; h) V62 within 4.0 of residues N527 and S530 of SEQ ID NO:1 ; i) E64 within 4.0 of residue N527 of SEQ ID NO:1 ; j) R108 within 4.0 of residues N493 and D560 of SEQ ID NO:1 ; k) L109 within 4.0 of residues S492 and D560 of SEQ ID NO:1 ; l) P110 within 4.0 of residues S492 and N493 of SEQ ID NO:1 ; m) Y111 within 4.0 of residues S492, F494, H515, T518 and L522 of SEQ ID NO:1 ; n) Y112 within 4.0 of residues T491, S492, H515, V517 and T518 of SEQ ID NO:1 ; and o) Y113 within 4.0 of residues Q520 and Q524 of SEQ ID NO:1 ; and wherein the complementary residue within V L is: p) F38 within 4.0 of residue Q520 of SEQ ID NO:1 ; q) Y56 within 4.0 of residues V517 and T518 of SEQ ID NO:1 ; and r) R66 within 4.0 of residue E582 of SEQ ID NO:1 , numbering according to IMGT.

[0089] In some embodiments, the BBB transporter comprises an HCDR as described herein inserted into a human heavy chain framework sequence derived from the human germline sequences IGVH3-23*05 or IGHV1-46*01 and IGHJ6_01. Exemplary humanized V H sequences generated with these human germline sequences are shown in Figure 3A and Figure 3B .

[0090] In some embodiments, the BBB transporter comprises an LCDR as described herein inserted into a human κ light chain framework sequence derived from the human germline sequences IGKV1-39*01 and IGKJ4-01. Exemplary humanized V L sequences generated with these human germline sequences are shown in Figure 3C .

[0091] In some embodiments, the BBB transporter comprises: a V containing any one of SEQ ID NOs: 17-21 or an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical thereto H ; and / or a V containing any one of SEQ ID NOs: 22-25 or an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical thereto L . In additional embodiments, the BBB transporter comprises Vs containing H and V L respectively: 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 NO: 21 and 24, SEQ ID NO: 20 and 24, SEQ ID NO: 19 and 24, SEQ ID NO: 18 and 24, SEQ ID NO: 17 and 24, SEQ ID NO: 21 and 23, SEQ ID NO: 20 and 23, SEQ ID NO: 19 and 23, SEQ ID NO: 18 and 23, SEQ ID NO: 17 and 23, SEQ ID NO: 21 and 22, SEQ ID NO: 20 and 22, SEQ ID NO: 19 and 22, SEQ ID NO: 18 and 22, SEQ ID NO: 17 and 22, or SEQ ID NO: 4 and 8, 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 the SEQ ID NO pairs listed above. In some embodiments, variant V H and V L sequences contain the HCDR and LCDR (defined by, for example, IMGT) from the SEQ ID NO pairs listed above, and sequence variations occur in the framework regions. In some embodiments, variant V H and V L retains the properties of its parental sequence with respect to TfR binding properties.

[0092] In additional embodiments, the BBB transporter comprises V H and V L : SEQ ID NO: 21 and 25, SEQ ID NO: 21 and 24, SEQ ID NO: 21 and 23, SEQ ID NO: 19 and 23, SEQ ID NO: 18 and 23, SEQ ID NO: 17 and 23, SEQ ID NO: 21 and 22, SEQ ID NO: 20 and 22, SEQ ID NO: 18 and 22, SEQ ID NO: 17 and 22, or SEQ ID NO: 4 and 8, or an amino acid sequence that is at least 90% identical (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the pairs of SEQ ID NOs listed above. In some embodiments, variant V H and V L sequences contain the HCDR and LCDR (defined, e.g., by IMGT) from the pairs of SEQ ID NOs listed above, and sequence variation occurs in the framework regions. In some embodiments, variant V H and V L retains the properties of its parental sequence with respect to TfR binding properties.

[0093] The percent identity between two amino acid sequences (or two nucleic acid sequences) can be obtained, e.g., by BLAST using default parameters (available on the website of the U.S. National Library of Medicine’s National Center for Biotechnology Information). The length of the reference sequence aligned for comparison purposes is at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, or 90% of the reference sequence). For V H 、V L 、HC or LC sequence identity or homology, percent identity and homology are calculated based on alignment of the full-length query and reference V H 、V L 、HC or LC sequences.

[0094] The present disclosure also encompasses BBB transporters structurally defined according to any of the above embodiments, having any combination of the functional properties described herein for BBB transporters. 2. Forms of the BBB Transporter

[0095] In some embodiments, the BBB transporter of the present disclosure takes the form of a full tetrameric antibody. The antibody can be of any immunoglobulin isotype, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The antibodies of the present disclosure preferably comprise a human IgG (e.g., IgG1) constant region. In some embodiments, the IgG constant region can comprise mutations that enhance the clinical potential of the antibody, such as mutations that reduce or eliminate antibody effector functions (ADCC and / or CDC) (see, e.g., Wang et al., Protein Cell (2018) 9(1):63-73). For example, the monospecific or multispecific antibodies of the present disclosure can comprise a human IgG1 constant region with the mutations L235E, the "LALA" mutation (L234A / L235A), or the "LALAGA" mutation (L234A / L235A / G237A) (Eu numbering). In some embodiments, the heavy chain comprises one, two, or all three of the mutations S298N, T299A, and Y300S (the "NNAS" mutation).

[0096] The IgG constant region can comprise mutations that increase the serum half-life of the antibody, such as the M428L mutation, the M252Y / S254T / T256E ("YTE" mutation), and the mutations described in WO 2019 / 147973.

[0097] To facilitate proper protein assembly of the BBB during manufacture, the antibody heavy chain (e.g., IgG1 heavy chain) can comprise a knobs-into-holes mutation (e.g., the IgG1 hole mutation is Y349C, T366S, L368A, and Y407V; and the IgG1 knob mutation is S354C and T366W). In some embodiments, the hole heavy chain comprises the H435R / Y436F ("RF") double mutation, which enables easy removal of hole-hole homodimers and hole half-IgG by-products during manufacture.

[0098] All such mutated human constant regions are still considered "human" constant regions herein. Unless otherwise indicated, all residue numbering in the IgG constant region is Eu numbering.

[0099] In some embodiments, the anti-TfR antibody comprises: an HC comprising 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 NO:32; and / or an LC comprising 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 NO:31. In some embodiments, the anti-TfR antibody comprises an HC comprising SEQ ID NO:32 and an LC comprising SEQ ID NO:31 (531v25 mAb). In some embodiments, the variant HC and LC sequences comprise the HCDR and LCDR in SEQ ID NOs:32 and 31 (as defined by, e.g., IMGT), and sequence variations occur in the framework region and / or the constant region. In some embodiments, the variant HC and LC retain the properties of their parental sequences with respect to TfR binding properties.

[0100] The BBB transporter can also be an antigen-binding fragment of a full antibody. In some embodiments, the transporter is monovalent for TfR (i.e., each transporter has only one binding site for TfR) and / or comprises a Fab. For example, the BBB transporter can be a Fab that comprises an HC composed of V H and C H1 (e.g., IgG C H1 ) and an LC composed of V L and C L (e.g., κC L ). This transporter is also referred to herein as a "Fab transporter". In some embodiments, the Fab transporter comprises: an HC comprising 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 NO:30; and / or an LC comprising 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 NO:31. In some embodiments, the Fab transporter comprises an HC comprising SEQ ID NO:30 and an LC comprising SEQ ID NO:31 (531v25Fab). In some embodiments, the Fab transporter comprises: an HC comprising V H as shown in SEQ ID NO:21 and an LC comprising V LLC. In some embodiments, the variant HC and LC sequences contain the HCDRs and LCDRs (defined, for example, by IMGT) in SEQ ID NO: 30 and 31 (or SEQ ID NO: 21 and 25), and sequence variations occur in the framework regions and / or constant regions. In some embodiments, the variant HC and LC retain the characteristics of their parental sequences in terms of TfR binding properties.

[0101] In some embodiments, the BBB transporter is similar to a full-length IgG antibody, except that it has only one TfR binding domain. Such a BBB transporter is a heterotrimer comprising an LC, a full-length antibody HC, and an Fc polypeptide that dimerizes with the constant region of the full-length 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 dimer structure formed by disulfide bonding of the CH2 and CH3 of an HC pair. "Fc polypeptide" as used herein refers to a polypeptide that can dimerize via disulfide bonds to form an Fc domain but does not have the V H region of a full-length antibody HC. In some embodiments, the Fc polypeptide contains the CH2 and CH3 of human IgG (e.g., human IgG1, IgG2, or IgG4) and at least a portion of the hinge region such that it can dimerize with a full-length human IgG (e.g., human IgG1, IgG2, or IgG4) heavy chain to form a human IgG1, IgG2, or IgG4 Fc domain.

[0102] In some embodiments, the Fc polypeptide and the full-length heavy chain may contain one or more of the mutations discussed above that enhance 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 the mutations described in WO 2019 / 147973), mutations that facilitate proper protein assembly of the BBB transporter (e.g., the mortar and pestle mutation), and mutations that improve the purification of the BBB transporter during manufacture (e.g., "RF" mutations).

[0103] Exemplary TfR-binding antibody fragments include the following molecules: (i) A Fab-FcOL TfR-binding antibody fragment comprising: an HC containing the V H as shown in SEQ ID NO: 21, an LC containing the V L as shown in SEQ ID NO: 25, and an Fc polypeptide (optionally as shown in SEQ ID NO: 34); (ii) A Fab-FcOL TfR-binding antibody fragment comprising: an HC containing SEQ ID NO:30, an LC containing SEQ ID NO:31, and an Fc polypeptide (optionally as shown in SEQ ID NO:34); (iii) A Fab-FcOL TfR-binding antibody fragment comprising: an HC containing SEQ ID NO:33, an LC containing SEQ ID NO:31, and an Fc polypeptide comprising SEQ ID NO:34 (531v25Fab-FcOL); and (iv) A Fab-FcOL TfR-binding antibody fragment comprising: an HC containing SEQ ID NO:56, an LC containing SEQ ID NO:31, and an Fc polypeptide comprising SEQ ID NO:34 (531v25Fab-FcOL with engineered cysteine).

[0104] Additional exemplary BBB transporters include the following molecules: (i) an antibody or an antigen-binding fragment thereof comprising: an HC containing HCDR1-3 as shown in SEQ ID NOs: 26, 28, and 7, respectively, and an LC containing LCDR1-3 as shown in SEQ ID NOs: 29, 10, and 11, respectively (and an optional Fc polypeptide); (ii) an antibody or an antigen-binding fragment thereof comprising a V as shown in SEQ ID NO:21 H and a V as shown in SEQ ID NO:25 L (and an optional Fc polypeptide); and (iii) an antibody or an antigen-binding fragment thereof comprising an HC containing SEQ ID NO:30 and an LC containing SEQ ID NO:31 (and an optional Fc polypeptide). These BBB transporters are referred to herein as "531v25 BBB transporters." The 531v25 Fab-FcOL BBB transporters having the Fab-FcOL form as defined herein are referred to as "531v25 Fab-FcOL BBB transporters." C. Cargo

[0105] The BBB transporters of the present invention (including 531v25 BBB transporters) can be used to transport a variety of cargoes across the BBB into the brain for diagnostic, prophylactic, and therapeutic purposes. The cargo can be chemically conjugated to the transporter, for example, through lysine or cysteine residues in the transporter. In some embodiments, the cargo can be chemically conjugated to engineered residues, such as engineered cysteine residues (THIOMAB TM technology) or engineered lysine residues.

[0106] Alternatively, if the cargo is a peptide or polypeptide, it can be recombinantly fused to a transporter. For example, when the transporter is a full 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 additional embodiments, the cargo can be fused to the C-terminus of one of the heavy chains (e.g., the pestle heavy chain or the mortar heavy chain).

[0107] In some embodiments, the transporter is a Fab transporter (such as 531v25Fab), and the cargo can be fused to the N-terminus or C-terminus of the light chain, or to the N-terminus or C-terminus of the heavy chain. In additional embodiments, the cargo can be fused to the C-terminus of the heavy chain. In certain embodiments, the cargo can be fused to the C-terminus of the light chain.

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

[0109] The cargo can be linked to an anti-TfR antibody or an antigen-binding fragment thereof via a peptide linker. In some embodiments, the peptide linker can mainly comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The length of the peptide linker can be sufficient to link the two molecules in such a way that they present the correct conformation relative to each other so that they maintain their respective desired activities. In some embodiments, the length of the linker is 1 to 50 (e.g., 1 to 30, 1 to 20, 1 to 10, or 1 to 5) amino acids. Useful linkers include glycine-serine polymers, including, for example, (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) or SGSGGGG (SEQ ID NO:43). Other exemplary linkers for linking antibody fragments or single-chain variable fragments can include AAEPKSS (SEQ ID NO:47), AAEPKSSDKTHTCPPCP (SEQ ID NO:48), GGGG (SEQ ID NO:42), or GGGGDKTHTCPPCP (SEQ ID NO:49).

[0110] Cargo that can be transported by the BBB transporters of the present invention (including the 531v25 BBB transporter) can be a diagnostic agent, such as an imaging agent for the brain.

[0111] Cargo that can be transported by the BBB transporters of the present invention (including the 531v25 BBB transporter) can be a therapeutic agent. Exemplary therapeutic agents are peptides / polypeptides and oligonucleotides.

[0112] In some embodiments, the therapeutic agent is an enzyme. In certain embodiments, the enzyme is a lysosomal enzyme. In additional 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 recombinant human GAA is alglucosidase alfa (Myozyme and Lumizyme ). In some embodiments, recombinant GAA comprises or consists of SEQ ID NO:35.

[0113] In some embodiments, the TfR-binding protein for transporting GAA of the present invention is a Fab-FcOL transporter, which comprises HC, LC, and Fc polypeptides, and a human GAA sequence fused to the C-terminus of (a) HC, (b) LC, or (c) the Fc polypeptide, wherein the heavy chain and the Fc polypeptide dimerize to form an Fc domain. In additional embodiments, the TfR-binding protein for transporting GAA of the present invention comprises HC, LC, Fc polypeptides, and a human GAA sequence fused to the C-terminus of the LC, wherein the heavy chain and the Fc polypeptide dimerize to form an Fc domain. In additional embodiments, 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 additional embodiments, the TfR-binding protein is a Fab-FcOL (531v25Fab-FcOL-LC-GAA) comprising three polypeptides respectively comprising SEQ ID NO:33, 36, and 34. In other embodiments, the TfR-binding protein for transporting GAA of the present invention comprises HC, LC, Fc polypeptides, 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 additional embodiments, the TfR-binding protein for transporting GAA is a 531v25 Fab-FcOLBBB transporter comprising HC, LC, and Fc polypeptides, wherein the human GAA sequence is fused to the C-terminus of the Fc polypeptide. In additional embodiments, the TfR-binding protein is a Fab-FcOL (531v25Fab-FcOL-GAA) comprising three polypeptides respectively comprising SEQ ID NO:33, 31, and 37.

[0114] In some embodiments, the TfR-binding protein for transporting GAA of the present invention comprises a Fab and a human GAA sequence fused to the C-terminus of the HC or LC of the Fab. In additional embodiments, the TfR-binding protein for transporting GAA of the present invention comprises a Fab and a human GAA sequence fused to the C-terminus of the LC of the Fab. In additional embodiments, the TfR-binding protein for transporting GAA is a 531v25 BBB transporter in the form of a Fab comprising HC and LC, wherein the human GAA sequence is fused to the C-terminus of the LC. In additional embodiments, the TfR-binding protein comprises two polypeptides respectively comprising SEQ ID NO:30 and 36 (531v25Fab-GAA).

[0115] In some embodiments, the TfR-binding protein for transporting GAA of the present invention comprises two HCs, two LCs, and a human GAA sequence fused to the C-terminus of one of the two HCs. In additional embodiments, the TfR-binding protein for transporting GAA of the present invention is the 531v25 BBB transporter, which comprises two HCs, two LCs, and a human GAA sequence fused to the C-terminus of one of the two HCs. In further embodiments, each of the two LCs comprises SEQ ID NO:31, one HC comprises SEQ ID NO:33, and the other HC comprises SEQ ID NO:37 (531v25mAb-GAA).

[0116] In some embodiments, the therapeutic agent is an oligonucleotide. In certain embodiments, the oligonucleotide is, for example, an antisense oligonucleotide or siRNA targeting a CNS gene.

[0117] In some embodiments, the TfR-binding protein for transporting an oligonucleotide of the present invention is a Fab-FcOL transporter comprising an HC, an LC, and an Fc polypeptide, wherein the heavy chain and the Fc polypeptide dimerize to form an Fc domain, and wherein 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 the THIOMAB TM technology, or an engineered lysine residue) for site-specific conjugation. In further embodiments, the TfR-binding protein for transporting an oligonucleotide is a 531v25 Fab-FcOL BBB transporter comprising an HC, an LC, and an Fc polypeptide, for example, wherein the oligonucleotide is conjugated to one of the transport polypeptides as described above. In additional embodiments, the TfR-binding protein is a Fab-FcOL comprising three polypeptides comprising SEQ ID NO:31, 34, and 56, respectively.

[0118] In some embodiments, the TfR-binding protein for transporting an oligonucleotide of the present invention comprises a Fab. In further embodiments, the TfR-binding protein for transporting an oligonucleotide is a 531v25 BBB transporter comprising an HC and an LC in Fab form, wherein 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 the THIOMAB TM technology, or an engineered lysine residue) for site-specific conjugation.

[0119] In some embodiments, the TfR-binding protein for transporting oligonucleotides of the present invention comprises two heavy chains (HCs), two light chains (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 the THIOMAB TM technology, or an engineered lysine residue) for site-specific conjugation. In additional embodiments, the TfR-binding protein for transporting oligonucleotides of the present invention is the 531v25 BBB transporter protein comprising two HCs, two LCs, and an oligonucleotide conjugated to one of the HCs and LCs as described above. In certain embodiments, each of the two LCs comprises SEQ ID NO:31, one HC comprises SEQ ID NO:33, and the other HC comprises SEQ ID NO:56. In certain embodiments, each of the two LCs comprises SEQ ID NO:31, and each of the two HCs comprises SEQ ID NO:56.

[0120] It should be understood that the linkage of the cargo to the BBB transporter protein herein does not affect the TfR-binding properties of the transporter protein. The cargo-loaded BBB transporter protein has the binding properties (e.g., TfR-binding affinity) as described in Part A above. II. Preparation of TfR binding protein

[0121] 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 biomolecule (e.g., a nucleic acid or polypeptide molecule) referred to herein as "isolated" or "purified" is one that (1) has been separated from the biomolecules of its source (e.g., nucleic acids of genomic DNA or cellular RNA, or polypeptides) and / or (2) those biomolecules that do not exist in nature. The coding sequence for each polypeptide chain can be cloned into a single vector or into separate vectors.

[0122] Methods for producing proteins, such as antibodies, are well known. The binding proteins of the present invention, such as antibodies, can be produced in mammalian host cells, for example, using appropriate expression constructs. Mammalian cell lines that can be used as expression hosts include many immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines particularly include 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 many other cell lines. Other cell lines that can be used are insect cell lines (such as Sf9 or Sf21 cells) and yeast cell lines. The cell line can be selected based on their expression level. The binding protein 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. III. Pharmaceutical composition and uses

[0123] The present disclosure also provides a pharmaceutical composition comprising the TfR binding protein herein. The pharmaceutical composition can comprise one or more pharmaceutically acceptable excipients, carriers, or diluents. As used herein, "pharmaceutically acceptable" with respect to a "carrier", "excipient", or "diluent" 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 can comprise buffers; surfactants; polyols; antioxidants; and / or chelating agents.

[0124] A pharmaceutical composition comprising the BBB transporter herein loaded with an enzyme (e.g., a lysosomal enzyme) can be used to treat human patients suffering from an enzyme deficiency (e.g., lysosomal enzyme deficiency) or at risk of developing an enzyme deficiency. In some embodiments, the enzyme or the activity of the enzyme is lacking in the patient (e.g., insufficient in quantity or even completely absent). In some embodiments, the GAA transporter herein can be used to treat patients in need of a higher level of GAA activity, such as patients congenitally lacking GAA. In some embodiments, the GAA BBB transporter can be used to restore GAA activity. For example, the GAA BBB transporter is used to treat human patients in need suffering from Pompe disease. Pompe disease (also known as acid α-glucosidase deficiency, acid maltase deficiency, glycogen storage disease type II, GSD II, and glycogenosis type II) is a hereditary disorder of glycogen metabolism caused by the lack or marked deficiency of the lysosomal enzyme GAA. The GAA transporter herein can be used to treat late-onset Pompe disease (LOPD) and / or infantile-onset Pompe disease (IOPD).

[0125] A pharmaceutical composition comprising a BBB transporter of the present invention loaded with an oligonucleotide (e.g., an ASO or siRNA) can be used to treat human patients who would benefit from the transport of the oligonucleotide to, for example, 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 the peripheral nervous system (e.g., the sciatic nerve), skeletal muscle, visceral organs (e.g., the heart or spleen), or other tissues. In the case where the oligonucleotide is an ASO or siRNA, the oligonucleotide-loaded BBB transporter can be used to treat human patients who would benefit from the knockdown of the target of the ASO or siRNA.

[0126] As used herein, the terms “treat,” “treatment,” and “treating” refer to the deliberate intervention in a physiological disease state such that the severity of the disease or disorder is reduced; the duration of the disease or disorder is decreased; one or more symptoms associated with the disease or disorder are improved or eliminated; or a beneficial effect is provided to a subject having the disease or disorder. Treatment does not require the cure of the underlying disease or disorder.

[0127] The pharmaceutical composition can be provided to a patient at a dosage strength and frequency determined by a healthcare provider as appropriate. A therapeutically effective amount is those amounts sufficient to improve one or more symptoms associated with the disease or affliction to be treated. The “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dose” of the binding protein of the present invention protects a subject from the onset of 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 disease-free symptom periods, or the prevention or delay of injury or disability caused by the disease affliction.

[0128] The present disclosure also provides the use of an antibody or antigen-binding fragment of the present invention for the manufacture of a drug comprising a therapeutic molecule, wherein the drug is capable of crossing the BBB. In some embodiments, the present disclosure provides the use of the enzyme-loaded TfR-binding protein of the present invention for the manufacture of a drug for treating a subject lacking an enzyme (e.g., a GAA-loaded TfR-binding protein for treating Pompe disease). In some embodiments, the present disclosure provides the use of an oligonucleotide-loaded TfR-binding protein in the manufacture of a drug for treating a subject in need thereof, e.g., wherein the oligonucleotide can be an ASO or siRNA.

[0129] The present disclosure also provides the use of the antibodies or antigen-binding fragments of the present invention in diagnostic procedures (e.g., in vitro or ex vivo). For example, the antibodies and antigen-binding fragments can be used to detect and / or measure the level of TfR in a biological sample (e.g., a tumor biopsy, a tissue sample, or a blood sample) from a patient. Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay, radioimmunoassay, and immunohistochemistry. The present disclosure also encompasses kits (e.g., diagnostic kits) comprising the antibodies or antigen-binding fragments described herein.

[0130] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and the embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises", or "comprising" shall be understood to imply the inclusion of the 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 many documents are cited herein, this citation does not mean that any of these documents constitutes a part of the common general knowledge in the art. As used herein, the term "about" or "approximately" as applied to one or more values of interest refers to a value similar to the stated reference value. In certain embodiments, unless otherwise stated or otherwise apparent from the context, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value.

[0131] According to the present disclosure, a back-reference in a dependent claim refers to a short writing of a direct and explicit disclosure of each combination of the claims indicated by the back-reference. Additionally, the headings herein are created for organizational convenience and are not intended to limit in any way the scope of the claimed invention.

[0132] To better understand the present invention, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way. Examples Example 1: Generation and Characterization of Mouse 531mAb A. m531 Design: 531 - 1L2_Mouse

[0133] The purpose of this study was to generate and identify antibodies that bind to TfR. To this end, mice were immunized with human tissues expressing human TfR. Hybridomas were then selected, and the sequences of their antibodies (designated 531 - 1L2_Mouse) were obtained by RT - PCR and sequencing. The V H and V L sequences are shown in SEQ ID NO:4 and 8, respectively. HCDR is shown in SEQ ID NO:5 - 7, and LCDR is shown in SEQ ID NO:9 - 11. B. Affinity of 531 - 1L2_Mouse Measured by SPR

[0134] The binding of 531 - 1L2_Mouse Ab to human, cynomolgus monkey, and mouse TfR was evaluated by SPR at pH 7.4. The experiment was run on a Bruker MASS - 2 instrument using HBS - EP+ as the running buffer and sample diluent (Cytiva Life Sciences, #BR100826). According to the provider's instructions, an HCA sensor chip (Bruker, #1862614) was immobilized with an anti - his antibody (His Capture Kit, Cytiva Life Sciences, #28995056). Approximately 10,000 RU of anti - his was obtained. The His - tagged extracellular domain of human transferrin receptor protein - SEQ ID NO:39, the extracellular domain of cynomolgus monkey TfR - SEQ ID NO:40, or the extracellular domain of mouse TfR - SEQ ID NO:41 (5 μg / ml) - was captured at 10 μL / min for 1 min. Then, a concentration series of 531 - 1L2_Mouse Ab was injected at 30 μL / min for 4 min on the surface of human transferrin receptor, and dissociation was monitored for 5 min. The surface was regenerated with a 1 - min pulse of 10 mM glycine - HCl at pH 1.5. Analysis was performed using Bruker Sierra Analyzer software: the sensorgrams were double - referenced with a reference surface (subtracting bulk and weak non - specific binding) and a blank (drift removal), and the curves were fitted with a 1:1 binding model.

[0135] This experiment confirmed the binding of 531 - 1L2_Mouse antibody to the extracellular domain of TfR. The binding affinities for TfR are shown in Table 1 below. Table 1. Affinity of 531_1L2 - Mouse for TfR Evaluated by SPR C. Competition between 531-1L2_ murine antibody and transferrin by SPR

[0136] The aim of this study was to determine whether the 531-1L2_ murine antibody affects the binding of transferrin to its receptor.

[0137] The experiment was carried out on a BIAcore TM T200 instrument using HBS-EP+ as the running buffer and sample diluent (Cytiva Life Sciences, #BR100826). Approximately 500 RU of the 531-1L2_ murine antibody was covalently immobilized on a C1 sensor chip (Cytiva Life Sciences, #BR100050) using an amine coupling kit (Cytiva Life Sciences, #BR100540). 50 nM of hTfR (R&D Systems, #2474-TR; SEQ ID NO:1 with the G142S SNP substitution associated with the Caucasian population) was injected onto the 531-1L2_ murine surface for 3 min, followed immediately by injection of 100 nM human transferrin (Sigma, #T4132; represented as SEQ ID NO:54 and with its peptide signal cleaved) at 10 μL / min for 3 min. Approximately 150 RU of hTfR bound to the 531-1L2_ murine antibody and approximately 100 RU of human transferrin bound to hTfR were measured. 50 nM of hTfR was injected onto the 531-1L2_ murine surface for 3 min, then immediately 100 nM of the 531-1L2_ murine antibody was injected at 10 μL / min for 3 min. Approximately 150 RU of hTfFR bound to the 531-1L2_ murine was measured and no binding of the 531-1L2_ murine antibody was detected (competition control). The surface was regenerated with 30 s pulses of 10 mM glycine-HCl (pH 2.5) at 10 μL / min.

[0138] We observed no competitive binding of the 531-1L2_ murine antibody and transferrin to their receptor. Example 2: Generation of humanized Fab antibodies

[0139] The aim of this study was to generate a humanized 531 variant (Fab form) with optimal binding properties for hTfR and cTfR (i.e., with approximately 1 log or less difference between the affinity for cTfR and the affinity for hTfR). A. Generation and expression of humanized Fab variants

[0140] The technique applied to design humanized Fab variants of the 531-1L2_ murine antibody was CDR grafting, in which the CDRs of the non-human antibody were grafted onto a human framework.

[0141] First, based on the positions of the CDRs identified within the V H and V L sequences (SEQ ID NOs: 4 and 8) of the 531-1L2_ murine antibody, residues crucial for CDR loop conformation, antibody structure, and affinity and specificity for human and cynomolgus monkey TfR were identified (e.g., cursor residues, contact residues, and anchor residues). Several human heavy chain germline (such as IGHV3-23*05 shown in SEQ ID NO: 12 and IGHV1-46*01 shown in SEQ ID NO: 13) and human light chain germline (such as IGKV1-39*01 shown in SEQ ID NO: 15) sequences that were considered to provide the closest murine framework and support the murine CDR structure were selected as recipients for CDR grafting.

[0142] Then, the sequences of the human IMGT CDRs were replaced with the residues of the murine IMGT CDRs. One or more of the above-identified key residues were backmutated (reverted to the murine sequence), and residues within one or more CDRs were modified as appropriate to avoid chemical instability. Five humanized V H sequences (SEQ ID NOs: 17-21) and four humanized V L sequences (SEQ ID NOs: 22-25) were generated and combined to produce humanized Fab variants.

[0143] The humanized anti-TfR variants in Fab-His tag form were expressed by transient transfection of HEK293 cells. The Fab proteins were purified by a two-step method (IMAC & SEC) to achieve a purity of at least 95%. The His tag was removed by cleavage with TEV protease prior to affinity measurement by SPR. B. Measuring the affinity of humanized Fab variants for TfR of different species by SPR

[0144] The binding of humanized Fab variants to human, cynomolgus monkey, and mouse TfR was evaluated using SPR at pH 7.4. The experiments were run on a Bruker MASS-2 instrument using HBS-EP+ as the running buffer and sample diluent (Cytiva Life Sciences, #BR100826). The HCA sensor chip (Bruker, #1862614) was immobilized with an anti-His antibody (His Capture Kit, Cytiva Life Sciences, #28995056) according to the manufacturer's instructions. Approximately 10,000 RU of anti-His was obtained. His-tagged extracellular domain of hTfR (SEQ ID NO:39), cTfR extracellular domain (SEQ ID NO:40), or mouse TfR extracellular domain (SEQ ID NO:41) (5 μg / ml) was captured at 10 μL / min for 1 min. Then, a concentration series of humanized Fab variants was injected at 30 μL / min for 4 min on the hTfR surface, and dissociation was monitored for 5 min. The surface was regenerated with a 1-min pulse of 10 mM glycine-HCl at pH 1.5. Analysis was performed using Bruker Sierra Analyzer software: the sensorgrams were double-referenced with a reference surface (subtracting bulk and weak non-specific binding) and a blank (drift removal), and the curves were fitted with a 1:1 binding model.

[0145] Variant candidates were selected based on their affinity for human and cynomolgus monkey TfR and the cynomolgus monkey / human ratio. This selection process yielded 10 variants (see Table 2; SEQ: SEQ ID NO; NB: no binding). None of the variants bound to mouse TfR (mTfR). Next, we determined the V H and V L sequences and their respective CDRs and combinations of CDRs of these 10 variants. One variant (531v25) was selected for subsequent experiments (see Example 3). Table 2. Binding affinities of mouse and humanized antibodies determined by SPR

[0146] The HC and LC of 531v25 Fab are shown in SEQ ID NO:30 and 31, respectively. The V H and V L sequences of 531v25 Fab are shown in SEQ ID NO:21 and 25, respectively. HCDR1-3 are shown in SEQ ID NO:26, 28, and 7, respectively, and LCDR1-3 are shown in SEQ ID NO:29, 10, and 11, respectively. Example 3: TfR binding properties of constructs derived from 531v25 A. Affinity measurement

[0147] The purpose of this study was to compare the TfR binding affinities of the following various 531v25-derived constructs: (1) 531v25Fab as in Example 2 and (2) 531v25Fab-FcOL (having an LC as shown in SEQ ID NO:31, an HC as shown in SEQ ID NO:33, and an Fc polypeptide as shown in SEQ ID NO:34).

[0148] The affinities (and cynomolgus / human ratios) of the constructs for human and cynomolgus TfR were evaluated using SPR and Biolayer Interferometry (BLI) (see Table 3). Table 3. Affinities for TfR Evaluated by SPR or BLI

[0149] Affinity was evaluated by SPR as described in Example 2B, except that 531v25Fab or 531v25Fab-FcOL was used instead of the humanized Fab variant.

[0150] The affinities obtained by BLI were evaluated using an Octet HTX instrument: The equilibrium dissociation constant (K D ) was determined in HBSP buffer (10 mM HEPES, 150 HEPES, 0.05% polysorbate 20, pH 7.4) on the Octet . A fixed concentration of ligand: hTfR or cTfR, both site-specifically biotinylated at the N-terminal Avitag, was immobilized using a streptavidin probe; in this experiment, hTfR consisted of an AviTag (SEQ ID NO:55) fused to the N-terminus of the extracellular domain of human TfR (residues 90 to 760 of SEQ ID NO:1), and cTfR consisted of an AviTag fused to the N-terminus of the extracellular domain of cTfR (amino acid residues 90 to 760 of SEQ ID NO:2). The association and dissociation of the test ligand with a 2-fold serial dilution series of analyte (531v25-Fab-FcOL) from 250 nM to 3.91 nM were tested. After subtracting the buffer and probe analyte interactions, a 1:1 binding model with global fitting was applied, with 0 - 90 second association and 0 - 60 dissociation windows.

[0151] SPR data confirmed that the 531v25Fab-FcOL construct retained the affinity characteristics of 531v25Fab (including the cynomolgus / human affinity ratio). In addition, BLI gave similar affinity measurements for 531v25Fab-FcOL for human and cynomolgus TfR, and thus a similar affinity ratio. B. No competition between 531v25Fab-FcOL and transferrin was observed by BLI

[0152] We attempted to use BLI to confirm the absence of binding competition (for TfR) with transferrin in the presence of saturation by holo-TF (holotransferrin) or 531v25Fab-FcOL. The 531v25Fab-FcOL molecule is detailed in Example 3A.

[0153] The equilibrium dissociation constant (K D ) was determined on the Octet in HBSP buffer (10 mM HEPES, 150 HEPES, 0.05% polysorbate 20, pH 7.4). A fixed concentration of the ligand hTfR, which was site-specifically biotinylated on the N-terminal AviTag fused to the N-terminus of the hTfR extracellular domain, was immobilized using a streptavidin probe. Subsequently, the immobilized hTfR was saturated with 500 nM human holotransferrin (#2914-HT, R&D Systems) or 531v25Fab-FcOL. Subsequently, the association and dissociation of potential competing ligands with a 2-fold serial dilution series of the blocking analyte (531v25Fab-FcOL) from 500 nM to 31.25 nM were tested. After subtracting the buffer and probe analyte interactions, a 1:1 binding model with global fitting was applied, with 0 - 90 s association and 0 - 60 dissociation windows. The association and dissociation of test ligands with a 2-fold serial dilution series of the analyte (531v25Fab-FcOL) from 250 nM to 3.91 nM were tested. After subtracting the buffer and probe analyte interactions, a 1:1 binding model with global fitting was applied. The data showed that transferrin binding was not affected by the pre-association of 531v25Fab-FcOL. Holotransferrin saturation of hTfR minimally affected the binding of 531v25Fab-FcOL to hTfR. These data also indicated that there was no competition for binding to the transferrin receptor between 531v25Fab-FcOL and transferrin (see Table 4).

[0154] Table 4. Competition of 531v25-Fab-FcOL with transferrin for binding to hTfR as determined by BLI C. Biodistribution of monovalent 531v25-Fab-FcOL in hTfR-KI mice after a single dose

[0155] ​hTfR-KI mice were dosed with 531v25-Fab-FcOL (as described above) or human IgG1 control Ab (Southern Biotech, Cat#0151K-14) by intravenous injection at a dose of 70 nmol / kg. Mice were terminally anesthetized at 1, 3, and 24 h post-dose. Blood was collected in EDTA for plasma separation, and then mice were perfused with Dulbecco's phosphate-buffered saline solution containing heparin and Ca / Mg (8 mL / min for 5 min). 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 concentration of anti-TfR construct in tissues (per wet tissue weight) was quantified by human IgG MSD (MSD human / NHP typing panel #K150JLD-1, Meso Scale Discovery) according to the manufacturer's protocol.

[0156] Compared to control IgG, monovalent 531v25-Fab-FcOL had increased brain and spinal cord biodistribution (see Table 5), as well as faster plasma clearance (data not shown), which confirmed that the 531v25 binding domain was able to reach the brain and CNS via the transferrin receptor. Table 5. Brain and spinal cord biodistribution of 531v25-Fab-FcOL compared to IgG1 Ratio to IgG (brain) Ratio to IgG (spinal cord) 1h 5.3 4.5 3h 8.1 9.2 24h 22.4 29.4 Example 4: Epitope and paratope mapping of 531v25 Fab

[0157] We attempted to determine the localization of the human and cynomolgus monkey TfR epitopes recognized by 531v25 Fab using cryo-electron microscopy (cryo-EM).

[0158] In this study, the purified complex of hTfR (SEQ ID NO:39) or cTfR (SEQ ID NO:40) with 531v25 Fab was further purified using a Superdex 200 3.3 / 300 column pre-equilibrated with PBS buffer. UltraAufoil R 0.6 / 1 on 300 gold grids was glow-discharged at 22 mA for 45 s, and 3 μL of each sample with concentrations of 1 and 0.64 mg / ml was added to the grids and plunge-frozen in liquid ethane. Data from the grids were collected on a Glacios TMOn a microscope (Thermo Fisher). EER format images were recorded with EPU at a nominal magnification of 240,000X, with a pixel size of 0.58 and a defocus range from -0.8 to -2.2. During an exposure time of 4.72 s, the dose on the camera was 60 e- / pixel², and the fraction for the last 54 frames was 162.

[0159] A total of 7000 and 4630 images were taken for each grid, respectively. Data analysis was performed using CryoSPARC v3. Using the value when the FSC curve was below 0.143, the final reconstruction resolutions for human and cynomolgus monkey TfR were estimated to be 2.5 and 2.73 Å, respectively. Two cryo-EM maps were sharpened using Phenix and then used to fit the atomic coordinates of TfR and 531v25 Fab. The atomic coordinates underwent several rounds of manual (on Coot) and real-space refinement (in Phenix). The amino acid residues of hTfR and cTfR with atoms within 4 Å of the 531v25 Fab atoms are shown in the figures of Figure 1A and Figure 1B These distances were initially measured using the UCSF Chimera software and subsequently using the PISA and UCSF ChimeraX software. The amino acid residues of hTfR and cTfR with atoms within 5 Å of the 531v25 Fab atoms (as measured using the UCSF Chimera software) are shown in the alignment below.

[0160] As Figure 1A and Figure 1B shown, 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 4 Å resolution distance as determined by the UCSF Chimera software. 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 4 Å resolution distance as determined by the PISA and UCSF ChimeraX software.

[0161] As shown in the following alignment, 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 5 resolution distance.

[0162] Interestingly, we observed that 531v25 Fab binds to the protease-like domain (horizontally) of TfR, which corresponds to a part of TfR with less variability between humans and cynomolgus monkeys. Two amino acid differences (at positions 520 and 521) between human and cynomolgus monkey TfR have been identified, which have been shown by Cryo-EM not to affect the interaction between 531v25 Fab and TfR.

[0163] Concurrent with the epitope residue determination, PISA and UCSF ChimeraX software were used to determine the 531v25 paratope residues and the interactions between these paratope residues and the epitope residues.

[0164] Thirteen V H residues and three V L residues were found within 4.0 of the epitope residues (human TfR): Y28, R36, Y37, D57, S59, V62, E64, R108, L109, P110, Y111, Y112 and Y113 in V H and F38, Y56 and R66 in V L (all residue positions are defined according to IMGT numbering). Twelve of these residues are located in the CDRs: Y28, R36 and Y37 are in HCDR1; D57, S59, V62 and E64 are in HCDR2; R108, L109, P110, Y111, Y112 and Y113 are in HCDR3; F38 is in LCDR1; and Y56 is in LCDR2. One of these residues, namely R66, is in LFR3.

[0165] Due to the flexibility of human TfR, two additional residues of V H (S26 in HFR1 and Y38 in HCDR1) were found within 4.0 within, depending on the conformation of the hTfR monomer. S26 and Y38 can be within 4.0 of the epitope residues in one hTfR monomer conformation and at a distance greater than 4.0 from the epitope residues in another hTfR monomer conformation slightly farther away. Thus, these two residues are considered to have a weak interaction with human TfR.

[0166] The interface residues within 4.0 were determined as follows: V of 531v25 H -Y28(531v25 V H ) is within 4.0 of residue K261 of human TfR (SEQ ID NO:1); -R36 is within 4.0 of residues K495, K531, and E533; -Y37 is within 4.0 of residues N493 and D560; -D57 is within 4.0 of residues N527 and K531; -S59 is within 4.0 of residues S530 and K531 -V62 is within 4.0 of residues N527 and S530; -E64 is within 4.0 of residue N527; -R108 is within 4.0 of residues N493 and D560; -L109 is within 4.0 of residues S492 and D560; -P110 is within 4.0 of residues S492 and N493; -Y111 is within 4.0 of residues S492, F494, H515, T518, and L522; -Y112 is within 4.0 of residues T491, S492, H515, V517, and T518; and -Y113 is within 4.0 of residues Q520 and Q524. V of 531v25 L : -F38(531v25 V L ​​​​​​​​​​​​​)Within 4.0 of residue Q520 of human TfR (SEQ ID NO:1) ; -Y56 within 4.0 of residues V517 and T518 ; and -R66 within 4.0 of residue E582 . Regarding two additional residues of V H , the interface residues within 4.0 are as follows: -S26(531v25 V H ) within 4.0 of residues K261 and K358 of human TfR (SEQ ID NO:1) ; -Y38 within 4.0 of residue D525 .

[0167] Among the interface residues within 4.0 as defined above , complementary site residues related to hydrogen bonds and salt bridges with epitope residues have been identified (hydrogen bonds and salt bridges are considered strong interactions between antibodies and antigens): R36, Y37, D57, S59, R108, Y111, and Y113 in V H , and Y56 and R66 in V L (residue positions are defined according to IMGT numbering). In addition, residues S26 and Y38 in V H also show formation of hydrogen bonds with epitope residues when within 4.0 . The following hydrogen bonds and salt bridges have been 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 can form 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; and -Y113 can form a hydrogen bond with residue Q524. V of 531v25 L -Y56(531v25 V L) can form hydrogen bonds with residue V517 of human TfR (SEQ ID NO:1) and with residue T518; and - R66 can form a salt bridge with residue E582. When within 4.0 of the epitope residue V H The hydrogen bonds of two other residues in are as follows: - S26 (531v25 V H ) can form a hydrogen bond with residue K261 of human TfR (SEQ ID NO:1); and - Y38 can form a hydrogen bond with residue D525. Example 5: Generation and Characterization of 531v25 - GAA Molecules

[0168] Four anti - TfR - GAA molecules were constructed, which have 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 A. Affinity Measurement and GAA Activity

[0169] Next, we attempted to compare the GAA activities and binding characteristics of these 4 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.

[0170] Specific (GAA) activity was measured by a fluorescence enzyme assay using the synthetic substrate (4-methylumbelliferyl α-D-glucopyranoside, M9766, Sigma). Briefly, standard curves (3.9 - 250 ng / mL) of GAA and the anti-TfR-GAA construct 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 terminated by adding 135 μL of 1 M glycine-NaOH buffer (pH 12.5). The fluorescence of the reaction solution (excitation at 360 nm and emission at 450 nm) was measured. A standard curve of 4-methylumbelliferone (M-1381, Sigma) (0.039 - 5 nmol / well) was fitted by linear regression to calculate the amount of product. The specific activity (μmol product / min / mg GAA) was calculated by dividing the amount of product by the reaction time and the amount of GAA.

[0171] The enzyme activities of all test molecules were in the range of 70% - 120% of the GAA activity. The assay also showed similar activity of the purified anti-TfR-GAA protein compared to GAA (Table 6).

[0172] The binding kinetics of four anti-TfR-GAA molecules to 300.19 cells expressing hTfR and 300.19 cells expressing cTfR were determined by flow cytometry. 300.19 is a murine pre-B cell line derived from lymphoma and was stably transfected by nucleoporation with plasmids expressing hTfR or cTfR (SEQ ID NO:1 and SEQ ID NO:2, respectively).

[0173] 300.19 cells expressing TFRC were plated at 5x10 5 cells / well in a 96-well high-binding plate (MSD L15XB-3), and 100 μL / well of the anti-TfR-GAA molecule was added and incubated at 4 °C for 45 min and washed three times with PBS 1% BSA. 100 μL / well of goat anti-human IgG conjugated to Alexa488 (Jackson ImmunoResearch, #109-545-098) was added and incubated at 4 °C for 45 min and washed three times with PBS 1% BSA. Antibody binding was evaluated by adding 200 μl / well PBS 1% BSA after centrifugation and resuspension of the cells, and read using a Guava easyCyte TM 8HT flow cytometry system. The EC 50 values were estimated using BIOSTAT@T-BINDING and reported in Table 6. Table 6. Characterization of TfR-binding agents loaded with GAA

[0174] The affinities (SPR) of 531v25Fab-FcOL-LC-GAA and 531v25Fab-FcOL-GAA were evaluated as in Example 3A and are reported in Table 7. The affinity of the 531v25-mAb-GAAx2 molecule (GAA sequence fused to two HCs) was also measured by SPR.

[0175] These data show that the affinity profiles and cynomolgus monkey / human ratios of these two (monovalent) 531v25 Fab-FcOL molecules are conserved in fusion constructs with GAA. FACS data also confirm that the 531v25 antibody is capable of binding hTfR and cTfR in its native conformation. The data obtained with the 531v25-mAb-GAAx2 molecule show the affinity and avidity for binding TfR as a result of two TfR-binding sites. Table 7. Binding affinities of 531v25Fab-FcOL-GAA, 531v25Fab-FcOL-LC-GAA, and 531v25-mAb-GAAx2 to TfR determined by SPR

[0176] We also attempted to evaluate the affinity of 531v25Fab-FcOL-GAA by BLI and compare it with 531v25-Fab-FcOL (i.e., without GAA). The affinity of 531v25Fab-FcOL-GAA was evaluated by BLI as in Example 3A, and the data are reported in Table 8. These data show that the affinity profiles and cynomolgus monkey / human ratios of the 531v25 molecule are conserved in fusion constructs with GAA. Table 8. Affinities of 531v25Fab-FcOL-GAA and 531v25Fab-FcOL for TfR compared by BLI B. No competition for binding to human TfR between 531v25-Fab-FcOL-GAA and transferrin was observed as determined by BLI

[0177] We attempted to confirm the absence of competition for binding to TfR with transferrin by BLI, in the presence of saturation with holo-TF (holotransferrin) or 531v25Fab-FcOL-GAA. This was determined as in Example 3B, and the data are reported in Table 9.

[0178] Data showed that transferrin binding was not affected by pre-association with 531v25Fab-FcOL-GAA. Saturation of human TfR with holo-transferrin minimally affected the binding of 531v25Fab-FcOL-GAA to hTfR. These data indicate that there is no competition for transferrin receptor binding between 531v25Fab-FcOL-GAA and transferrin. Table 9. Competition for binding to hTfR with transferrin determined by BLI Analyte <![CDATA[K D (nM)]]> 531v25Fab - FcOL - GAA 17.5 500 nM holo - Tf saturated, then 531v25 - Fab - FcOL - GAA 30 Holo - TF 1.2 500 nM 531v25 - Fab - FcOL - GAA saturated, then holo - TF 1.0 Example 6: Evaluation of 531v25-GAA fusion molecule in mice

[0179] Next, we compared four anti-TfR-GAA molecules in a mouse model of Pompe disease that was knocked out for the endogenous GAA gene and knocked in for the extracellular domain of the human TfR gene. Glycogen levels in various organs (brain, spinal cord, heart, and four muscles) were evaluated after administration of anti-TfR-GAA molecules in mice ( Figure 4A - Figure 4G 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.

[0180] Pompe disease mice (GAA-KO 6neo / 6neo, GAAtm1 Rabn) (Raben et al., J Biol Chem. (1998) 273:19086 - 92) were crossed with humanized TfR knock-in (KI) mice (hTfR1-KI mice) to become double homozygotes for both alleles. The hTfR1-KI mice [C57BL / 6-TfR-tm2618(TfR)Arte(Taconic)] express a chimeric TfR consisting of the human extracellular domain fused to the murine transmembrane and cytoplasmic domains (exons 4 - 19 of murine TfR were replaced with the corresponding human sequences in C57BL / 6NTAc ES cells without disrupting the murine 3' untranslated region). Expression driven by the endogenous murine TfR1 promoter was determined by qPCR and whole-tissue immunoblotting to be at comparable levels to TfR1 in wild-type mice in all tissues.

[0181] Four weekly doses of 180 nmol / kg of the test article were administered to 9- to 10-month-old hTfR-KI-Pompe disease mice. This is the molar equivalent of 20 mg / kg of alglucosidase alfa (GAA). Each test group contained 7 mice / treatment (3M, 4F). Antihistamine diphenhydramine was administered to all animals starting from the second dose to mitigate hypersensitivity to the administered human protein. Animals were euthanized 7 days after the last dose, perfused with PBS and target tissues were collected. At 4 °C, tissues were homogenized using a bead beater at 1:10 or 1:50 volume / weight excess of water / tissue. Glycogen content was biochemically quantified using a commercial colorimetric / fluorometric kit (BioVision, Milpitas, California) according to the manufacturer's instructions. Samples treated with non-hydrolytic enzymes were used for each sample to correct for glucose background. All values were back-calculated to mg glycogen / gr initial tissue.

[0182] Compared to vehicle control in hTfR-KI-Pompe disease mice, alglucosidase alfa (GAA) that does not cross the blood-brain barrier did not result in changes in glycogen levels in the brain and spinal cord. In contrast, treatment with all anti-TfR-GAA constructs resulted in significant glycogen clearance in the CNS ( Figure 4A and Figure 4B ). As shown in Table 10 below, the percent reduction was calculated relative to vehicle treatment or GAA treatment.

[0183] Data showed that 531v25Fab-LC-GAA and 531v25Fab-FcOL-GAA were the most effective in the brain, with glycogen reduction of 84% and 74%, respectively. For the myocardium, all anti-TfR-targeted GAA constructs were more effective in reducing tissue glycogen, with Fab-LC-GAA and Fab-FcOL-GAA showing excellent effects ( Figure 4C and Table 10). In all skeletal muscles, anti-TfR-GAA constructs with an Fc domain appeared to be more effective than Fab-LC-GAA, where mAb-GAA was equal to or better than Fab-FcOL-GAA ( Figure 4D - Figure 4G and Table 10). Table 10. Glycogen levels in the Pompe disease mouse model *Veh: Vehicle. Example 7: Pharmacokinetics (PK) of anti-TfR-GAA molecules in human TfR knock-in mice

[0184] Next, we attempted 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-Fc OL-LC-GAA.

[0185] Experiments were conducted in hTfR-KI mice (see Example 6). At the start of the study, all mice were male and female, first-time treated, between 4 and 6 months of age. For dosing, the anti-TfR-GAA molecules were prepared in a formulation buffer of 10 mM histidine (pH 6), 150 mM NaCl and administered as a single intravenous dose of 70 nmol / kg at a dose volume of 10 mL / kg into the tail vein. During the 7-day study duration, using the terminal sampling method (0.25, 2, 5, 24, 48, 72, and 168 hours), a total of 3 animals were evaluated for each compound at each sampling time. Blood, quadriceps muscle, and brain samples were collected at the sampling times. Blood samples were centrifuged at 1500 g for 10 minutes at 4 °C, and the separated plasma was stored at -80 °C until analysis. Prior to collection, the brain was rinsed in situ with saline solution to avoid blood contamination and then homogenized in lysis buffer (1% NP-40). The brain and quadriceps muscle were immediately frozen at -80 °C after collection.

[0186] After tissue sampling, 5 volumes of lysis buffer were added to tubes containing ceramic beads for cerebral hemispheres and metal beads for quadriceps muscle. Homogenization was achieved using Precellys For the brain, one cycle consisted of: 2 times at 5500 rpm for 20 seconds, pause for 10 seconds; and for the quadriceps muscle, 2 cycles consisted of: 4 times at 7500 rpm for 20 seconds, pause for 10 seconds. After centrifugation at 1400 rpm for 30 seconds at 4 °C, the tubes were placed on a rotator at 4 °C for 1 hour and aliquoted in low-binding tubes at a volume of 100 μL and stored at -80 °C until analysis. The concentration of each anti-TfR-GAA molecule at each time point was determined by an immunoassay method using the MSD platform (QuickPlex SQ120). This assay was based on the GAA recognition property of rabbit anti-GAA antibody coated on a microtiter plate (standard MSD 96-well partition plate) (GAA capture) and detection by electrochemiluminescence using a goat anti-mouse κ ruthenium tracer (FabκLC-detection). Samples (standards, quality controls, and study samples) were diluted 10-fold in PBS-Tween 0.1% BSA buffer and dispensed into 96-well microtiter plates. All analyses were performed in duplicate, and the quantification range was 0.0078 to 1000 ng / mL.

[0187] Tables 11, 12, and 13 summarize the PK parameters of the anti-TfR-GAA molecules in hTfR-KI mice.

[0188] Compared to other constructs, 531v25Fab-GAA exhibited the highest clearance rate. All constructs were rapidly eliminated 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 in the case of the 531v25Fab-GAA construct (Table 12). Table 11. Single-dose PK in hTfR-KI mice (plasma)

[0189] Finally, in the quadriceps muscle, the highest exposure was observed for the two 531v25Fab-FcOL constructs (Table 12). The shortest elimination half-life was observed in the case of 531v25Fab-GAA (Table 13). Table 12. Single-dose PK in hTfR-KI mice (quadriceps muscle) Table 13. Single-dose PK in hTfR-KI mice (brain)

[0190] In summary, these results indicate that compared to 531v25Fab-GAA and 531v25mAb-GAA, both the 531v25Fab-FcOL-LC-GAA and 531v25Fab-FcOL-GAA molecules exhibited the most favorable PK behavior, with the highest plasma exposure, the lowest clearance rate, and the highest distribution in the brain and quadriceps muscle. Example 8: Knockdown of MALAT1 mRNA using an anti-hTfR 531v25-Fab-FcOL-anti-MALAT-1 ASO oligonucleotide conjugate in hTfR-KI mice

[0191] To evaluate whether other moieties could effectively target the brain and skeletal muscle, we evaluated the ASO-mediated knockdown of MALAT1 (a nuclear RNA expressed in most tissues). For this study, the following reagents were prepared.

[0192] Transiently express 531v25 Fab-FcOL (composed of SEQ ID NO: 31, 34, and 56) in Expi293 cells, and then purify it using a protein A capture step and a size exclusion chromatography purification step. SEQ ID NO: 56 is similar to SEQ ID NO: 33 but has an A129C (by IMGT numbering; A114C by Kabat numbering) mutation (THIOMA B TM technology) for site-specific conjugation of MALAT1 ASO. To generate the antibody-ASO conjugate (anti-hTfR-MALAT1-ASO conjugate), reduce 531v25 Fab-FcOL with 50x molar excess of TCEP at room temperature for 3 hours, and then desalt it on a HiPrep 26 / 10 desalting column. Allow the sample to re-oxidize overnight, and then mix it with 3 molar excess of mouse MALAT1-ASO (GCATTCTAATAGCAGC; SEQ ID NO: 57) with an SMCC linker. The MALAT1-ASO sequence is shown below and has the following modifications: 5'-(SMCC)(NHC6)GbsCbsAbsdTsdTs(5MdC)sdTsdAsdAsdTsdAsdGs(5MdC)sAbsGbsCb-3' Nb: LNA residues (including LNA-5MeC and LNA T / LNA-5MeU) dN: DNA residues (5MdC): 5-methyl DNA C s: phosphorothioate backbone modification (NHC6): aminohexyl linker (SMCC): succinimidyl-trans-4-(N-maleimidomethyl)cyclohexane-1-carboxylate Monitor the conjugation process using an SDS-PAGE gel until completion, and then purify the conjugated complex from the excess unreacted ASO using size exclusion chromatography.

[0193] To generate ASO alone, inactivate the maleimide in the SMCC linker with 10 mM cysteine at room temperature for 30 min.

[0194] To evaluate the tissue-specific knockdown of MALAT1 mRNA, hTfR-KI mice were administered four times (twice a week, with a 3-4 day dosing interval) with 400 nmol / kg of inactivated SMCC-MALAT1 ASO (N = 5), or 400 nmol / kg of the ASO in the form of an anti-hTfR-MALAT1-ASO conjugate (DAR1) (N = 3), or saline vehicle (N = 5). Three days (72 hours) after the fourth dosing, the mice were anesthetized with ketamine / xylazine and perfused transcardially with heparinized DBPS with Ca / Mg. Tissues (brain, heart, gastrocnemius, quadriceps, spleen, and sciatic nerve) were harvested and weighed for quantitative PCR of MALAT1, with β-actin serving as the housekeeping gene. RNA was isolated from the frozen tissue samples. Tissues were homogenized in TRIzol / chloroform in a 2 mL tube containing 2.8 mm ceramic beads using a bead mill homogenizer. Subsequently, RNA was isolated according to the Qiagen’s RNeasy 96 QIAcube HT kit instructions. cDNA was generated using the Applied Biosystems TM High-Capacity cDNA Reverse Transcription Kit. Finally, qPCR was run on a QuantStudio TM 7Flex using the 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; ACCCCCAGCCATGTA: SEQ ID NO:61); mouse MALAT1: MALAT1 Mm01227912_s1 FAM-MGB mouse, from ThermoFisher TaqMan MGB probe.

[0195] Our data show that the anti-hTfR-MALAT1-ASO conjugate significantly knocked down MALAT1 mRNA in the brain, heart, gastrocnemius and quadriceps muscles, as well as in the spleen and sciatic nerve, compared to mice treated with equimolar amounts of free MALAT1-ASO or vehicle administered intravenously. Significance was determined using one-way ANOVA and Dunnet multiple comparisons (GraphPad v9.5.0) (* = p < 0.05, ** = p < 0.005, *** p < 0.0001 compared to vehicle)( Figure 6 ).

[0196] These findings suggest that 531v25 Fab-FcOL can be used to target oligonucleotides such as MALAT1-ASO for gene knockdown in the brain, sciatic nerve, and skeletal muscle tissues in vivo. These data provide evidence that when a series of moieties, including enzymes and oligonucleotides, are conjugated to our 531v25-anti-TfR moiety, these moieties can effectively target brain and muscle cells and have functional roles such as replenishing enzyme function (GAA) or reducing gene expression levels (MALAT1). Sequences

[0198] The sequences described in this disclosure are summarized in the table below (SEQ: SEQ ID NO).

Claims

1. 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 containing GYTFTRYY (SEQ ID NO:26) or GYTFTRYW (SEQ ID NO:27) or DYTFTRYW (SEQ ID NO:5), An HCDR2 containing IDPSVSET (SEQ ID NO:28) or IDPSVSEC (SEQ ID NO:6), and an HCDR3 containing SQIRLPYYYAMDS (SEQ ID NO:7); and A light chain CDR (LCDR) 1 containing QDISSF (SEQ ID NO:29) or QDINSF (SEQ ID NO:9), an LCDR2 containing YTS (SEQ ID NO:10), and Optionally, an LCDR3 containing QQGNTLPRT (SEQ ID NO:11).

2. The antibody or antigen-binding fragment according to claim 1, wherein The HCDR1-3 respectively comprise SEQ ID NO:26, 28 and 7, SEQ ID NO:27, 28 and 7, or SEQ ID NO:5, 6 and 7; and / or The LCDR1-2 respectively comprise SEQ ID NO:29 and 10, or SEQ ID NO:9 and 10; and / or The LCDR3 comprises SEQ ID NO:

11.

3. The antibody or antigen-binding fragment according to claim 1, the antibody or antigen-binding fragment comprises (i) HCDR1-3 respectively comprising SEQ ID NO:26, 28 and 7, and LCDR1-2 respectively comprising SEQ ID NO:29 and 10, (ii) HCDR1-3 respectively comprising SEQ ID NO:5-7, and LCDR1-2 respectively comprising SEQ ID NO:9 and 10, (iii) HCDR1-3 respectively comprising SEQ ID NO:26, 28 and 7, and LCDR1-3 respectively comprising SEQ ID NO:29, 10 and 11, or (iv) HCDR1-3 respectively comprising SEQ ID NO:5-7, and LCDR1-3 respectively comprising SEQ ID NO:9-11.

4. The antibody or antigen-binding fragment according to claim 1, the antibody or antigen-binding fragment comprises Heavy chain variable domain (V) comprising any one of SEQ ID NO: 17-21 H ), and / or A light chain variable domain (V) comprising any one of SEQ ID NO: 22-25 L ).

5. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises V H and V L , wherein the V H and V L each comprise SEQ ID NO:21 and 25, SEQ ID NO:21 and 24, SEQ ID NO:21 and 23, SEQ ID NO:19 and 23, SEQ ID NO:18 and 23, SEQ ID NO:17 and 23, SEQ ID NO:21 and 22, SEQ ID NO:20 and 22, SEQ ID NO:18 and 22, SEQ ID NO:17 and 22, or SEQ ID NO:4 and 8.

6. The antibody according to any one of claims 1-5, wherein the antibody is of the human IgG1, IgG2 or IgG4 isotype subtype, optionally comprising one or more mutations selected from: mutations that reduce antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), optionally comprising S298N, T299A and Y300S, mutations that improve the serum half-life of the antibody, optionally comprising M252Y, S254T and T256E, proximal and distal mutations, optionally comprising Y349C, T366S, L368A and Y407V for the distal mutation; and S354C and T366W for the IgG1 proximal mutation; and mutations that facilitate purification, optionally comprising H435R and Y436F (Eu numbering).

7. The antibody according to claim 6, wherein the antibody comprises a heavy chain and a light chain comprising SEQ ID NO:32 and 31, respectively.

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

9. The antigen-binding fragment according to claim 8, the antigen-binding fragment comprising a heavy chain and a light chain comprising SEQ ID NO:30 and 31, respectively.

10. The antigen-binding fragment according to claim 8, wherein 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, optionally wherein the heavy chain and the Fc polypeptide are of the human IgG1, G2 or IgG4 isotype subtype.

11. The antigen-binding fragment according to claim 10, wherein the heavy chain and the Fc polypeptide comprise one or more mutations selected from: mutations that reduce ADCC and / or CDC, optionally comprising S298N, T299A and Y300S, mutations that improve the serum half-life of the antibody, optionally comprising M252Y, S254T and T256E, proximal and distal mutations, optionally comprising Y349C, T366S, L368A and Y407V for the distal mutation; and S354C and T366W for the IgG1 proximal mutation; and mutations that facilitate purification, optionally comprising H435R and Y436F (Eu numbering).

12. The antigen-binding fragment according to 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. A TfR-binding protein, the TfR-binding protein comprising (i) an antibody or antigen-binding fragment according to any one of the preceding claims, and (ii) a cargo linked to the antibody or antigen-binding fragment, optionally wherein the cargo is a therapeutic compound, further optionally wherein the therapeutic compound is a protein.

14. The TfR-binding protein according to claim 13, wherein the cargo is optionally linked to the antibody or antigen-binding fragment via a peptide linker A heavy chain, optionally at the C-terminus of the heavy chain, an Fc polypeptide, if present, optionally at the N- or C-terminus of the Fc polypeptide, or a light chain, optionally at the C-terminus of the light chain, Further optionally, wherein the peptide linker comprises SEQ ID NO: 42 or 43.

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

35.

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

17. The TfR-binding protein according to claim 16, the TfR-binding protein comprising three polypeptides respectively comprising: SEQ ID NO: 33, 36, and 34, or SEQ ID NO: 33, 31, and 37.

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

19. The TfR-binding protein according to claim 18, the TfR-binding protein comprising two polypeptides respectively comprising SEQ ID NO: 30 and 36.

20. The TfR-binding protein according to claim 16, the TfR-binding protein comprising two HCs, two LCs, and a human GAA sequence fused to the C-terminus of one of the two HCs, optionally, wherein 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.

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

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

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

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

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

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

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

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

29. A TfR-binding protein according to claim 15 for treating a subject lacking the enzyme or its activity.

30. Use of a TfR-binding protein according to claim 15 in the manufacture of a medicament for treating a subject lacking the enzyme or its activity.

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

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

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

34. Use of an antibody or antigen-binding fragment according to any one of claims 1-12 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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