Transferrin receptor binding domains and proteins comprising same

Abstract

Provided herein are transferrin receptor binding domains, polypeptides comprising transferrin receptor binding domains, and antibodies and antibody fragments comprising transferrin receptor binding domains, and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 342,313 filed on May 16, 2022; the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically under the XML file name "47364-0022WO1". The size of the XML file, created on May 15, 2023, is 254,608 bytes. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to polypeptides and protein constructs comprising an antigen binding domain that specifically binds to transferrin receptor. Background Art

[0006] The human brain is a highly vascularized organ containing about 400 miles of blood vessels. These blood vessels are arranged by tightly connected endothelial cells to form a blood-brain barrier (BBB), which protects the brain from toxins by regulating the transfer of proteins, nutrients and waste products. Attempts have been made to deliver therapeutic agents directly into the CNS; however, this represents a highly invasive method, and distribution is generally limited to areas close to the site of administration. The delivery of therapeutic agents across the BBB is a less invasive treatment modality, but there are its own challenges due to the relative impermeability of the BBB to macromolecules. Therefore, compositions containing brain permeabilizing agents or causing the expression of brain permeabilizing agents and methods of treating using brain permeabilizing agents will represent a major advance in the effective treatment of the targeting CNS.

[0007] The transferrin receptor is a homologous receptor for transferrin that, among other functions, is required for importing iron into cells and is regulated in response to intracellular iron concentrations. The transferrin receptor is expressed on the endothelium of the blood-brain barrier and can mediate transcytosis of its ligands across the blood-brain barrier. Protein therapeutics containing binding domains that specifically and reversibly interact with the transferrin receptor can achieve higher brain exposure than standard biotherapeutic molecules. Summary of the invention

[0008] Provided herein are transferrin receptor binding proteins comprising a camelid heavy chain variable domain. In some embodiments, the transferrin receptor binding protein has a K of about 1 nM to about 6 μM (e.g., about 1 nM to about 1 mM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, or about 1 nM to about 10 nM). DBinds to human transferrin. In some embodiments, the transferrin receptor binding protein binds to human transferrin with a K of about 1 nM to about 2.5 mM (e.g., about 1 nM to about 1 mM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, or about 1 nM to about 10 nM). D Binds to cynomolgus monkey transferrin.

[0009] The present invention also provides a transferrin receptor binding domain, which comprises: (a) a first sequence G-X1-T-X2-X3-X4-X5-X6-M-X7 (SEQ ID NO: 1), wherein X1 is L or F, X2 is S or F, X3 is D or S or E, X4 is T or S, X5 is G or Y, X6 is G or A, or X7 is G or S; (b) a second sequence AI-X8-X9-X 10 -X 11 -X 12 -X 13 -TX 14 -YADSVKG (SEQ ID NO: 2), wherein X8 is T or S, X9 is W or S or G or F or Y, X 10 Is S or N, X 11 It is G or A, X 12 It is R or S or G, X 13 It is H or A or X 14 is L or Y; and (c) a third sequence AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -DY (SEQ ID NO: 3), wherein X 15 It is L or R, X 16 Is V or A, X 17 Is V or A, X 18 It is G or A, X 19 Is I or A, X 20 It is G or A, X 21 Is I or A, X 22 It is E or A, X 23 V or A, X 24 It is Q or A, X 25 It is T or A or X 26 It is Y or F.

[0010] In some embodiments, the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GLTSDTGGMG (SEQ ID NO:4), GFTFS TGGMG (SEQ ID NO:12), GFTSDTGGMG (SEQ ID NO:5), GL TFDTGGMG (SEQ ID NO:6), GLTSSTGGMG (SEQ ID NO:7), GLTSETGGMG (SEQ ID NO:8), GLTSDTYGMG (SEQ ID NO:9), GLTSDTGAMG (SEQ ID NO:10), GLTSDTGGMS (SEQ ID NO:11), GFTFSTGGMS (SEQ ID NO:13), and GFTFSSGGMS (SEQ ID NO:14); (b) a second sequence selected from the group consisting of AITWSGRHTLY ADSVKG (SEQ ID NO:15), AISWSGRHTLYADSVKG (SEQ ID NO:16), AITSSGRHTLYADSVKG (SEQ ID NO:17), AITGSGRH TLYADSVKG (SEQ ID NO: 18), AITWNGRHTLYADSVKG (SEQ ID NO: 19), AITWSGSHTLYADSVKG (SEQ ID NO: 20), AITWSGGHTLYADSVKG (SEQ ID NO: 21), AITWSGRHTYYADSVKG (SEQ ID NO: 22), AITWSARHTLYADSVKG (SEQ ID NO: 23), AI TWSGRATLYADSVKG (SEQ ID NO:24), AITFSGRHTLYADSVKG (SEQ ID NO:25) and AITYSGRHTLYADSVKG (SEQ ID NO:26);and (c) a third sequence selected from the group consisting of ALDVVGIGIEVQTYDY (SEQ ID NO:27), ARDVVGIGIEVQTYDY (SEQ ID NO:28), ALDAVGIGIEVQTYDY (SEQ ID NO:29), ALDVAGIGIEVQTYDY (SEQ ID NO:30), ALDVVAIGIEVQTYDY (SEQ ID NO:31), ALDVVGAGIEVQTYDY (SEQ ID NO:32), ALDVVGIAIEVQTYDY (SEQ ID NO:33), ALDVVGIGAEVQTYDY (SEQ ID NO:34), ALDVVGIGIAVQTYDY (SEQ ID NO:35), ALDVVGIGIEAQTYDY (SEQ ID NO:36), ALDVVGIGIEVATYDY (SEQ ID NO:37), ALDVVGIGIEVQAYDY (SEQ ID NO:38), ALDVVGI NO:38) and ALDVVGIGIEVQTFDY (SEQ ID NO:39). ;

[0011] Also provided herein is a transferrin receptor binding domain comprising: (a) a first sequence G-X1-TLD-X2-X3-AI-X4 (SEQ ID NO: 183), wherein X1 is S or F, wherein X2 is D, H or Y, X3 is Y or F, and X4 is G or A; (b) a second sequence CIS-X5-X6-DG-X7-T-X8-Y-X9-DX 10 -VKG (SEQ ID NO: 184), wherein X5 is S or R, X6 is S or G, X7 is I or R, X8 is F or Y, X9 is A or G, and X 10 is F or S; and (c) a third sequence AX 11 -X 12 -X 13 -GPNX 14 -CRGWLWX 15 -PX 16 -X 17 -SGS (SEQ ID NO: 185), wherein X 11 A or S, X 12 Is K or H, X 13 is Y or D, X 14 Is I or V, X 15 Is V or E, X 16 is P or Q, and X 17 is V, I or L (SEQ ID NO: 185).

[0012] In some embodiments, the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GSTLDYAIG (SEQ ID NO: 186), GSTLDHYAIG (SEQ ID NO: 187), and GFTLDYFAIA (SEQ ID NO: 188); (b) a second sequence selected from the group consisting of CISSSDGITFYGDFVKG (SEQ ID NO: 189), CISRSDGITYYADSVKG (SEQ ID NO: 190), and CISSGDGRTFYADSVKG (SEQ ID NO: 191); and (c) a third sequence selected from the group consisting of AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192), AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193), and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

[0013] Also provided herein is a transferrin receptor binding domain comprising: (a) a first sequence G-X1-T-X2-X3-X4-X5-X6-M-X7 (SEQ ID NO: 195), wherein X1 is L or F, X2 is S or F, X3 is D or S or E, X4 is T or S, X5 is G or Y, X6 is G or A, and X7 is G or S; (b) a second sequence AI-X8-X9-X 10 -X 11 -X 12 -X 13 -TX 14 -YADSVKG (SEQ ID NO: 196), wherein X8 is T or S, X9 is W, S, G, F or Y, X 10 Is S or N, X 11 It is G or A, X 12 is R, S or G, X 13 It is H or A or X 14 is L or Y; and (c) a third sequence AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -DY (SEQ ID NO: 197), wherein X15 It is L or R, X 16 Is V or A, X 17 Is V or A, X 18 It is G or A, X 19 Is I or A, X 20 It is G or A, X 21 Is I or A, X 22 It is E or A, X 23 V or A, X 24 It is Q or A, X 25 is T or A, and X 26 It is Y or F.

[0014] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain is a VHH domain. In some embodiments, the VHH domain is humanized.

[0015] Also provided herein is a polypeptide comprising any transferrin receptor binding domain as described herein. In some embodiments, the polypeptide also comprises an Fc polypeptide. In some embodiments of any polypeptide as described herein, the polypeptide further comprises a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of: enzymes, hormones, growth factors, cytokines, chemokines, glycolipids, lipids, soluble proteins, nucleic acids and other antigen binding domains.

[0016] In some embodiments of any of the polypeptides described herein, the polypeptide comprises a linker between the fusion partner and the transferrin receptor binding domain. In some embodiments of any of the polypeptides described herein, the fusion partner is directly adjacent to the transferrin receptor binding domain.

[0017] In some embodiments of any polypeptide described herein, the polypeptide is a single polypeptide. In some embodiments, the single polypeptide comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 216-221. In some embodiments, the single polypeptide comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 216-221. In some embodiments, the single polypeptide comprises the sequence of any one of SEQ ID NOs: 216-221.

[0018] In some embodiments of any of the polypeptides described herein, the polypeptide is part of a protein complex.

[0019] Also provided herein are antibodies and antibody fragments comprising any transferrin receptor binding domain described herein. In some embodiments, the antibody or antibody fragment comprises a VHH domain. In some embodiments, the antibody is humanized.

[0020] In some embodiments, the antibody or antibody fragment further comprises a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of: an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

[0021] In some embodiments, the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain. In some embodiments, the fusion partner is directly adjacent to the transferrin receptor binding domain.

[0022] Also provided herein are compositions comprising (i) any transferrin receptor binding domain described herein, any polypeptide described herein, any antibody described herein, or any antibody fragment described herein, and (ii) a pharmaceutically acceptable carrier.

[0023] Also provided herein are kits comprising any of the compositions described herein.

[0024] Also provided herein is a nucleic acid encoding any transferrin receptor binding domain described herein, any polypeptide described herein, any antibody described herein, or any antibody fragment described herein. Also provided herein is an expression vector comprising any nucleic acid described herein. Also provided herein is a host cell transfected with any nucleic acid described herein or transduced with any expression vector described herein.

[0025] Provided herein are methods for producing a transferrin receptor binding domain, polypeptide, antibody, or antibody fragment, the method comprising: (a) culturing any host cell described herein in a culture medium under conditions sufficient to allow production of the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment; and (b) harvesting the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment from the host cell or the culture medium.

[0026] In some embodiments of any of the methods described herein, the method further comprises isolating the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment.

[0027] In some embodiments of any of the methods described herein, the method further comprises formulating the isolated transferrin receptor binding domain, polypeptide, antibody, or antibody fragment.

[0028] Provided herein is a transferrin receptor binding domain comprising a sequence at least 80% identical to any one of SEQ ID NOs: 40-88, 147, and 210-215. In some embodiments, the transferrin receptor binding domain comprises a sequence at least 90% identical to any one of SEQ ID NOs: 40-88, 147, and 210-215. In some embodiments, the transferrin receptor binding domain comprises the sequence of any one of SEQ ID Nos: 40-88, 147, and 210-215.

[0029] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain is a VHH domain. In some embodiments, the VHH domain is humanized.

[0030] Provided herein are polypeptides comprising any transferrin receptor binding domains as described herein. In some embodiments, the polypeptide also comprises an Fc polypeptide. In some embodiments of any polypeptide as described herein, the polypeptide further comprises a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of: enzymes, hormones, growth factors, cytokines, chemokines, glycolipids, lipids, soluble proteins, nucleic acids and other antigen binding domains.

[0031] In some embodiments of any of the polypeptides described herein, the polypeptide comprises a linker between the fusion partner and the transferrin receptor binding domain. In some embodiments of any of the polypeptides described herein, the fusion partner is directly adjacent to the transferrin receptor binding domain.

[0032] In some embodiments of any of the polypeptides described herein, the polypeptide is a single polypeptide.

[0033] In some embodiments, a single polypeptide comprises a sequence at least 80% identical to any one of SEQ ID Nos: 216-221. In some embodiments, a single polypeptide comprises a sequence at least 90% identical to any one of SEQ ID Nos: 216-221. In some embodiments, a single polypeptide comprises the sequence of any one of SEQ ID Nos: 216-221.

[0034] In some embodiments of any of the polypeptides described herein, the polypeptide is part of a protein complex.

[0035] Also provided herein are antibodies comprising any of the transferrin receptor binding domains described herein.

[0036] In some embodiments, the antibodies are humanized.

[0037] In some embodiments of any of the antibodies described herein, the antibody further comprises a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

[0038] In some embodiments of any of the antibodies described herein, the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain. In some embodiments of any of the antibodies described herein, the fusion partner is directly adjacent to the transferrin receptor binding domain.

[0039] Also provided herein are antibody fragments comprising any of the transferrin receptor binding domains described herein.In some embodiments, the antibody fragment comprises a VHH domain.

[0040] In some embodiments of any of the antibody fragments described herein, the antibody fragment is humanized.

[0041] In some embodiments of any antibody fragment described herein, the antibody fragment further comprises a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of: an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

[0042] In some embodiments of any of the antibody fragments described herein, the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain. In some embodiments of any of the antibody fragments described herein, the fusion partner is directly adjacent to the transferrin receptor binding domain.

[0043] Also provided herein are compositions comprising (i) any transferrin receptor binding domain described herein, any polypeptide described herein, any antibody described herein, or any antibody fragment described herein, and (ii) a pharmaceutically acceptable carrier.

[0044] Also provided herein are kits comprising any of the compositions described herein.

[0045] Also provided herein are nucleic acids encoding any transferrin receptor binding domain described herein, any polypeptide described herein, any antibody described herein, or any antibody fragment described herein.

[0046] Also provided herein are expression vectors comprising any of the nucleic acids described herein. Also provided herein are host cells transfected with any of the nucleic acids described herein or transduced with any of the expression vectors described herein.

[0047] Also provided herein is a method for producing any transferrin receptor binding domain described herein, any polypeptide described herein, any antibody described herein, or any antibody fragment described herein, the method comprising: (a) culturing any host cell described herein in a culture medium under conditions sufficient to allow production of the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment; and (b) harvesting the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment from the host cell or the culture medium.

[0048] In some embodiments of any of the methods described herein, the method further comprises isolating the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment.

[0049] In some embodiments of any of the methods described herein, the method further comprises formulating the isolated transferrin receptor binding domain, polypeptide, antibody, or antibody fragment.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0051] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A series of fluorescent images are shown showing the uptake of polypeptides comprising an exemplary antigen binding domain that specifically binds to the transferrin receptor after culturing HEK293T cells (human transferrin receptor positive cells), CHO:cyTfR cells (cyno transferrin receptor positive cells), and CHO cells (human and cyno transferrin receptor negative cells) in the presence of 4.0 nM to 1000 nM of the polypeptide. Detection was performed using a secondary antibody conjugated to a fluorophore that specifically binds to the polypeptide.

[0053] Figure 2A Shown are a series of representative cellular uptake / binding curves for exemplary transferrin receptor binding domains generated as described herein. Cellular uptake / binding curves were generated in HEK293T cells (human transferrin receptor positive cells).

[0054] Figure 2BShown are a series of representative cellular uptake / binding curves for exemplary transferrin receptor binding domains generated as described herein. Cellular uptake / binding curves were generated in CHO:cyTfR cells (cyno transferrin receptor positive cells). DETAILED DESCRIPTION

[0055] Provided herein are transferrin receptor binding proteins comprising a camelid heavy chain variable domain.

[0056] Also provided are polypeptides, antibodies or antibody fragments comprising any transferrin receptor binding domain described herein. In some embodiments, the polypeptide is a single polypeptide. In other embodiments, the polypeptide is part of a protein complex.

[0057] Also provided herein are compositions comprising any transferrin receptor binding domain, polypeptide, antibody or antibody fragment as described herein. Also provided herein are kits comprising any compositions as described herein. Also provided herein are nucleic acids encoding any transferrin receptor binding domain, polypeptide, antibody or antibody fragment as described herein. Also provided are host cells comprising any nucleic acid as described herein. Also provided are methods for producing any transferrin receptor binding domain, polypeptide, antibody or antibody fragment as described herein, the methods comprising cultivating any host cell as described herein.

[0058] In some embodiments of any of the antibodies, antibody fragments, or polypeptides described herein, a total of about 50 to about 250 amino acids, about 100 to about 300 amino acids, about 150 to about 350 amino acids, about 200 to about 400 amino acids, about 600 to about 1,500 amino acids, about 650 to about 1,400 amino acids, about 700 to about 1,300 amino acids, about 750 to about 1,200 amino acids, about 800 to about 1,100 amino acids, or about 900 to about 1,000 amino acids can be included.

[0059] The terms "a" and "an" refer to one or more (ie, at least one) grammatical objects of the article. For example, "a polypeptide" encompasses one or more polypeptides.

[0060] As used herein, the terms "about" and "approximately," when used to modify an amount specified in a value or range, represent the value and reasonable deviations from the value known to those skilled in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the value.

[0061] Unless otherwise indicated, a "nucleotide sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and therefore encode the same amino acid sequence.

[0062] The term "exogenous" refers to any material introduced or derived from outside a cell, tissue or organism that is not produced by or derived from the same cell, tissue or organism into which it is introduced.

[0063] The terms "transduction", "transfection" or "transformation" refer to the process of introducing or transferring exogenous nucleic acid into a cell. A "transduced", "transfected" or "transformed" cell (e.g., a mammalian cell, a hepatocyte) is a cell that has been transduced, transfected or transformed with an exogenous nucleic acid (e.g., an expression vector) comprising an exogenous nucleic acid encoding a polypeptide.

[0064] The term "subject" is intended to include any mammal. In some embodiments, the subject is a cat, dog, goat, human, non-human primate, rodent (e.g., mouse or rat), pig, or sheep. In some embodiments, the subject suffers from or is at risk of suffering from a CNS disorder or disease. In some embodiments, the subject has previously been identified or diagnosed as suffering from a CNS disorder or disease.

[0065] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a combination thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses complementary sequences as well as sequences explicitly indicated. In some embodiments of any nucleic acid described herein, the nucleic acid is DNA. In some embodiments of any nucleic acid described herein, the nucleic acid is RNA.

[0066] Modification can be introduced into the nucleotide sequence by standard techniques known in the art (such as site-directed mutagenesis and polymerase chain reaction (PCR) mediated mutagenesis). Conservative amino acid substitutions are amino acid substitutions in which amino acid residues are substituted by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include having basic side chains (e.g., arginine, lysine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine and tryptophan), non-polar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline and valine), β-branched side chains (e.g., isoleucine, threonine and valine) and aromatic side chains (e.g., histidine, phenylalanine, tryptophan and tyrosine) and aromatic side chains (e.g., histidine, phenylalanine, tryptophan and tyrosine).

[0067] The term "antigen binding domain" is used to refer to one or more antibody variable domains (e.g., formed by amino acids from a single polypeptide or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that are capable of specific binding to one or more different antigens. In some examples, the antigen binding domain may bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain may be an antibody or fragment thereof. In some embodiments, the antigen binding domain may include an alternative scaffold. Non-limiting examples of antigen binding domains are described herein. Additional examples of antigen binding domains are known in the art.

[0068] The term "antibody" refers to a protein with an immunoglobulin fold that specifically binds to an antigen via its variable region. As used herein, the term "antibody" may refer to a camelid (e.g., camel, llama, alpaca, dromedary) heavy chain antibody lacking a light chain. These heavy chain antibodies interact with antigens through a single variable domain referred to as VHH, VHH domain or VHH antibody. The antigen binding site of the VHH domain is similar to the antigen binding site of the heavy chain variable domain of a conventional antibody, with differences in the framework region and the complementary determining region. The target antigen typically has many binding sites, also referred to as epitopes, which are identified by CDRs (complementary determining regions) on multiple antibodies. Each antibody that specifically binds to different epitopes has a different structure. Therefore, an antigen may have more than one corresponding antibody. The antibody may, for example, comprise a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen binding site of an antigen that immunospecifically binds to a target of interest or a portion thereof (e.g., transferrin receptor). The immunoglobulins disclosed herein can be of any subclass of immunoglobulin molecules (eg, IgG1, IgG2, IgG3).

[0069] Antibodies bound to various types of molecules such as polyethylene glycol (PEG) can be used as modified antibodies. Methods for modifying antibodies are already established in the art.

[0070] The term "antibody fragment" refers to a portion of a full-length antibody or a polypeptide comprising a portion of a full-length antibody, which portion retains antigen-binding activity via its variable region. As used herein, the term "antibody fragment" may refer to a portion of a full-length camelid heavy chain antibody that retains antigen-binding activity via its variable region. Non-limiting examples of antibody fragments comprising camelid heavy chain antibodies that retain their antigen-binding activity include VHH domains, single domain antibodies, nanobodies, single domain antibodies fused to an Fc domain, and VHH domains fused to an Fc domain (Bannas et al., Front. Immunol., 8: doi: 10.3389 / fimmu.2017.01603 (2017)), and further non-limiting examples of antibody fragments include diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4): 315-323), fragments produced by a Fab expression library, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0071] The term "variable region" or "variable domain" refers to a domain in an antibody heavy chain or a domain in an antibody light chain that is encoded by a combination of germline variable (V) gene segments, diversity (D) gene segments, or joining (J) gene segments, and confers to the antibody its specificity for binding to an antigen. As described herein, a camelid heavy chain antibody includes a single variable heavy chain domain (e.g., a VHH domain) encoded by a combination of germline variable (V) gene segments that confers to the antibody its specificity for binding to an antigen (e.g., transferrin receptor).

[0072] The term "complementarity determining region" or "CDR" refers to one of the three hypervariable regions (or HVRs) known to confer (at least in part) antigen binding specificity in each antibody light chain and each antibody heavy chain variable domain (e.g., VHH domain). The three CDRs in the antibody heavy chain and antibody light chain interrupt the four framework regions in the heavy chain variable domain and the light chain variable domain. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, which are numbered sequentially from the N-terminus and are usually also identified by the chain in which the specific CDR is located. Therefore, VH CDR3 or CDR-H3 is the third CDR located in the heavy chain variable domain, and VL CDR1 or CDR-L1 is the first CDR from the light chain variable domain. As used herein, VH CDR1 can be used interchangeably with "first sequence", VH CDR2 can be used interchangeably with "second sequence", and VH CDR3 can be used interchangeably with "third sequence".

[0073] The "framework region" or "FR" of the heavy chain immunoglobulin chain serves to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences.

[0074] The amino acid sequences of the CDR and framework regions can be determined using various definitions well known in the art (eg, Kabat).

[0075] The term "protein complex" means a complex of two or more polypeptides (e.g., identical or different polypeptides) associated by non-covalent bonds. For example, a protein complex may comprise at least one antigen binding domain. Non-limiting examples and aspects of protein complexes are described herein. Additional examples and aspects of protein complexes are known in the art. In some embodiments, a protein complex comprises one or more single polypeptides.

[0076] The term "treating," "treating," "treating," "treating," "treating," and "treatment" means reducing the number, frequency, severity, and / or duration of one or more (e.g., two, three, four, five, or six) symptoms of a disease or disorder in a subject (e.g., any subject described herein), and / or causing a decrease in the rate of development and / or worsening of one or more symptoms of a disease or disorder in a subject over time.

[0077] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired biological site of action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, colon delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the compositions described herein are administered intravenously.

[0078] The term "promoter" means a DNA sequence recognized by an enzyme / protein in a cell (e.g., a mammalian cell, a hepatocyte) that is required to initiate transcription of an operably linked coding sequence (e.g., a nucleic acid encoding a polypeptide (e.g., any exemplary polypeptide described herein)). A promoter generally refers to, for example, a nucleotide sequence to which RNA polymerase and / or any associated factors bind and initiate transcription. A promoter may be constitutive, inducible, or tissue-specific (e.g., a liver-specific promoter).

[0079] The term "enhancer" refers to a nucleotide sequence that can increase the transcription of an operably linked nucleic acid (e.g., a nucleic acid encoding a polypeptide (e.g., any of the exemplary polypeptides described herein)). An enhancer can increase the level of transcription by providing additional binding sites for transcription-related proteins (e.g., transcription factors). Unlike a promoter, an enhancer can function at a greater distance (e.g., compared to a promoter) from the start site of transcription.

[0080] In the context of two or more polypeptide sequences, the terms "same" or "percent identity" means that two or more sequences or subsequences are identical or have a specified percentage of amino acid residues, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, when compared and aligned for maximum correspondence over a comparison window or designated region when measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0081] In order to carry out the sequence comparison of polypeptide, usually an amino acid sequence serves as a reference sequence and is compared with the candidate sequence. The various methods available to those skilled in the art can be used, for example, visual alignment or using publicly available software using a known algorithm for realizing maximum alignment to perform comparison. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters for comparison to realize maximum alignment can be determined by those skilled in the art. For the purpose of this application, in order to carry out the sequence comparison of polypeptide sequences, the BLASTP algorithm standard protein BLAST is used to compare two protein sequences with default parameters.

[0082] The term "affinity" refers to the strength of the sum of all non-covalent interactions between an antigen binding site and its antigen. Unless otherwise indicated, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between an antigen binding domain and an antigen. Surface plasmon resonance (SPR) techniques (e.g., ) or biolayer interferometry (e.g. ) Affinity is measured. Additional methods for determining the affinity of an antigen binding domain and its antigen are known in the art.

[0083] Transferrin receptor binding domain and peptides

[0084] Provided herein are transferrin receptor binding proteins comprising a camelid heavy chain variable domain. In some embodiments, the protein has a K of about 0.1 nM to about 1 μM (e.g., about 0.1 nM to about 1 μM, about 0.5 nM to about 500 nM, about 1 nM to about 250 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, or about 1 nM to about 10 nM). DIn some embodiments, the protein binds to human transferrin with a K of about 1 nM to about 2.5 μM (e.g., about 1 nM to about 1 μM, about 1 nM to about 500 nM, about 1 nM to about 250 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, or about 1 nM to about 10 nM). D Binds to cynomolgus monkey transferrin.

[0085] The present invention also provides a transferrin receptor binding domain, which comprises: (a) a first sequence G-X1-T-X2-X3-X4-X5-X6-M-X7 (SEQ ID NO: 1), wherein X1 is L or F, X2 is S or F, X3 is D or S or E, X4 is T or S, X5 is G or Y, X6 is G or A, or X7 is G or S; (b) a second sequence AI-X8-X9-X 10 -X 11 -X 12 -X 13 -TX 14 -YADSVKG (SEQ ID NO: 2), wherein X8 is T or S, X9 is W or S or G or F or Y, X 10 Is S or N, X 11 It is G or A, X 12 It is R or S or G, X 13 It is H or A or X 14 is L or Y; and (c) a third sequence AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -DY (SEQ ID NO: 3), wherein X 15 It is L or R, X 16 Is V or A, X 17 Is V or A, X 18 It is G or A, X 19 Is I or A, X 20 It is G or A, X 21 Is I or A, X 22 It is E or A, X 23 V or A, X 24 It is Q or A, X 25 It is T or A or X 26 It is Y or F.

[0086] In some embodiments, the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GLTSDTGGMG (SEQ ID NO:4), GFTSD TGGMG (SEQ ID NO:5), GLTFDTGGMG (SEQ ID NO:6), GL TSSTGGMG (SEQ ID NO:7), GLTSETGGMG (SEQ ID NO:8), GLTSDTYGMG (SEQ ID NO:9), GLTSDTGAMG (SEQ ID NO:10), GLTSDTGGMS (SEQ ID NO:11), GFTFSTGGMG (SEQ ID NO:12), GFTFSTGGMS (SEQ ID NO:13), and GFTSSSGGMS (SEQ ID NO:14); (b) a second sequence selected from the group consisting of AITWSGRHTLYADSVKG (SEQ ID NO:15), AISWSGRHTLYADSVKG (SEQ ID NO:16), AITSSGRHTLYADSVKG (SEQ ID NO:17), AITGSGRHT LYADSVKG (SEQ ID NO: 18), AITWNGRHTLYADSVKG (SEQ ID NO: 19), AITWSGSHTLYADSVKG (SEQ ID NO: 20), AITWSGGHTLYADSVKG (SEQ ID NO: 21), AITWSGRHTYYADSVKG (SEQ ID NO: 22), AITWSARHTLYADSVKG (SEQ ID NO: 23), AI TWSGRATLYADSVKG (SEQ ID NO:24), AITFSGRHTLYADSVKG (SEQ ID NO:25) and AITYSGRHTLYADSVKG (SEQ ID NO:26);and (c) a third sequence selected from the group consisting of ALDVVGIGIEVQTYDY (SEQ ID NO:27), ARDVVGIGIEVQTYDY (SEQ ID NO:28), ALDAVGIGIEVQTYDY (SEQ ID NO:29), ALDVAGIGIEVQTYDY (SEQ ID NO:30), ALDVVAIGIEVQTYDY (SEQ ID NO:31), ALDVVGAGIEVQTYDY (SEQ ID NO:32), ALDVVGIAIEVQTYDY (SEQ ID NO:33), ALDVVGIGAEVQTYDY (SEQ ID NO:34), ALDVVGIGIAVQTYDY (SEQ ID NO:35), ALDVVGIGIEAQTYDY (SEQ ID NO:36), ALDVVGIGIEVATYDY (SEQ ID NO:37), ALDVVGIGIEVQAYDY (SEQ ID NO:38), ALDVVGI NO:38) and ALDVVGIGIEVQTFDY (SEQ ID NO:39). ;

[0087] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain comprises: (a) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (b) GFTSDTGGMG (SEQ ID NO:5), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTY DY (SEQ ID NO:27); (c) GLTFDTGGMG (SEQ ID NO:6), AIT WSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (d) GLTSSTGGMG (SEQ ID NO:7), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); NO: 27); (e) GLTSETGGMG (SEQ ID NO: 8), AITWSGR HTLYADSVKG (SEQ ID NO: 15) and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (f) GLTSDTYGMG (SEQ ID NO: 9), AITWSGRHT LYADSVKG (SEQ ID NO: 15) and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); NO:27); (g) GLTSDTGAMG (SEQ ID NO:10), AITWSGRHTLY ADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (h) GLTSDTGGMS (SEQ ID NO:11), AITWSGRHTLYAD SVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:11) NO:27); (i)GLTSDTGGMG (SEQ ID NO:4), AISWSGRHTLYADSVKG (SEQ ID NO:16) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (j) GLTSDTGGMG (SEQ ID NO:4), AITSSGRHTLYADSVKG (SEQ ID NO:17) and ALDVVGIGIEVQTYDY (SEQ ID NO:27);(k) GLTSDTGGMG (SEQ ID NO:4), AITGSGRHTLYADSVKG (SEQ ID NO:18), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (l) GLTSDTGGMG (SEQ ID NO:4), AITWNGRHTLYADSVKG (SEQ ID NO:19), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (m) GLTSD TGGMG (SEQ ID NO:4), AITWSGSHTLYADSVKG (SEQ ID NO:20), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (n) GLTSDTGG MG (SEQ ID NO:4), AITWSGGHTLYADSVKG (SEQ ID NO:21), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (o) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTYYADSVKG (SEQ ID NO:22), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (p) GLTSDTGGMG (SE Q ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ARD VVGIGIEVQTYDY (SEQ ID NO:28); (q) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDAVGIGIEVQTYDY (SEQ ID NO:29); (r) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ IDNO:15), and ALDVAGI GIEVQTYDY (SEQ ID NO:30); (s) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVAIGIEV QTYDY (SEQ ID NO:31); (t) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGAGIEVQT YDY (SEQ ID NO:32); (u) GLTSDTGGMG (SEQ ID NO:4), AI TWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIAIEVQTYD Y (SEQ ID NO:33);(v) GLTSDTGGMG (SEQ ID NO:4), AITW SGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGAEVQTYDY (SEQ ID NO:34); (w) GLTSDTGGMG (SEQ ID NO:4), AITWS GRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIAVQTYDY (SEQ ID NO:35); (x) GLTSDTGGMG (SEQ ID NO:4), AITWSGR HTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEAQTYDY (SEQID NO:36); (y) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHT LYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVATYDY (SEQ ID NO:37); (z) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLY ADSVKG (SEQ ID NO:15), and ALDVVGIGIEVQAYDY (SEQ ID NO:38); (aa) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYAD SVKG (SEQ ID NO:15), and ALDVVGIGIEVQTFDY (SEQ ID NO:39); (bb) GLTSDTGGMG (SEQ ID NO:4), AITWSARHTLYADSV KG (SEQ ID NO:23), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (cc) GLTSDTGGMG (SEQ ID NO:4), AITWSGRATLYADSVK G (SEQ ID NO:24), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (dd) GLTSDTGGMG (SEQ ID NO:4), AITFSGRHTLYADSVKG (SEQ ID NO:25), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (ee) GFTFSTGGMG (SEQ ID NO:12), AITWSGRHTLYADSVKG (SEQID NO:15), and ALDVVGIGIEVQTYDY (SEQ ID NO:27);(ff)GFTFSTGGMS (SEQ ID NO:13), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (gg)GFT FSSGGMS (SEQ ID NO:14), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:15) OR IDNO:27). ;

[0088] Also provided herein is a transferrin receptor binding domain comprising: (a) a first sequence G-X1-TLD-X2-X3-AI-X4 (SEQ ID NO: 183), wherein X1 is S or F, wherein X2 is D, H or Y, X3 is Y or F, and X4 is G or A; (b) a second sequence CIS-X5-X6-DG-X7-T-X8-Y-X9-DX 10 -VKG (SEQ ID NO: 184), wherein X5 is S or R, X6 is S or G, X7 is I or R, X8 is F or Y, X9 is A or G, and X 10 is F or S; and (c) a third sequence AX 11 -X 12 -X 13 -GPNX 14 -CRGWLWX 15 -PX 16 -X 17 -SGS (SEQ ID NO: 185), wherein X 11 A or S, X 12 Is K or H, X 13 is Y or D, X 14 Is I or V, X 15 It is V or E, X 16 is P or Q, and X 17 is V, I or L (SEQ ID NO: 185).

[0089] In some embodiments, the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GSTLDYAIG (SEQ ID NO: 186), GSTLDHYAIG (SEQ ID NO: 187), and GFTLDYFAIA (SEQ ID NO: 188); (b) a second sequence selected from the group consisting of CISSSDGITFYGDFVKG (SEQ ID NO: 189), CISRSDGITYYADSVKG (SEQ ID NO: 190), and CISSGDGRTFYADSVKG (SEQ ID NO: 191); and (c) a third sequence selected from the group consisting of AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192), AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193), and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

[0090] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain comprises: (a) GSTLDDYAIG (SEQ ID NO: 186), CISSSDGITFYGDFVKG (SEQ ID NO: 189) and AAKYGPNICR GWLWVPPVSGS (SEQ ID NO: 192); (b) GSTLDHYAIG (SEQ ID NO: 187), CISRSDGITYYADSVKG (SEQ ID NO: 190) and AAHYGP NVCRGWLWEPPISGS (SEQ ID NO: 193); or (c) GFTLDYFAIA (SEQ ID NO: 188), CISSGDGRTFYADSVKG (SEQ ID NO: 191) and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

[0091] Also provided herein is a transferrin receptor binding domain comprising: (a) a first sequence G-X1-T-X2-X3-X4-X5-X6-M-X7 (SEQ ID NO: 195), wherein X1 is L or F, X2 is S or F, X3 is D or S or E, X4 is T or S, X5 is G or Y, X6 is G or A, and X7 is G or S; (b) a second sequence AI-X8-X9-X 10 -X 11 -X 12 -X 13 -TX 14-YADSVKG (SEQ ID NO: 196), wherein X8 is T or S, X9 is W, S, G, F or Y, X 10 Is S or N, X 11 It is G or A, X 12 is R, S or G, X 13 It is H or A or X 14 is L or Y; and (c) a third sequence AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -DY (SEQ ID NO: 197), wherein X 15 It is L or R, X 16 Is V or A, X 17 Is V or A, X 18 It is G or A, X 19 Is I or A, X 20 It is G or A, X 21 Is I or A, X 22 It is E or A, X 23 V or A, X 24 It is Q or A, X 25 is T or A, and X 26 is Y or F. In some embodiments, the transferrin receptor binding domain comprises: GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199), and ALDVVGIGIEVQT YDY (SEQ ID NO: 200).

[0092] Provided herein is a transferrin receptor binding domain comprising: ESAFSLNAIG (SEQ ID NO: 201), GIGTDGITTYYADFVKD (SEQ ID NO: 202), and NAGSWRTVLSGTHVSRS (SEQ ID NO: 203).

[0093] Provided herein is a transferrin receptor binding domain comprising: GRDYNHFQRA (SEQ ID NO: 204), RITWSGTITYNESVKG (SEQ ID NO: 205), and ALKTQPPLSQDAGDYTY (SEQ ID NO: 206).

[0094] Provided herein is a transferrin receptor binding domain comprising: GSTLDDYAIG (SEQ ID NO: 186), CISSSDGITFYGDFVKG (SEQ ID NO: 189), and AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192).

[0095] Provided herein is a transferrin receptor binding domain comprising: GRSLSTYVMG (SEQ ID NO: 210), ARNGMSTYYTDSVKD (SEQ ID NO: 211), and AGDRSWSRLLRGEYEY (SEQ ID NO: 212).

[0096] Provided herein is a transferrin receptor binding domain comprising: GSTLDHYAIG (SEQ ID NO: 187), CISRSDGITYYADSVKG (SEQ ID NO: 190), and AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193).

[0097] Provided herein is a transferrin receptor binding domain comprising: GFTLDYFAIA (SEQ ID NO: 188), CISSGDGRTFYADSVKG (SEQ ID NO: 191), and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

[0098] Provided herein is a transferrin receptor binding domain comprising: GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199), and ALDVVGIGIEVQTYDY (SEQ ID NO: 200).

[0099] In some embodiments of any transferrin receptor binding domain described herein, the transferrin receptor binding domain is a humanized transferrin receptor binding domain.In some embodiments of any antibody described herein comprising a transferrin receptor binding domain, the first, second, and third sequences are humanized.

[0100] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain is a VHH domain. In some embodiments, the VHH domain is humanized.

[0101] Provided herein is a transferrin receptor binding domain comprising a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to any one of SEQ Id NOs: 40-88, 147 and 210-215.

[0102] Also provided herein are polypeptides comprising any of the transferrin receptor binding domains described herein. In some embodiments, the polypeptide further comprises an Fc polypeptide.

[0103] In some embodiments, the polypeptide further comprises a fusion partner. In some embodiments, the fusion partner is selected from the group consisting of an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

[0104] In some embodiments, the polypeptide includes a joint between a fusion partner and a transferrin receptor binding domain. In some embodiments, the joint includes a total of about 1 amino acid to about 50 amino acids (e.g., about 1 amino acid to about 40 amino acids, about 1 amino acid to about 30 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 10 amino acids, or about 1 amino acid to about 5 amino acids). Non-limiting examples of joints include: (G4S)n (SEQ ID NO: 153), wherein n is an integer between 1 and 10, including GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGS (SEQ ID NO: 156) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 157).

[0105] In some embodiments, the fusion partner is directly adjacent to the transferrin receptor binding domain.

[0106] In some embodiments, the polypeptide is a single polypeptide. In some embodiments, the single polypeptide comprises a transferrin receptor binding domain, and the transferrin receptor binding domain comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100%) identical to any one of SEQ ID No: 40-88, 147 and 210-215. In some embodiments, the single polypeptide comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100%) identical to any one of SEQ ID No. 89-138 and 216-221.

[0107] In some embodiments of any single polypeptide described herein, the single polypeptide can be or include a Nanobody, a Nanobody-HSA, a Camelid heavy chain antibody BiTE, a single variable domain, a monomeric VHH domain, a polypeptide comprising two or more VHH domains, a polypeptide comprising a VHH domain and a second functional domain, VHH-scFv, IgG-VHH-scFv, or Fc polypeptide-VHH-scFv.

[0108] In some embodiments of any of the single polypeptides described herein, the transferrin receptor binding domain is fused to an Fc polypeptide.

[0109] In some embodiments of any single polypeptide described herein, the single polypeptide further comprises a fusion partner (e.g., a heterologous fusion partner). In some embodiments, the fusion partner is an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, or another antigen binding domain (e.g., any exemplary antigen binding domain described herein that specifically binds to a transferrin receptor (e.g., human transferrin receptor), or an antigen binding domain that binds to a different antigen). In some embodiments, the fusion partner is glucosylceramidase β (GBA). In some embodiments, the fusion partner is progranulin or pregranulin (PGRN).

[0110] In some embodiments of any single polypeptide comprising GBA as a fusion partner, the single polypeptide comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 179. In some embodiments of any single polypeptide comprising GBA as a fusion partner, the single polypeptide comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID Nos: 139-144.

[0111] In some embodiments of any single polypeptide comprising PGRN as a fusion partner, the single polypeptide comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 181. In some embodiments of any single polypeptide comprising PGRN as a fusion partner, the single polypeptide comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 145, 146, or 148. In some embodiments of any single polypeptide comprising PGRN as a fusion partner, the single polypeptide comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 147.

[0112] In some embodiments, the single polypeptide further comprises a joint (e.g., any exemplary joint described herein) between a fusion partner and a transferrin receptor binding domain. In some embodiments, the fusion partner and the transferrin receptor binding domain are directly adjacent to each other. In some embodiments, the single polypeptide comprises from the N-terminus to the C-terminus: a transferrin receptor binding domain, a joint, and a fusion partner. In some embodiments, the single polypeptide comprises from the N-terminus to the C-terminus: a fusion partner, a joint, and a transferrin receptor binding domain. In some embodiments, the joint comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any sub-range of this range described herein). Non-limiting examples of linkers include: (G4S)n (SEQ ID NO: 153), wherein n is an integer between 1 and 10, including GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGS (SEQ ID NO: 156), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 157).

[0113] In some embodiments of any of the single polypeptides described herein, the single polypeptide does not comprise any additional targeting domains.

[0114] In some embodiments, the polypeptide is part of a protein complex.In some embodiments, the protein complex comprises at least a first polypeptide comprising any of the exemplary transferrin receptor binding domains described herein.

[0115] In some embodiments, the protein complex may include at least a first polypeptide comprising a transferrin receptor binding domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 40-88, 147, and 210-215. In some embodiments, the protein complex may include at least a first polypeptide comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs. 89-138 and 216-221.

[0116] In some embodiments of any protein complex described herein, the first polypeptide and / or the second polypeptide further comprises a fusion partner (e.g., a heterologous fusion partner). In some embodiments, the fusion partner is an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, or another antigen binding domain (e.g., any exemplary antigen binding domain described herein that specifically binds to a transferrin receptor (e.g., human transferrin receptor), or an antigen binding domain that binds to a different antigen). In some embodiments, the fusion partner is glucosylceramidase β (GBA). In some embodiments, the fusion partner is progranulin or pregranulin (PGRN). In some embodiments of any protein complex described herein, the protein complex comprises a polypeptide containing a transferrin receptor binding domain and a fusion partner, wherein the polypeptide is optionally fused to an Fc domain. In some embodiments, the polypeptide may comprise a linker (e.g., any exemplary linker described herein) between the polypeptide and the Fc domain.

[0117] In some embodiments of any protein complex having a first and / or second polypeptide comprising GBA as a fusion partner, the first polypeptide and / or the second polypeptide may comprise a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 179. In some embodiments, the first polypeptide and / or the second polypeptide comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID Nos: 139-144.

[0118] In some embodiments of any protein complex having a first and / or second polypeptide comprising PGRN as a fusion partner, the first polypeptide and / or the second polypeptide may comprise a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 181. In some embodiments, the first polypeptide and / or the second polypeptide may comprise a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 145, 146, or 148.

[0119] In some embodiments of any protein complex having a first and / or second polypeptide comprising a fusion partner (e.g., a GBA polypeptide or a PGRN polypeptide), the first polypeptide and / or the second polypeptide may further comprise a linker between the fusion partner and the transferrin receptor binding domain (e.g., any exemplary linker described herein). In some embodiments, the fusion partner and the transferrin receptor binding domain are directly adjacent to each other. In some embodiments, the first polypeptide and / or the second polypeptide comprise, from N-terminus to C-terminus: a transferrin receptor binding domain, a linker, and a fusion partner (e.g., a GBA polypeptide or a PGRN polypeptide). In some embodiments, the first polypeptide and / or the second polypeptide comprise, from N-terminus to C-terminus: a fusion partner (e.g., a GBA polypeptide or a PGRN polypeptide), a linker, and a transferrin receptor binding domain.

[0120] In some embodiments of any protein complex having a first and / or second polypeptide comprising a fusion partner (e.g., a GBA polypeptide or a PGRN polypeptide), the linker comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any subrange of this range described herein). Non-limiting examples of linkers include: (G4S)n (SEQ ID NO: 153), wherein n is an integer between 1 and 10, including GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGS (SEQ ID NO: 156), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 157).

[0121] In some embodiments of any of the protein complexes described herein, the protein complex comprises: a camelid heavy chain antibody, a nanobody, a nanobody-HSA, a humanized camelid heavy chain antibody, a bispecific humanized camelid heavy chain antibody, a camelid heavy chain antibody BiTE, a single variable domain, a monomeric VHH domain, a dimer of VHH domains, a bispecific dimer of VHH domains, a dimer containing one VHH domain and a second functional domain, VHH-Fc, VHH-IgG, IgG-VHH, a knobs-in-holes assembly, VHH-scFv, IgG-VHH-scFv, or Fc-VHH-scFv.

[0122] In some embodiments, the first polypeptide and the second polypeptide form a human or humanized antibody.In some embodiments of any of the protein complexes described herein, the complex does not comprise any additional targeting domains.

[0123] In some embodiments of any protein complex described herein, protein complex comprises at least a first polypeptide, and the first polypeptide comprises a transferrin receptor binding domain. Some embodiments of these protein complexes further comprise a second polypeptide, and the second polypeptide comprises a fusion partner (e.g., any exemplary fusion partner described herein, e.g., GBA or PGRN polypeptide). In such embodiments, the first polypeptide and / or the second polypeptide can be fused or connected to the Fc domain.

[0124] In some embodiments of any of the protein complexes described herein, the protein complex can be human or humanized.

[0125] Also provided herein is a nucleic acid encoding transferrin receptor binding domains as described herein or polypeptide.An expression vector is also provided herein, and it includes any nucleic acid as described herein, and the nucleic acid encodes any transferrin receptor binding domains as described herein or polypeptide.In some examples, the expression vector as described herein can further include a promoter (for example, any exemplary promoter as described herein or known in the art) and / or an enhancer (for example, any exemplary enhancer as described herein or known in the art), wherein promoter and / or enhancer are operably connected to the nucleic acid sequence encoding any transferrin receptor binding domains as described herein or polypeptide.

[0126] Also provided herein are nucleic acids encoding each polypeptide in any protein complex described herein.Also provided herein are pairs or sets of nucleic acids that together encode each polypeptide in any protein complex described herein.

[0127] Also provided herein is an expression vector comprising any nucleic acid described herein.An expression vector pair or expression vector group is also provided, which together encodes each polypeptide in any protein complex described herein.In some examples, the expression vector described herein may further include a promoter (e.g., any exemplary promoter described herein or known in the art) and / or an enhancer (e.g., any exemplary enhancer described herein or known in the art), wherein the promoter and / or enhancer are operably connected to a nucleic acid sequence encoding any polypeptide described herein.Other aspects and examples of expression vectors are described herein.

[0128] Antibodies and Antibody Fragments

[0129] Provided herein are antibodies or antibody fragments comprising any transferrin receptor binding domains described herein. In some embodiments, the transferrin receptor binding domain is a humanized transferrin receptor binding domain. In some embodiments, the first, second, and third sequences of the transferrin receptor binding domain are humanized.

[0130] In some embodiments, the antibody or antibody fragment may comprise a transferrin receptor binding domain having a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100%) identical to any one of SEQ ID NOs. 40-88, 147 and 210-215.

[0131] In some embodiments, the antibody or antibody fragment comprises a VHH domain. In some embodiments, the antibody or antibody fragment may comprise one or more additional single variable domains (e.g., VHH domains) comprising an antigen binding domain (transferrin receptor binding domain, e.g., any exemplary transferrin receptor binding domain described herein) that specifically binds to a transferrin receptor. In some embodiments, the antibody or antibody fragment comprises one or more additional single variable domains that bind to different antigens. In some embodiments, the antibody or antibody fragment does not comprise any other targeting domain.

[0132] In some embodiments, the antibody may be or may include an antibody, a camelid heavy chain antibody, a nanobody, a nanobody-HSA, a humanized camelid heavy chain antibody, a bispecific humanized camelid heavy chain antibody, a camelid heavy chain antibody BiTE, a single variable domain, a monomeric VHH domain, a dimer of VHH domains, a bispecific dimer of VHH domains, a dimer containing one VHH domain and a second functional domain, VHH-Fc, VHH-IgG, IgG-VHH, a knob-in-hole assembly, VHH-scFv, IgG-VHH-scFv or Fc-VHH-scFv. In some embodiments, the antibody fragment can be or comprise: a camelid heavy chain antibody, a nanobody, a nanobody-HSA, a humanized camelid heavy chain antibody, a bispecific humanized camelid heavy chain antibody, a camelid heavy chain antibody BiTE, a single variable domain, a monomeric VHH domain, a dimer of VHH domains, a bispecific dimer of VHH domains, a dimer containing one VHH domain and a second functional domain, VHH-Fc, VHH-IgG, IgG-VHH, a knob-in-hole assembly, VHH-scFv, IgG-VHH-scFv or Fc-VHH-scFv.

[0133] In some embodiments, the antibody or antibody fragment comprises an antigen binding domain (e.g., a transferrin receptor binding domain) that specifically binds to a transferrin receptor, wherein the antigen binding domain is fused to an Fc domain. In some embodiments, the antibody or antibody fragment may comprise a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100%) identical to any one of SEQ ID NO: 89-138 or 216-221.

[0134] In some embodiments, the antibody or antibody fragment may be a human or humanized antibody or antibody fragment.

[0135] In some embodiments, the antibody or antibody fragment further comprises a fusion partner (e.g., a heterologous fusion partner) (e.g., any exemplary fusion partner described herein). In some embodiments, the fusion partner is an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, or another antigen binding domain (e.g., any exemplary antigen binding domain described herein that specifically binds to a transferrin receptor (e.g., a human transferrin receptor), or an antigen binding domain that binds to a different antigen). In some embodiments, the fusion partner is a glucosylceramidase β (GBA) polypeptide. In some embodiments, the fusion partner is a progranulin or pregranulin (PGRN) polypeptide.

[0136] In some embodiments, the antibody or antibody fragment comprises a GBA polypeptide as a fusion partner, wherein the GBA polypeptide has a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 179. In some embodiments, the antibody or antibody fragment comprises a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 139-144.

[0137] In some embodiments, the antibody or antibody fragment comprises a PGRN polypeptide as a fusion partner, wherein the PGRN polypeptide has a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 181. In some embodiments, the antibody or antibody fragment comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NO: 145, 146, or 148.

[0138] In some embodiments, the antibody or antibody fragment further comprises a joint (e.g., any exemplary joint described herein) between a fusion partner and a transferrin receptor binding domain. In some embodiments, the fusion partner and the transferrin receptor binding domain are directly adjacent to each other. In some embodiments, the antibody or antibody fragment comprises from the N-terminus to the C-terminus: a transferrin receptor binding domain, a joint, and a fusion partner (e.g., a GBA polypeptide or a PGRN polypeptide). In some embodiments, the antibody or antibody fragment comprises from the N-terminus to the C-terminus: a fusion partner (e.g., a GBA polypeptide or a PGRN polypeptide), a joint, and a transferrin receptor binding domain.

[0139] In some embodiments, the linker comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any subrange of this range described herein). Non-limiting examples of linkers include: (G4S)n (SEQ ID NO: 153), wherein n is an integer between 1 and 10, including GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGS (SEQ ID NO: 156), or GGGGSGG GGSGGGGSGGGGS (SEQ ID NO: 157).

[0140] Also provided herein are nucleic acids encoding any antibody or antibody fragment described herein. Also provided herein are expression vectors comprising any nucleic acids described herein encoding any antibody or antibody fragment described herein. In some examples, the expression vectors described herein may further comprise a promoter (e.g., any exemplary promoter described herein or known in the art) and / or an enhancer (e.g., any exemplary enhancer described herein or known in the art), wherein the promoter and / or enhancer are operably linked to a nucleic acid sequence encoding any antibody or antibody fragment described herein. Other aspects and examples of expression vectors are described herein.

[0141] Additional Exemplary Aspects

[0142] The transferrin receptor binding domains, single polypeptides, protein complexes, antibodies and antibody fragments described herein comprise at least one transferrin receptor binding domain. An exemplary human wild-type transferrin receptor protein may comprise a sequence of SEQ ID NO: 149, 150, 151 or 152.

[0143] In some embodiments, the transferrin receptor binding domain is present in an amount less than about 1 x 10 -5 M, less than about 1x 10 -6 M or less than about 1x 10 -7 M (e.g., less than about 1 x 10 -8 M, less than about 1x 10 -9 M, less than about 1x 10 -10 M, less than about 1x 10 -11 M, less than about 1x10 -12 M or less than about 1x 10 -13 The dissociation constant (K D ) binds to a transferrin receptor (e.g., human transferrin receptor). In some embodiments, the affinity of the transferrin receptor binding domain is determined by surface plasmon resonance (e.g., in phosphate buffered saline).

[0144] In some embodiments in which an antibody, antibody fragment, single polypeptide or protein complex comprises a transferrin receptor binding domain fused to an Fc polypeptide or Fc domain, the antibody, antibody fragment or single polypeptide may comprise a linker between the transferrin receptor binding domain and the Fc region (e.g., any exemplary linker described herein). In some embodiments, the linker comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any sub-range of this range described herein). Non-limiting examples of linkers include: (G4S)n (SEQ ID NO: 153), wherein n is an integer between 1 and 10, including GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGG GGSGGGGS (SEQ ID NO: 156) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 157). In some embodiments, the transferrin receptor binding domain and the Fc domain are directly adjacent.

[0145] Non-limiting examples of sequences that specifically bind to human transferrin receptor include SEQ Id NOs: 40-88, 147, and 89-138.

[0146] In some embodiments, any of the transferrin receptor binding domains, single polypeptides, protein complexes, antibodies, and antibody fragments described herein may comprise a modified Fc region (e.g., a modified CH2 and / or CHD3 domain of a human Fc region (e.g., a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, or a human IgG4 Fc region)).

[0147] Expression vector

[0148] Non-limiting examples of expression vectors include plasmids and viral vectors. In some embodiments, the expression vector is a plasmid, an adeno-associated virus (AAV) vector, a lentiviral vector, a Sindbis virus vector, an alphavirus-based vector or an adenoviral vector. AAV vectors are generally described in, for example, Asokan et al., Mol. Ther. 20: 699-708, 2012 and BJ Carter, see: "Handbook of Parvoviruses", editor: P. Tijsser, CRC Press, pages 155-168, 1990. Adenoviral vectors are generally described in, for example, Wold and Toth, Curr. Gene Ther. 13 (6): 421-433, 2013; Baron et al., Curr. Opin. Virol. 29: 1-7, 2018; and Barry, Expert Rev. Vaccines 17 (2): 163-173, 2018. Lentiviral vectors are generally described in, for example, Milone and O'Doherty, Leukemia 32(7): 1529-1541, 2018, Zheng et al., Anat. Rec. 301(5): 825-836, 2018; and Cai et al., Curr. Gene Ther. 16(3): 194-206, 2016.

[0149] Some embodiments of any of the expression vectors described herein can include a promoter and / or enhancer operably linked to a nucleic acid encoding a transferrin receptor binding domain (e.g., any exemplary transferrin receptor binding domain described herein), a polypeptide (e.g., any exemplary polypeptide described herein), an antibody (e.g., any exemplary antibody described herein), an antibody fragment (e.g., any exemplary antibody fragment described herein), or a protein complex (e.g., any exemplary protein complex described herein).

[0150] In some embodiments, the expression vector can be an AAV vector. For example, the AAV vector can be selected from the group consisting of: AAV2 vector, AAV5 vector and AAV8 vector, AAV1 vector, AAV7 vector, AAV9 vector, AAV3 vector, AAV6 vector, AAV10 vector and AAV11 vector. In any exemplary AAV vector described herein, the AAV vector may include AAV8 capsid protein, AAV5 capsid protein, AAV-LK03 capsid protein, AAV-NK59 capsid protein, AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV6 capsid protein, AAV7 capsid protein and AAV9 capsid protein.

[0151] In some embodiments, the nucleic acid further comprises a promoter and / or an enhancer, which is operably connected to a sequence encoding any transferrin receptor binding domain, antibody, antibody fragment, single polypeptide or protein complex described herein. In some embodiments, the promoter is constitutive. In some embodiments, the promoter is inducible. In some embodiments, the promoter is a tissue-specific promoter. Exemplary promoters of constitutive, inducible and / or tissue-specific are known in the art. Non-limiting examples of promoters include CMV promoter, CAG promoter, human α-1-antitrypsin (hAAT) promoter (e.g., SEQ ID NO: 167), HLP promoter (e.g., SEQ ID NO: 166), HCB promoter (e.g., SEQ ID NO: 168) and transthyretin promoter (e.g., SEQ ID NO: 169). Non-limiting examples of enhancers are apolipoprotein E (ApoE) enhancer (e.g., SEQ ID NO: 174) and serine protease inhibitor enhancer (e.g., SEQ ID NO: 175). Other promoters and enhancers are described herein.

[0152] In some embodiments, the nucleic acid comprises a promoter operably linked to a nucleic acid sequence encoding a fusion partner (e.g., any exemplary fusion partner described herein, e.g., a heterologous fusion partner). In some embodiments, the nucleic acid comprises an enhancer operably linked to a nucleic acid sequence encoding a fusion partner (e.g., any exemplary fusion partner described herein). In some embodiments, the nucleic acid comprises an enhancer and a promoter operably linked to a nucleic acid sequence encoding a fusion partner (e.g., any exemplary fusion partner described herein).

[0153] In some embodiments, a nucleic acid comprising a promoter and / or enhancer operably linked to a nucleic acid encoding a first polypeptide, a second polypeptide, or both (e.g., any exemplary first polypeptide described herein, any exemplary second polypeptide described herein) is flanked at its 5' and 3' ends by viral inverted terminal repeat (ITR) sequences (e.g., any exemplary ITR sequences described herein or known in the art). In some embodiments, a nucleic acid comprises a promoter and / or enhancer operably linked to a nucleic acid sequence encoding a first polypeptide, a second polypeptide, or both (e.g., any exemplary first polypeptide described herein, any exemplary second polypeptide described herein), and the nucleic acid is flanked at its 5' and 3' ends by viral ITR sequences (e.g., any viral ITR sequences described herein or known in the art).

[0154] Exemplary ITR sequences are described in BJ Carter, in: "Handbook of Parvoviruses", ed. P. Tijsser, CRC Press, pp. 155-168, 1990, and U.S. Pat. No. 9,150,882 (incorporated herein by reference). Typical AAV ITR sequences are about 80 to about 200 nucleotides in length (e.g., about 80 to about 200 nucleotides, about 80 to about 180 nucleotides, about 80 to about 160 nucleotides, about 80 to about 140 nucleotides, about 80 to about 120 nucleotides, about 80 to about 100 nucleotides, about 100 to about 200 nucleotides, about 100 to about 180 nucleotides, about 100 to about 160 nucleotides, about 100 to about 150 nucleotides, about 100 to about 14 ... about 120 nucleotides, about 120 to about 200 nucleotides, about 120 to about 180 nucleotides, about 120 to about 160 nucleotides, about 120 to about 150 nucleotides, about 120 to about 140 nucleotides, about 140 to about 200 nucleotides, about 140 to about 180 nucleotides, about 140 to about 160 nucleotides, about 140 to about 150 nucleotides, about 160 to about 200 nucleotides, about 160 to about 180 nucleotides, or about 180 to about 200 nucleotides).

[0155] The ability to modify ITR sequences is within the skill of the art. See, for example, texts such as Sambrook et al., "Molecular Cloning: A Laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory, New York, 1989; and Fischer et al., J. Virol. 70:520-532, 1996). Additional examples of ITR sequences can be obtained from any known AAV (AAV ITR). In some examples, the AAV ITR sequence can be an AAV2 ITR sequence or a functional variant thereof. In some embodiments of any AAV vector described herein, the ITR sequence is a self-complementary ITR (scITR) sequence.

[0156] In some embodiments, the nucleic acid encoding a polypeptide, an antibody, an antibody fragment, or a protein complex comprises a single antigen binding domain that specifically binds to the transferrin receptor. For example, in some embodiments, the nucleic acid sequence encoding a polypeptide, an antibody fragment, an antibody, or a protein complex encodes a single binding domain that specifically binds to the transferrin receptor, without encoding another binding domain.

[0157] In some embodiments, the expression vector does not comprise any other targeting domains or amino acid sequences.In some embodiments of any of the expression vectors described herein, the expression vector comprises only a first sequence, a linker, and a second sequence.

[0158] In some embodiments, the expression vectors described herein may comprise a nucleic acid encoding a protein tag sequence (e.g., a human influenza hemagglutinin (HA) tag, a c-Myc tag, or any other protein tag sequence known in the art). The presence of the HA tag in the cells or CNS of a subject can be detected by protein binding assays (e.g., Western blot, immunohistochemistry, radioimmunoassay (RIA) or chemiluminescence).

[0159] In some embodiments, the expression vectors described herein comprise one or more (e.g., two, three, four, five, or six) promoters (e.g., any promoter described herein or known in the art), enhancers (e.g., any enhancers described herein or known in the art), Kozak sequences (e.g., any Kozak sequences described herein or known in the art), RNA splicing sequences, polyadenylation (poly(A)) signal sequences (e.g., any poly(A) signals described herein or known in the art), and internal ribosome entry site (IRES) sequences (e.g., any IRES sequences described herein or known in the art).

[0160] Methods for producing transferrin receptor binding domains, polypeptides, antibodies or antibody fragments

[0161] Also provided herein is a method for producing any transferrin receptor binding domain, polypeptide, antibody or antibody fragment described herein, the method comprising: (a) culturing a cell (e.g., any cell described herein) containing any nucleic acid encoding any transferrin receptor binding domain, polypeptide, antibody or antibody fragment described herein, or any expression vector described herein containing a nucleic acid encoding a transferrin receptor binding domain, polypeptide, antibody or antibody fragment described herein in a culture medium under conditions sufficient to allow the production of transferrin receptor binding domain, polypeptide, antibody or antibody fragment; and (b) harvesting transferrin receptor binding domain, polypeptide, antibody or antibody fragment from host cells or culture medium. In some embodiments of any of the methods described herein, the method further comprises separating transferrin receptor binding domain, polypeptide, antibody or antibody fragment (e.g., by performing one or more column chromatography steps, ultrafiltration / diafiltration and / or viral inactivation). In some embodiments of any of the methods described herein, the method further comprises formulating the separated transferrin receptor binding domain, polypeptide, antibody or antibody fragment into a composition (e.g., a pharmaceutical composition).

[0162] Any transferrin receptor binding domain, polypeptide, antibody or antibody fragment described herein can be produced by any cell (e.g., mammalian cell). Non-limiting examples of mammalian cells include: human cells, rodent cells (e.g., rat cells or mouse cells), rabbit cells, dog cells, cat cells, pig cells or non-human primate cells. For example, the host cell can be a CHO cell or a HEK cell.

[0163] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that are conducive to cell proliferation, cell growth and / or cell differentiation. For example, cells can be cultured by contacting cells (e.g., any cells described herein) with a cell culture medium containing supplemental growth factors to support cell viability and cell growth.

[0164] Methods for introducing nucleic acids (e.g., any exemplary nucleic acids described herein) and / or expression vectors (e.g., any exemplary expression vectors described herein (e.g., AAV vectors)) into cells (e.g., mammalian cells) are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids (e.g., any exemplary nucleic acids described herein) and / or expression vectors (e.g., any exemplary expression vectors described herein (e.g., AAV vectors)) include: electroporation, lipofection, transfection, microinjection, calcium phosphate transfection, dendrimer-based transfection, anionic polymer transfection, cationic polymer transfection, transfection using highly branched organic compounds, cell squeezing, sonoporation, optical transfection, magnetofection, particle-based transfection (e.g., nanoparticle transfection), transfection using liposomes (e.g., cationic liposomes), and viral transduction (e.g., lentiviral transduction, adenoviral transduction).

[0165] Also provided herein are methods further comprising isolating the transferrin receptor binding domain, polypeptide, antibody or antibody fragment from cell culture medium or from cells (e.g., mammalian cells) using techniques well known in the art (e.g., ion exchange chromatography (anion or cation), metal affinity chromatography, ligand affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography and precipitation (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation)).

[0166] cell

[0167] Also provided herein are cells (e.g., peripheral mammalian cells, mammalian hepatocytes, e.g., human hepatocytes) comprising any expression vector, expression vector pair, nucleic acid or nucleic acid pair described herein. Also provided are cells (e.g., mammalian cells, mammalian hepatocytes, e.g., human hepatocytes) transduced with any expression vector or expression vector pair described herein, edited using lentivirus or CRISPR technology, or otherwise engineered or modified to express any polypeptide or protein complex described herein. It will be appreciated by those skilled in the art that the expression vectors and nucleic acids described herein can be introduced into any cell (e.g., any mammalian cell, any hepatocyte), and a variety of techniques can be used to modify the genome of a cell (e.g., a mammalian cell). Non-limiting examples of methods for introducing expression vectors and nucleic acids into cells (e.g., any mammalian cell, any hepatocyte, e.g., human hepatocytes) are described herein.

[0168] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell, a rodent cell (e.g., a rat cell or a mouse cell), a rabbit cell, a dog cell, a cat cell, a pig cell, or a non-human primate cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is present in a subject (e.g., a mammalian subject, a human subject). In some embodiments, the cell is an autologous cell obtained from a subject (e.g., a mammalian subject, a human subject) and cultured in vitro. In some embodiments, the cell is in vitro.

[0169] Without further description, it is believed that one of ordinary skill in the art can use the preceding description and the following illustrative examples to make and utilize the expression vectors of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention and should not be construed as limiting the remainder of the present disclosure in any way.

[0170] Compositions and kits

[0171] Composition (for example, pharmaceutical composition) is also provided herein, it comprises any transferrin receptor binding domain as described herein, polypeptide, antibody, antibody fragment, expression vector, nucleic acid and the cell containing them.Any pharmaceutical composition can comprise any transferrin receptor binding domain as described herein, polypeptide, antibody or antibody fragment and one or more (for example, 1,2,3,4 or 5 kinds) pharmaceutically or physiologically acceptable carrier, diluent or excipient.In some embodiments, any pharmaceutical composition as described herein can comprise one or more buffers (for example, neutral buffered saline, phosphate buffered saline (PBS)), one or more carbohydrates (for example, glucose, mannose, sucrose, dextran or mannitol), one or more proteins, polypeptides or amino acids (for example, glycine), one or more antioxidants, one or more chelating agents (for example, glutathione or EDTA), one or more preservatives and / or pharmaceutically acceptable carriers (for example, PBS, saline or antibacterial water).

[0172] In some embodiments, any pharmaceutical composition described herein may further comprise one or more (e.g., 1, 2, 3, 4, or 5) agents (e.g., liposomes or cationic lipids) that facilitate entry of any expression vector or nucleic acid described herein into cells (e.g., mammalian cells, hepatocytes).

[0173] In some embodiments, any expression vector or nucleic acid described herein can be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that can be included in any pharmaceutical composition described herein can include, but are not limited to, poloxamers, chitosans, dendrimers, and poly(lactic-co-glycolic acid) (PLGA) polymers.

[0174] The pharmaceutical compositions provided herein can, for example, be formulated to be compatible with its intended route of administration. In some embodiments, the compositions are formulated for subcutaneous, intramuscular, intravenous or intrahepatic administration. In some examples, the compositions include any polypeptide, protein complex or expression vector described herein in a therapeutically effective amount. Single or multiple administrations of any pharmaceutical composition described herein can be given (e.g., administered) to a subject, depending on, for example, the frequency and dosage required and tolerated by the subject. The dosage of the pharmaceutical composition comprising any polypeptide described herein, any protein complex described herein or any expression vector described herein should provide a sufficient amount to effectively improve or treat symptoms, illness or disease.

[0175] Also provided is a kit comprising any composition described herein (e.g., a pharmaceutical composition), comprising any nucleic acid described herein, any transferrin receptor binding domain, any polypeptide, any antibody, any antibody fragment, any expression vector, or any cell containing them. In some embodiments, the kit may comprise a solid composition (e.g., a lyophilized composition comprising any expression vector, polypeptide, or protein complex described herein) and a liquid for dissolving the lyophilized composition.

[0176] In some embodiments, the kit may include at least one dose of any composition described herein (e.g., any pharmaceutical composition). In some embodiments, the kit may include a preloaded syringe containing any pharmaceutical composition described herein. In some embodiments, the kit includes a vial containing any pharmaceutical composition described herein (e.g., formulated as an aqueous pharmaceutical composition). In some embodiments, the kit may include instructions for performing any method described herein.

[0177] Example

[0178] Example 1: Generation of camelid antibodies against human transferrin receptor

[0179] A set of experiments was conducted to generate camelid antibodies against human transferrin receptor. In these experiments, Creative Biolabs (Shirley, New York) performed llama immunization, peripheral blood mononuclear cell (PBMC) separation and VHH phage library construction. In brief, llama was immunized with recombinant human transferrin receptor ("TfR")-apical domain every three weeks during three months. Serum titers were measured for human TfR-apical domain to confirm immune response. On the last day of the immunization schedule (after final serum titer confirmation), PBMC was separated to purify total RNA, thereby constructing VHH phage library in pComb3xss phagemid.

[0180] Phage screening to isolate anti-TfR VHH antibodies

[0181] A set of experiments was performed to isolate anti-TfR VHH antibodies from a VHH phage library. The first round of phage panning using a VHH immune phage library was performed on human TfR (full length), followed by a second round of phage panning on human TfR-top or cynomolgus monkey ("cyno") TfR-top domain. After two rounds of phage panning, TG1 Escherichia coli was infected with phage eluates and plated overnight to isolate single E. coli colonies. Phages from single colonies were grown in 96-well plates by adding M13 helper phages in the culture medium and incubating overnight at 30°C. Phage ELISA was performed on human and cyno TfR (full length) and human TfR top domains. Counter screening was also performed in parallel to remove any nonspecific binding to streptavidin. Sanger sequencing was performed on sequence VHH from clones with positive binding to human and cyno TfR.

[0182] Generation of VHH antibodies

[0183] The variable heavy chain domains of TfR binders identified from phage panning of immune llama libraries were subcloned into the Fc domain of human IgG1. The expression plasmid contained a CMV promoter driving expression and a signal peptide for secretion. The heavy chain plasmid was transfected into Expi293 cells. The supernatant was collected five days later and the monoclonal antibody was purified by protein A chromatography. Affinity variants and humanized variants of B07 were subcloned into the Fc domain of human IgG1. These variants were expressed and purified in the same manner. A selected group of B07 and humanized B07 variants were expressed only as VHH domains. These variants were also expressed and purified in the same manner.

[0184] Example 2: Cell binding and uptake experiments after treatment with VHH antibodies

[0185] A set of experiments was performed to evaluate the cellular binding and uptake of a panel of VHH antibodies. In these experiments, HEK293T, CHO:cyTfR and CHO cells were plated at 40,000 cells / well in standard growth medium (DMEM (Gibco TMCatalog number: 11995073) + 10% FBS (VWR catalog number: 89510-188) + 1X penicillin / streptomycin (Gibco catalog number: 15140122) in a 96-well plate. Approximately 24 hours later, the molecules were diluted into standard growth medium warmed to 37°C. The old medium was removed from the cells and the diluted molecules were added to the cells. The cells were incubated at 37°C for 45 minutes. The cells were then washed with PBS and then fixed in 4% PFA (Electron Microscopy Sciences catalog number: 15714-S) for 10 minutes. The cells were washed with PBS and then blocked with 5% BSA, 0.3% Triton X100 in PBS for 30 minutes. Cells were stained with anti-human IgG-488 (1:1000; Jackson ImmunoResearch catalog number: 109-545-003), cell mask (1:10,000; Thermo Fisher catalog number: H32721), and DAPI (1:2000; Thermo Fisher catalog number: D1306) diluted in 1% BSA, 0.3% Triton X100 in PBS for at least 30 minutes. Cells were washed with PBS, imaged on OperaPhenix, and images were analyzed using Harmony software. Figure 2A A and B illustrate exemplary hTfR binding curves generated by VHH antibodies generated as described in Example 1.

[0186] Example 3: Evaluation of Binding Affinity of VHH-Fc Fusions

[0187] A set of experiments was performed to assess the binding affinity of VHH-Fc fusions to the human and cyno TfR apical domains. In these experiments, Biacore TM Binding affinity was determined by surface plasmon resonance on a Biacore 8K instrument. TMSeries S CM5 sensor chips were treated with a human antibody capture kit (GE Healthcare catalog number: BR100839). VHH-Fc fusions were captured on each flow cell and serial 3-fold dilutions of human and cyno TfR apical domains (300, 100, 33, 11, 3.7 and 0 nM) were injected using a multi-cycle kinetic method at a flow rate of 30 μL / min. Each sample was analyzed with 270 seconds association and 10 minutes dissociation. Some VHH-Fc fusions were assayed with serial 3-fold dilutions of human and cyno TfR apical domains (100, 33, 11 and 0 nM) with 300 seconds association and 10 minutes dissociation. After each cycle, the chip was regenerated for 30 seconds using 3M magnesium chloride at 50 μL / min. The binding response was corrected by subtracting RU from the reference flow cell. Biacore TM 8K evaluation software, k on and k off A 1:1 Langmuir model was fitted simultaneously for kinetic analysis.Table 1 shows the binding of various VHH clones generated as described in Example 1 (huTfR-AD = human transferrin receptor apical domain; cyTfR-AD = cyno transferrin receptor apical domain).

[0188] Table 1.

[0189]

[0190] Example 4: Generation of affinity variants of B07

[0191] To generate affinity variants of B07, selected residues in the CDR regions were mutated with mutations listed in Table 2 with Kabat numbering. The variable domains containing the mutations were subcloned into the corresponding constant heavy chain domains of human IgG1. The heavy chain plasmids were transfected into Expi293 cells. The supernatants were collected five days later and the monoclonal antibodies were purified by protein A chromatography. To evaluate the affinity of the B07 mutants for the human and cyno TfR apical domains, Biacore was used as described herein. TM Binding affinity was determined by surface plasmon resonance on an 8K instrument. Table 2 shows the effect of each mutation in B07 on the binding affinity of the human and cyno TfR apical domains.

[0192] Table 2.

[0193]

[0194] NB = No Binding

[0195] Example 5: Humanization of B07

[0196] A set of experiments was conducted to assess the impact of humanization on the B07 VHH antibody. In these experiments, B07 VHH was humanized by first comparing the heavy chain variable domain with the common framework of human antibody germline. The human H3 consensus sequence was identified as having the highest homology with the B07 framework, so the H3 sequence was used as the starting point for humanization. The B07 CDR was transplanted into the human H3 common framework and then designed to contain other variants, in which the combination of specific amino acid positions was backmutated into the original llama residues (37F, 44E, 45R, 47F, 73K, 78V; Kabat numbering). As previously mentioned, all variants were generated as fusions with human IgG1 Fc domains. The variants were recombinantly expressed and tested for their combination with people and cyno TfR. Table 3 shows the impact of humanization on the B07 VHH antibody.

[0197] Table 3.

[0198]

[0199] NB = No Binding

[0200] NT = Not Tested

[0201] hB07v17 was further humanized in the CDR H1 region. Additional humanized variants were generated containing CDR H1 residues that had a common H1 sequence with human VH3. Mutations at positions 47 and 52a (Kabat numbering) were also tested. Table 4 shows a summary of the mutations produced for hB07v17. As described herein, all variants were generated as fusions with human IgG1 Fc domains. The variants were recombinantly expressed and tested for binding to human and cyno TfR. Table 4 shows the effects of humanization in the CDR H1 region on the binding affinity of hB07v17 to human and cyno TfR.

[0202] Table 4.

[0203]

[0204] Example 6: Generation of B07-GBA fusion polypeptide

[0205] A set of experiments was performed to evaluate fusion polypeptides comprising a B07 VHH domain or variants thereof fused to a glucosylceramidase β (GBA) polypeptide. In these experiments, the B07 VHH domain or humanized B07 VHH domain was fused to the N-terminus or C-terminus of human GBA. These constructs were used in in vitro GBA activity experiments. Additional combinations of B07 variants fused to GBA with various linker lengths were also generated.

[0206] Non-limiting examples of nucleic acid sequences encoding human GBA polypeptides include SEQ ID NO: 178. Non-limiting examples of amino acid sequences of GBA include SEQ ID NO:179.

[0207] For the in vitro GBA activity assay, a GBA polypeptide comprising SEQ ID NO: 179 was used as a control.

[0208] In a non-limiting example, a B07-GBA fusion polypeptide was produced. The amino acid sequence of an exemplary B07-PGRN fusion polypeptide comprises SEQ ID NO:139.

[0209] In a non-limiting example, a B07-GBA fusion polypeptide was produced with a 2XGGGGS linker between the B07 and GBA domains. The amino acid sequence of an exemplary B07-GBA fusion polypeptide comprises SEQ ID NO:140.

[0210] In a non-limiting example, constructs were also generated with N-terminal GBA in a GBA-B07 fusion polypeptide. The amino acid sequence of an exemplary GBA-B07 fusion polypeptide comprises SEQ ID NO:141.

[0211] In a non-limiting example, a GBA-B07 fusion polypeptide was generated with a 2X GGGGS linker between GBA and B07. The amino acid sequence of the GBA-B07 fusion polypeptide comprises SEQ ID NO:142.

[0212] In a non-limiting example, a humanized B07-GBA fusion polypeptide was generated. The amino acid sequence of an exemplary humanized B07-GBA fusion polypeptide comprises SEQ ID NO:143.

[0213] In a non-limiting example, a GBA humanized B07 fusion polypeptide was generated. The amino acid sequence of an exemplary GBA humanized B07 fusion polypeptide comprises SEQ ID NO:144.

[0214] Example 7: Generation of B07-progranulin fusion polypeptide

[0215] A set of experiments was performed to evaluate fusion polypeptides comprising a B07 VHH domain or variant thereof fused to a progranulin (progranulin or PGRN) polypeptide. In these experiments, the B07 domain was fused to the N-terminus of human PGRN using two GGGGS linkers.

[0216] A non-limiting example of a nucleic acid sequence encoding a human progranulin or progranulin (PRGN) polypeptide comprises SEQ ID NO: 180. A non-limiting example of an amino acid sequence of human PGRN comprises SEQ ID NO: 181. In a non-limiting example, a B07-PGRN construct was generated. The amino acid sequence of an exemplary B07-PGRN fusion polypeptide comprises SEQ ID NO: 145.

[0217] In a non-limiting example, a 6xHis-Tev-B07-PGRN construct was generated. Adding a 6xHis tag enables purification by nickel chromatography. The amino acid sequence of an exemplary 6xHis-Tev-B07-PGRN fusion polypeptide comprises SEQ ID NO: 146.

[0218] In a non-limiting example, a B07(N68T)-PGRN construct was generated. The N68T mutation in B07 restores the protein A binding site and enables purification by protein A chromatography. The amino acid sequence of an exemplary B07(N68T)-PGRN fusion polypeptide comprises SEQ ID NO: 148.

[0219] Other Implementations

[0220] It should be understood that although the invention has been described in conjunction with the specific description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0221] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, section headings, materials, methods, and examples are illustrative only and not restrictive.

[0222] Sequence Appendix

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Claims

1. A transferrin receptor binding protein comprising a camelid heavy chain variable domain.

2. The transferrin receptor binding protein of claim 1, wherein the protein has a K of about 1 nM to about 6 μM. D Binds to human transferrin receptor.

3. The transferrin receptor-binding protein of claim 1 or 2, wherein the protein is present in a K of about 1 nM to about 2.5 mM. D Binds to the cynomolgus monkey transferrin receptor.

4. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: (a) the first sequence G-X1-T-X2-X3-X4-X5-X6-M-X7 (SEQ ID NO: 1), wherein X1 is L or F, X2 is S or F, X3 is D or S or E, X4 is T or S, X5 is G or Y, X6 is G or A, or X7 is G or S; (b) Second sequence AI-X8-X9-X 10 -X 11 -X 12 -X 13 -TX 14 -YADSVKG (SEQ ID NO: 2), wherein X8 is T or S, X9 is W or S or G or F or Y, X 10 Is S or N, X 11 It is G or A, X 12 It is R or S or G, X 13 It is H or A or X 14 is L or Y; and (c) The third sequence AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -DY (SEQ ID NO: 3), wherein X 15 It is L or R, X 16 Is V or A, X 17 Is V or A, X 18 It is G or A, X 19 Is I or A, X 20 It is G or A, X 21 Is I or A, X 22 It is E or A, X 23 V or A, X 24 It is Q or A, X 25 It is T or A or X 26 It is Y or F.

5. The transferrin receptor binding domain of claim 4, wherein the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GLTSDTGGMG (SEQ ID NO:4), GFTFSTGGMG (SEQ ID NO:12), GFTSDTGGMG (SEQ ID NO:5), GLTFDTGGMG (SEQ ID NO:6), GLTSSTGGMG (SEQ ID NO:7), GLTSETGGMG (SEQ ID NO:8), GLTSDTYGMG (SEQ ID NO:9), GLTSDTGAMG (SEQ ID NO:10), GLTSDTGGMS (SEQ ID NO:11), GFTFSTGGMS (SEQ ID NO:13), and GFTSSSGGMS (SEQ ID NO:14); (b) a second sequence selected from the group consisting of AITWSGRHTLYADSVKG (SEQ ID NO: 15), AISWSGRHTLYADSVKG (SEQ ID NO: 16), AITSSGRHTLYADSVKG (SEQ ID NO: 17), AITGSGRHTLYADSV KG (SEQ ID NO: 18), AITWNGRHTLYADSVKG (SEQ ID NO: 19), AITWSGSHTLYADSVKG (SEQ ID NO: 20), AITWSGGHTLY ADSVKG (SEQ ID NO: 21), AITWSGRHTYYADSVKG (SEQ ID NO: 22), AITWSARHTLYADSVKG (SEQ ID NO: 23), AITWSGRATLYADSVKG (SEQ ID NO: 24), AITFSGRHTLYADSVKG (SEQ ID NO: 25), and AITYSGRHTLYADSVKG (SEQ ID NO: 26); and (c) a third sequence selected from the group consisting of ALDVVGIGIEVQTYDY (SEQ ID NO:27), ARDVVGIGIEVQTYDY (SEQ ID NO:28), ALDAVGIGIEVQTYDY (SEQ ID NO:29), ALDVAGIGIEVQTYDY (SEQ ID NO:30), ALDVVAIGIEVQTYDY (SEQ ID NO:31), ALDVVGAGIEVQTYDY (SEQ ID NO:32), ALDVVGIAIEVQTYDY (SEQ ID NO:33), ALDVVGIGAEVQTYDY (SEQ ID NO:34), ALDVVGIGIAVQTYDY (SEQ ID NO:35), ALDVVGIGIEAQTYDY (SEQ ID NO:36), ALDVVGIGIEVATYDY (SEQ ID NO:37), ALDVVGIGIEVQAYDY (SEQ ID NO:38), ALDVVGI NO:38) and ALDVVGIGIEVQTFDY (SEQ ID NO:39).

6. The transferrin receptor binding domain of claim 4 or 5, wherein the transferrin receptor binding domain comprises: (a) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15) and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (b) GFTFSTGGMG (SEQ ID NO: 12), AITWSGRHTLYADSVKG (SEQ ID NO: 15) and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (c) GFTSDTGGMG (SEQ ID NO:5), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (d) GLTFDTGGMG (SEQ ID NO:6), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (e) GLTSSTGGMG (SEQ ID NO:7), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (f) GLTSETGGMG (SEQ ID NO:8), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (g) GLTSDTYGMG (SEQ ID NO:9), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (h) GLTSDTGAMG (SEQ ID NO:10), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (i) GLTSDTGGMS (SEQ ID NO:11), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (j) GLTSDTGGMG (SEQ ID NO:4), AISWSGRHTLYADSVKG (SEQ ID NO:16), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (k) GLTSDTGGMG (SEQ ID NO:4), AITSSGRHTLYADSVKG (SEQ ID NO:17), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (l) GLTSDTGGMG (SEQ ID NO:4), AITGSGRHTLYADSVKG (SEQ ID NO:18), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (m) GLTSDTGGMG (SEQ ID NO:4), AITWNGRHTLYADSVKG (SEQ ID NO:19), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (n) GLTSDTGGMG (SEQ ID NO:4), AITWSGSHTLYADSVKG (SEQ ID NO:20), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (o) GLTSDTGGMG (SEQ ID NO:4), AITWSGGHTLYADSVKG (SEQ ID NO:21), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (p) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTYYADSVKG (SEQ ID NO:22), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (q) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ARDVVGIGIEVQTYDY (SEQ ID NO:28); (r) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDAVGIGIEVQTYDY (SEQ ID NO:29); (s) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVAGIGIEVQTYDY (SEQ ID NO:30); (t) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVAIGIEVQTYDY (SEQ ID NO:31); (u) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGAGIEVQTYDY (SEQ ID NO:32); (v) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIAIEVQTYDY (SEQ ID NO:33); (w) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGAEVQTYDY (SEQ ID NO:34); (x) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIAVQTYDY (SEQ ID NO:35); (y) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEAQTYDY (SEQ ID NO:36); (z) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVATYDY (SEQ ID NO:37); (aa) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVQAYDY (SEQ ID NO:38); (bb) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTFDY (SEQ ID NO:39); (cc) GLTSDTGGMG (SEQ ID NO:4), AITWSARHTLYADSVKG (SEQ ID NO:23) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (dd) GLTSDTGGMG (SEQ ID NO:4), AITWSGRATLYADSVKG (SEQ ID NO:24) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (ee) GLTSDTGGMG (SEQ ID NO: 4), AITFSGRHTLYADSVKG (SEQ ID NO: 25) and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (ff) GFTFSTGGMS (SEQ ID NO: 13), AITWSGRHTLYADSVKG (SEQ ID NO: 15) and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (gg)GFTFSSGGMS (SEQ ID NO:14), AITWSGRHTLYADSVKG (SEQ ID NO:15) and ALDVVGIGIEVQTYDY (SEQ ID NO:27); (hh)GFTFSTGGMS (SEQ ID NO:13), AITFSGRHTLYADSVKG (SEQ ID NO:25), and ALDVVGIGIEVQTYDY (SEQ ID NO:27); or (ii) GFTFSTGGMS (SEQ ID NO:13), AITYSGRHTLYADSVKG (SEQ ID NO:26) and ALDVVGIGIEVQTYDY (SEQ ID NO:27).

7. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: (a) a first sequence G-X1-TLD-X2-X3-AI-X4 (SEQ ID NO: 183), wherein X1 is S or F, wherein X2 is D, H or Y, X3 is Y or F, and X4 is G or A; (b) Second sequence CIS-X5-X6-DG-X7-T-X8-Y-X9-DX 10 -VKG (SEQ ID NO: 184), wherein X5 is S or R, X6 is S or G, X7 is I or R, X8 is F or Y, X9 is A or G, and X 10 is F or S; and (c) The third sequence AX 11 -X 12 -X 13 -GPNX 14 -CRGWLWX 15 -PX 16 -X 17 -SGS (SEQ ID NO: 185), wherein X 11 A or S, X 12 Is K or H, X 13 is Y or D, X 14 Is I or V, X 15 Is V or E, X 16 is P or Q, and X 17 is V, I or L (SEQ ID NO: 185).

8. The transferrin receptor binding domain of claim 7, wherein the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GSTLDYAIG (SEQ ID NO: 186), GSTLDHYAIG (SEQ ID NO: 187) and GFTLDYFAIA (SEQ ID NO: 188); (b) a second sequence selected from the group consisting of CISSSDGITFYGDFVKG (SEQ ID NO: 189), CISRSDGITYYADSVKG (SEQ ID NO: 190), and CISSGDGRTFYADSVKG (SEQ ID NO: 191); and (c) a third sequence selected from the group consisting of AAKYGPNICRGWLWVP PVSGS (SEQ ID NO: 192), AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193), and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

9. The transferrin receptor binding domain of claim 7 or 8, wherein the transferrin receptor binding domain comprises: (a) GSTLDDYAIG (SEQ ID NO: 186), CISSSDGITFYGDFVKG (SEQ ID NO: 189) and AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192); (b) GSTLDHYAIG (SEQ ID NO: 187), CISRSDGITYYADSVKG (SEQ ID NO: 190), and AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193); or (c) GFTLDYFAIA (SEQ ID NO:188), CISSGDGRTFYADSVKG (SEQ ID NO:191) and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO:194).

10. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: (a) a first sequence G-X1-T-X2-X3-X4-X5-X6-M-X7 (SEQ ID NO: 195), wherein X1 is L or F, X2 is S or F, X3 is D or S or E, X4 is T or S, X5 is G or Y, X6 is G or A, and X7 is G or S; (b) Second sequence AI-X8-X9-X 10 -X 11 -X 12 -X 13 -TX 14 -YADSVKG (SEQ ID NO: 196), wherein X8 is T or S, X9 is W, S, G, F or Y, X 10 Is S or N, X 11 It is G or A, X 12 is R, S or G, X 13 It is H or A or X 14 is L or Y; and (c) The third sequence AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 -DY (SEQ ID NO: 197), wherein X 15 It is L or R, X 16 Is V or A, X 17 Is V or A, X 18 It is G or A, X 19 Is I or A, X 20 It is G or A, X 21 Is I or A, X 22 It is E or A, X 23 V or A, X 24 It is Q or A, X 25 is T or A, and X 26 It is Y or F.

11. The transferrin receptor binding domain of claim 10, wherein the transferrin receptor binding domain comprises: GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199) and ALDVVGIGIEVQT YDY (SEQ ID NO: 200).

12. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: ESAFSLNAIG (SEQ ID NO: 201), GIGTDGITTYYA DFVKD (SEQ ID NO: 202) and NAGSWRTVLSGTHVSRS (SEQ ID NO: 203).

13. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: GRDYNHFQRA (SEQ ID NO: 204), RITWSGTITYN ESVKG (SEQ ID NO: 205) and ALKTQPPLSQDAGDYTY (SEQ ID NO: 206).

14. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: GSTLDDYAIG (SEQ ID NO: 186), CISSSDGITFYGD FVKG (SEQ ID NO: 189) and AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192).

15. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: GRSLSTYVMG (SEQ ID NO: 210), ARNGMSTYYT DSVKD (SEQ ID NO: 211) and AGDRSWSRLLRGEYEY (SEQ ID NO: 212).

16. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: GSTLDHYAIG (SEQ ID NO: 187), CISRSDGITYYADSVKG (SEQ ID NO: 190) and AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193).

17. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: GFTLDYFAIA (SEQ ID NO: 188), CISSGDGRTFYADSVKG (SEQ ID NO: 191) and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

18. A transferrin receptor binding domain, wherein the transferrin receptor binding domain comprises: GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLY ADSVKG (SEQ ID NO: 199) and ALDVVGIGIEVQTYDY (SEQ ID NO: 200).

19. The transferrin receptor binding domain of any one of claims 1 to 18, wherein the transferrin receptor binding domain is a VHH domain.

20. The transferrin receptor binding domain of claim 19, wherein the VHH domain is humanized.

21. A polypeptide comprising the transferrin receptor binding domain of any one of claims 1 to 20.

22. The polypeptide of claim 21, wherein the polypeptide further comprises a fusion partner.

23. The polypeptide of claim 22, wherein the fusion partner is selected from the group consisting of an Fc domain, an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

24. The polypeptide of claim 22 or 23, wherein the polypeptide comprises a linker between the fusion partner and the transferrin receptor binding domain.

25. The polypeptide of claim 22 or 23, wherein the fusion partner is directly adjacent to the transferrin receptor binding domain.

26. The polypeptide of any one of claims 21 to 25, wherein the polypeptide is a single polypeptide.

27. The polypeptide of claim 26, wherein the single polypeptide comprises a sequence at least 80% identical to any one of SEQ ID NOs: 216-221.

28. The polypeptide of claim 27, wherein the single polypeptide comprises a sequence at least 90% identical to any one of SEQ ID NOs: 216-221.

29. The polypeptide of claim 28, wherein the single polypeptide comprises the sequence of any one of SEQ ID NOs: 216-221.

30. The polypeptide of any one of claims 21 to 29, wherein the polypeptide is part of a protein complex.

31. An antibody comprising the transferrin receptor binding domain of any one of claims 1 to 20.

32. The antibody of claim 31 , wherein the antibody is humanized.

33. The antibody of claim 31 or 32, further comprising a fusion partner.

34. The antibody of claim 33, wherein the fusion partner is selected from the group consisting of an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

35. The antibody of claim 33 or 34, wherein the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain.

36. The antibody of claim 33 or 34, wherein the fusion partner is directly adjacent to the transferrin receptor binding domain.

37. An antibody fragment comprising the transferrin receptor binding domain of any one of claims 1 to 20.

38. The antibody fragment of claim 37, wherein the antibody fragment comprises a VHH domain.

39. The antibody fragment of claim 37 or 38, wherein the antibody fragment is humanized.

40. The antibody fragment of any one of claims 37 to 39, further comprising a fusion partner.

41. The antibody fragment of claim 40, wherein the fusion partner is selected from the group consisting of an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

42. The antibody fragment of claim 40 or 41, wherein the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain.

43. The antibody fragment of claim 40 or 41, wherein the fusion partner is directly adjacent to the transferrin receptor binding domain.

44. A composition comprising (i) a transferrin receptor binding domain as described in any one of claims 1 to 20, a polypeptide as described in any one of claims 21 to 30, an antibody as described in any one of claims 31 to 36, or an antibody fragment as described in any one of claims 37 to 43, and (ii) a pharmaceutically acceptable carrier.

45. A kit comprising the composition of claim 44.

46. ​​A nucleic acid encoding the transferrin receptor binding domain of any one of claims 1 to 20, the polypeptide of any one of claims 21 to 30, the antibody of any one of claims 31 to 36, or the antibody fragment of any one of claims 37 to 43.

47. An expression vector comprising the nucleic acid of claim 46.

48. A host cell comprising the nucleic acid of claim 46 or the expression vector of claim 47.

49. A method for producing a transferrin receptor binding domain, a polypeptide, an antibody or an antibody fragment, the method comprising: (a) culturing the host cell of claim 48 in a culture medium under conditions sufficient to allow production of the transferrin receptor binding domain, the polypeptide, the antibody or the antibody fragment; and (b) harvesting the transferrin receptor binding domain, the polypeptide, the antibody or the antibody fragment from the host cell or the culture medium.

50. The method of claim 49, further comprising isolating the transferrin receptor binding domain, the polypeptide, the antibody or the antibody fragment.

51. The method of claim 50, further comprising formulating the isolated transferrin receptor binding domain, polypeptide, antibody or antibody fragment.

52. A transferrin receptor binding domain comprising a sequence at least 80% identical to any one of SEQ ID NOs: 40-88, 147, and 210-215.

53. The transferrin receptor binding domain of claim 52, wherein the transferrin receptor binding domain comprises a sequence that is at least 90% identical to any one of SEQ ID Nos: 40-88, 147, and 210-215.

54. The transferrin receptor binding domain of claim 53, wherein the transferrin receptor binding domain comprises a sequence that is any one of SEQ ID Nos: 40-88, 147, and 210-215.

55. The transferrin receptor binding domain of any one of claims 52 to 54, wherein the transferrin receptor binding domain is a VHH domain.

56. The transferrin receptor binding domain of claim 55, wherein the VHH domain is humanized.

57. A polypeptide comprising the transferrin receptor binding domain of any one of claims 52 to 56.

58. The polypeptide of claim 57, wherein the polypeptide further comprises a fusion partner.

59. The polypeptide of claim 58, wherein the fusion partner is selected from the group consisting of an Fc domain, an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

60. The polypeptide of claim 58 or 59, wherein the polypeptide comprises a linker between the fusion partner and the transferrin receptor binding domain.

61. The polypeptide of claim 58 or 59, wherein the fusion partner is directly adjacent to the transferrin receptor binding domain.

62. The polypeptide of any one of claims 57 to 61, wherein the polypeptide is a single polypeptide.

63. The polypeptide of claim 62, wherein the single polypeptide comprises a sequence at least 80% identical to any one of SEQ ID NOs: 216-221.

64. The polypeptide of claim 63, wherein the single polypeptide comprises a sequence at least 90% identical to any one of SEQ ID NOs: 216-221.

65. The polypeptide of claim 64, wherein the single polypeptide comprises the sequence of any one of SEQ ID NOs: 216-221.

66. The polypeptide of any one of claims 57 to 61, wherein the polypeptide is part of a protein complex.

67. An antibody comprising the transferrin receptor binding domain of any one of claims 52 to 56.

68. The antibody of claim 67, wherein the antibody is humanized.

69. The antibody of claim 67 or 68, further comprising a fusion partner.

70. The antibody of claim 69, wherein the fusion partner is selected from the group consisting of an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

71. The antibody of claim 69 or 70, wherein the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain.

72. The antibody of claim 69 or 70, wherein the fusion partner is directly adjacent to the transferrin receptor binding domain.

73. An antibody fragment comprising the transferrin receptor binding domain of any one of claims 52 to 56.

74. The antibody fragment of claim 73, wherein the antibody fragment comprises a VHH domain.

75. The antibody fragment of claim 73 or 74, wherein the antibody fragment is humanized.

76. The antibody fragment of any one of claims 73 to 75, further comprising a fusion partner.

77. The antibody fragment of claim 76, wherein the fusion partner is selected from the group consisting of an enzyme, a hormone, a growth factor, a cytokine, a chemokine, a glycolipid, a lipid, a soluble protein, a nucleic acid, and an additional antigen binding domain.

78. The antibody fragment of claim 76 or 77, wherein the fusion partner further comprises a linker between the fusion partner and the transferrin receptor binding domain.

79. The antibody fragment of claim 76 or 77, wherein the fusion partner is directly adjacent to the transferrin receptor binding domain.

80. A composition comprising (i) a transferrin receptor binding domain as described in any one of claims 52 to 56, a polypeptide as described in any one of claims 57 to 66, an antibody as described in any one of claims 67 to 72, or an antibody fragment as described in any one of claims 73 to 79, and (ii) a pharmaceutically acceptable carrier.

81. A kit comprising the composition of claim 80.

82. A nucleic acid encoding the transferrin receptor binding domain of any one of claims 52 to 56, the polypeptide of any one of claims 57 to 66, the antibody of any one of claims 67 to 72, or the antibody fragment of any one of claims 73 to 79.

83. An expression vector comprising the nucleic acid of claim 82.

84. A host cell comprising the nucleic acid of claim 82 or the expression vector of claim 82.

85. A method for producing a transferrin receptor binding domain, a polypeptide, an antibody or an antibody fragment, the method comprising: (a) culturing the host cell of claim 84 in a culture medium under conditions sufficient to allow production of the transferrin receptor binding domain, the polypeptide, the antibody or the antibody fragment; as well as (b) harvesting the transferrin receptor binding domain, the polypeptide, the antibody or the antibody fragment from the host cell or the culture medium.

86. The method of claim 85, further comprising isolating the transferrin receptor binding domain, the polypeptide, the antibody or the antibody fragment.

87. The method of claim 86, further comprising formulating the isolated transferrin receptor binding domain, polypeptide, antibody or antibody fragment.

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