Transferrin receptor binding domain and protein containing same

Abstract

Provided herein are transferrin receptor binding domains, polypeptides comprising the transferrin receptor binding domain, antibodies and antibody fragments comprising the transferrin receptor binding domain, and uses thereof. In some embodiments, the transferrin receptor binding proteins bind to the human transferrin receptor with a KD 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).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 342,313, filed May 16, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically under the XML filename "47364-0022WO1". The size of the XML file created on May 15, 2023 is 254,608 bytes. The material in the XML file is incorporated herein by reference in its entirety.

[0003] The present disclosure relates generally to polypeptides and protein constructs that include an antigen-binding domain that specifically binds to the transferrin receptor. [Background technology]

[0004] The human brain is a highly vascular organ that contains approximately 400 miles of blood vessels. These blood vessels are lined with tightly connected endothelial cells, thereby forming the blood-brain barrier (BBB). The BBB protects the brain from toxins by regulating the movement of proteins, nutrients, and waste products. Attempts have been made to deliver therapeutics directly to the CNS, but this represents a highly invasive approach, often limiting distribution to areas close to the site of administration. Delivering therapeutics through the BBB is a minimally invasive therapeutic modality, but presents its own challenges, as the BBB is relatively impermeable to large molecules. Thus, compositions containing or resulting in the expression of brain-penetrating therapeutics, and therapeutic methods using such compositions, would be a major advance in effective CNS-targeted therapy.

[0005] The transferrin receptor is the cognate receptor for transferrin, which, among other functions, is required for cellular uptake of iron and is regulated according to intracellular iron concentration. The transferrin receptor is expressed on the endothelium of the blood-brain barrier and can mediate transcytosis of its ligands at the blood-brain barrier. Protein therapeutics that contain binding domains that specifically and reversibly interact with the transferrin receptor may have the potential to achieve greater brain exposure than standard biotherapeutic molecules. Summary of the Invention

[0006] Provided herein is a transferrin receptor binding protein 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). D In some embodiments, the transferrin receptor binding protein binds to the human transferrin receptor 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 and binds to the cynomolgus monkey transferrin receptor.

[0007] Also provided herein is a transferrin receptor binding domain comprising: (a) GX 1 -TX 2 -X 3 -X 4 -X 5 -X 6 -MX 7 (SEQ ID NO:1), wherein X 1 is L or F, and X 2 is S or F, and X 3 is D, S, or E, and X 4 is T or S, and X 5 is G or Y, and X 6is G or A, or X 7 is G or S; (b) AIX 8 -X 9 -X 10 -X 11 -X 12 -X 13 -TX 14 - a second sequence which is YADSVKG (SEQ ID NO: 2), wherein X 8 is T or S, and X 9 is W or S or G or F or Y, and X 10 is S or N, and X 11 is G or A, and X 12 is R, S, or G, and X 13 is H or A, or X 14 is L or Y; and (c) AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 - a third sequence which is DY (SEQ ID NO: 3), wherein X 15 is L or R, and X 16 is V or A, and X 17 is V or A, and X 18 is G or A, and X 19 is I or A, and X 20 is G or A, and X 21 is I or A, and X 22 is E or A, and X 23 is V or A, and X 24 is Q or A, and X 25 is T or A, or X 26 is Y or F.

[0008] In some embodiments, the transferrin receptor binding domain is selected from the group consisting of (a) 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 GFTFSSGGMS (SEQ ID NO: 14). 4); (b) a first sequence selected from the group consisting of AITWSGRHTLYADSVKG (SEQ ID NO: 15), AISWSGRHTLYADSVKG (SEQ ID NO: 16), AITSSGRHTLYADSVKG (SEQ ID NO: 17), AITGSGRHTLYADSVKG (SEQ ID NO: 18), AITWWNGRHTLYADSVKG (SEQ ID NO: 19), AITWSGSHTLYADSVKG (SEQ ID NO: 20), AITWSGGHTLYADSVKG (SEQ ID NO: 21), AITWSSGRHTYYADSVKG (SEQ ID NO: 22), AITWS A second sequence selected from the group consisting of ARHTLYADSVKG (SEQ ID NO: 23), AITWSGRATLYADSVKG (SEQ ID NO: 24), AITFSGRHTLYADSVKG (SEQ ID NO: 25), and AITYSGRHTLYADSVKG (SEQ ID NO: 26); and (c) ALDVVGIGIEVQTYDY (SEQ ID NO: 27), ARDVVGIGIEVQTYDY (SEQ ID NO: 28), ALDAVGIGIEVQTYDY (SEQ ID NO: 29), ALDVAGIGIEVQTYDY (SEQ ID NO: 30), ALDVVAIGIEV and a third sequence selected from the group consisting of QTYDY (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), and ALDVVGIGIEVQTFDY (SEQ ID NO: 39).

[0009] Also provided herein is a transferrin receptor binding domain comprising: (a) GX 1 -TLDX 2 -X 3 -AIX 4 (SEQ ID NO: 183), wherein X 1 is S or F, and X 2 is D, H, or Y, and X 3 is Y or F, and X 4 is G or A; (b) CISX 5 -X 6 -DGX 7 -TX 8 -YX 9 -DX 10 - a second sequence which is VKG (SEQ ID NO: 184), wherein X 5 is S or R, and X 6 is S or G, and X 7 is I or R, and X 8 is F or Y, and X 9 is A or G, and X 10 is F or S; and (c) AX 11 -X 12 -X 13 -GPNX 14 -CRGWLWX 15 -PX 16 -X 17 - a third sequence which is SGS (SEQ ID NO: 185), wherein X 11 is A, S, or X 12 is K, H, or X 13 is Y or D, X 14 is I, V, X 15 V, E, X 16 P or Q, and X 17 is V, I, or L (SEQ ID NO: 185).

[0010] In some embodiments, the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GSTLDDYAIG (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).

[0011] Also provided herein is a transferrin receptor binding domain comprising: (a) GX 1 -TX 2 -X 3 -X 4 -X 5 -X 6 -MX 7 (SEQ ID NO: 195), wherein X 1 is L or F, and X 2 is S or F, and X 3 is D, S, or E, and X 4 is T or S, and X 5 is G or Y, and X 6 is G or A, and X 7 is G or S; (b) AIX 8 -X 9 -X 10 -X 11 -X 12 -X 13 -TX 14 - a second sequence which is YADSVKG (SEQ ID NO: 196), wherein X 8 is T or S, and X 9 is W, S, G, F, or Y, and X 10 is S or N, and X 11 is G or A, and X12 is R, S, or G, and X 13 is H or A, or X 14 is L or Y; and (c) AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 - a third sequence which is DY (SEQ ID NO: 197), wherein X 15 is L or R, and X 16 is V or A, and X 17 is V or A, and X 18 is G or A, and X 19 is I or A, and X 20 is G or A, and X 21 is I or A, and X 22 is E or A, and X 23 is V or A, and X 24 is Q or A, and X 25 is T or A, and X 26 is Y or F.

[0012] 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.

[0013] Also provided herein are polypeptides comprising any of the transferrin receptor binding domains described herein. In some embodiments, the polypeptide also comprises an Fc polypeptide. In some embodiments of any of the polypeptides described herein, 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.

[0014] 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 immediately adjacent to the transferrin receptor binding domain.

[0015] In some embodiments of any of the polypeptides 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.

[0016] In some embodiments of any of the polypeptides described herein, the polypeptide is part of a protein complex.

[0017] Also provided herein are antibodies and antibody fragments comprising any of the transferrin receptor binding domains described herein. In some embodiments, the antibody or antibody fragment comprises a VHH domain. In some embodiments, the antibody is humanized.

[0018] In some embodiments, the antibody or antibody fragment further comprises a fusion partner, hi 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.

[0019] 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 immediately adjacent to the transferrin receptor binding domain.

[0020] Also provided herein are compositions comprising (i) any of the transferrin receptor binding domains described herein, any of the polypeptides described herein, any of the antibodies described herein, or any of the antibody fragments described herein; and (ii) a pharma- ceutically acceptable carrier.

[0021] Also provided herein are kits that contain any of the compositions described herein.

[0022] Also provided herein are nucleic acids encoding any of the transferrin receptor binding domains described herein, any of the polypeptides described herein, any of the antibodies described herein, or any of the antibody fragments described herein. 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.

[0023] Provided herein are methods for producing a transferrin receptor binding domain, polypeptide, antibody, or antibody fragment, the methods comprising: (a) culturing any of the host cells 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 culture medium.

[0024] 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.

[0025] 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.

[0026] Provided is a transferrin receptor binding domain comprising a sequence that is 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 that is 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 a sequence that is any one of SEQ ID NOs: 40-88, 147, and 210-215.

[0027] 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.

[0028] Provided herein is a polypeptide comprising any of the transferrin receptor binding domains described herein. In some embodiments, the polypeptide also comprises an Fc polypeptide. In some embodiments of any of the polypeptides described herein, 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.

[0029] 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 immediately adjacent to the transferrin receptor binding domain.

[0030] In some embodiments of any of the polypeptides described herein, the polypeptide is a single polypeptide.

[0031] 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.

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

[0033] Also provided herein are antibodies comprising any of the transferrin receptor binding domains described herein.

[0034] In some embodiments, the antibody is humanized.

[0035] In some embodiments of any of the antibodies described herein, the antibody further comprises a fusion partner, hi 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.

[0036] 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 immediately adjacent to the transferrin receptor binding domain.

[0037] 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.

[0038] In some embodiments of any of the antibody fragments described herein, the antibody fragment is humanized.

[0039] In some embodiments of any of the antibody fragments described herein, the antibody fragment further comprises a fusion partner, hi 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.

[0040] 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 immediately adjacent to the transferrin receptor binding domain.

[0041] Also provided herein are compositions comprising (i) any of the transferrin receptor binding domains described herein, any of the polypeptides described herein, any of the antibodies described herein, or any of the antibody fragments described herein; and (ii) a pharma- ceutically acceptable carrier.

[0042] Also provided herein are kits that contain any of the compositions described herein.

[0043] Also provided herein is a nucleic acid encoding any of the transferrin receptor binding domains described herein, any of the polypeptides described herein, any of the antibodies described herein, or any of the antibody fragments described herein.

[0044] Also provided herein is an expression vector comprising any of the nucleic acids described herein. Also provided herein is a host cell transfected with any of the nucleic acids described herein or transduced with any of the expression vectors described herein.

[0045] Also provided herein is a method of producing any of the transferrin receptor binding domains described herein, any of the polypeptides described herein, any of the antibodies described herein, or any of the antibody fragments described herein, the method comprising: (a) culturing any of the host cells 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 culture medium.

[0046] 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.

[0047] 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.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein. Other suitable methods and materials known in the art can 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 by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0049] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0050] [Figure 1] 1 shows a series of fluorescent images showing uptake of a polypeptide 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 (cynomolgus monkey transferrin receptor positive cells), and CHO cells (human and cynomolgus monkey transferrin receptor negative cells) in the presence of 4.0 nM to 1000 nM of the polypeptide. Detection was performed using a fluorophore-conjugated secondary antibody that specifically binds to the polypeptide. [Figure 2A]

[0023] Figure 1 shows a series of representative cellular uptake / binding curves for exemplary transferrin receptor binding domains generated as described herein. The cellular uptake / binding curves were generated in HEK293T cells (human transferrin receptor positive cells). [Figure 2B]

[0023] Figure 1 shows a series of representative cellular uptake / binding curves for exemplary transferrin receptor binding domains generated as described herein. The cellular uptake / binding curves were generated in CHO:cyTfR cells (cynomolgus monkey transferrin receptor positive cells). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Provided herein is a transferrin receptor binding protein comprising a camelid heavy chain variable domain.

[0052] Also provided are polypeptides, antibodies, or antibody fragments comprising any of the transferrin receptor binding domains described herein. In some embodiments, the polypeptide is a single polypeptide. In other embodiments, the polypeptide is part of a protein complex.

[0053] Also provided herein are compositions comprising any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments described herein. Also provided herein are kits comprising any of the compositions described herein. Also provided herein are nucleic acids encoding any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments described herein. Also provided are host cells comprising any of the nucleic acids described herein. Also provided are methods of producing any of the transferrin receptor binding domains, polypeptides, antibodies, and antibody fragments described herein, the methods comprising culturing any of the host cells described herein.

[0054] In some embodiments, any of the antibodies, antibody fragments, or polypeptides described herein may contain 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.

[0055] The terms "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. By way of example, "a polypeptide" includes one or more polypeptides.

[0056] As used herein, when used to modify a quantity specified in a numerical value or range, the terms "about" and "approximately" indicate numerical values ​​as well as reasonable deviations from the values ​​known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value.

[0057] Unless otherwise specified, a "nucleotide sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.

[0058] The term "exogenous" refers to any substance introduced from or derived from outside a cell, tissue, or organism and that is not produced by or derived from the same cell, tissue, or organism into which it is introduced.

[0059] The terms "transduced," "transfected," or "transformed" refer to the process by which exogenous nucleic acid is introduced or transferred into a cell. A "transduced," "transfected," or "transformed" cell (e.g., a mammalian cell, a hepatic cell) is a cell that has been transduced, transfected, or transformed with an exogenous nucleic acid (e.g., an expression vector containing an exogenous nucleic acid encoding a polypeptide).

[0060] The term "subject" is intended to include any mammal. In some embodiments, the subject is a cat, a dog, a goat, a human, a non-human primate, a rodent (e.g., a mouse or a rat), a pig, or a sheep. In some embodiments, the subject has or is at risk of developing a CNS disorder or disease. In some embodiments, the subject has previously been identified or diagnosed as having a CNS disorder or disease.

[0061] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in either single-stranded or double-stranded form, or a combination thereof. Unless otherwise limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the referenced nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also the complementary sequence. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0062] Modifications can be introduced into nucleotide sequences by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with 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), nonpolar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, and valine), beta-branched side chains (e.g., isoleucine, threonine, and valine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine).

[0063] The term "antigen-binding domain" is used to refer to one or more antibody variable domains (e.g., a domain formed from amino acids from a single polypeptide or from two or more polypeptides (e.g., the same or different polypeptides) that can specifically bind to one or more different antigens). In some examples, an antigen-binding domain can bind to an antigen or epitope with similar specificity and affinity as a naturally occurring antibody. In some embodiments, the antigen-binding domain may be an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain may comprise an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are known in the art.

[0064] The term "antibody" refers to a protein with an immunoglobulin fold that specifically binds to an antigen through its variable region(s). As used herein, the term "antibody" may refer to heavy chain antibodies of camelids (e.g., camels, llamas, alpacas, dromedaries) that lack light chains. These heavy chain antibodies interact with antigens through one single variable domain called VHH, VHH domain, or VHH antibody. The antigen binding site of a VHH domain is similar to the antigen binding site of the heavy chain variable domain of a conventional antibody, although the framework regions and complementarity determining regions are different. A target antigen generally has multiple binding sites, also called epitopes, recognized by CDRs (complementarity determining regions) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have two or more corresponding antibodies. Antibodies can include, for example, full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen of a target of interest or a portion thereof (e.g., the transferrin receptor). Immunoglobulins disclosed herein include any subclass of immunoglobulin molecule (e.g., IgG 1 , IgG 2 , IgG 3 )

[0065] The modified antibody can be an antibody bound to various types of molecules such as polyethylene glycol (PEG). Methods for modifying antibodies have already been established in the art.

[0066] The term "antibody fragment" refers to a portion of a full-length antibody or a polypeptide comprising a portion of a full-length antibody that retains antigen-binding activity through its variable region(s). 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 through its variable region(s). 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)). Additional 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.

[0067] The term "variable region" or "variable domain" refers to a domain within an antibody heavy chain or a domain within an antibody light chain that is encoded by a combination of germline variable (V), diversity (D), or joining (J) gene segments and that confers to the antibody its specificity for binding to an antigen. As described herein, a camelid heavy chain antibody contains a single variable heavy chain domain (e.g., a VHH domain) that is encoded by a combination of germline variable (V) gene segments that confers to the antibody its specificity for binding to an antigen (e.g., the transferrin receptor).

[0068] The term "complementarity determining region" or "CDR" refers to one of three hypervariable regions (or HVRs) known to confer (at least in part) antigen-binding specificity within each antibody light chain variable domain and each antibody heavy chain variable domain (e.g., VHH domain). The three CDRs in an antibody heavy chain and an antibody light chain separate 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, numbered sequentially from the N-terminus, and are also usually identified by the chain in which a particular CDR is located. Thus, the VH CDR3 or CDR-H3 is the third CDR located in the heavy chain variable domain, and the VL CDR1 or CDR-L1 is the first CDR in the light chain variable domain. As used herein, VH CDR1 is used interchangeably with "first sequence," VH CDR2 is used interchangeably with "second sequence," and VH CDR3 is used interchangeably with "third sequence."

[0069] The "framework region" or "FR" of an immunoglobulin heavy chain serves to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences.

[0070] The amino acid sequences of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat.

[0071] The term "protein complex" refers to a complex of two or more polypeptides (e.g., the same or different polypeptides) that are associated through non-covalent bonds. For example, a protein complex can include at least one antigen-binding domain. Non-limiting examples and aspects of protein complexes are described herein. Further examples and aspects of protein complexes are known in the art. In some embodiments, a protein complex includes one or more single polypeptides.

[0072] The term "treating" 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 of the subjects described herein) and / or resulting in a decrease in the rate of progression and / or worsening of one or more symptoms of a disease or disorder in a subject over time.

[0073] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired site of biological action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, colonic, rectal, or intraperitoneal delivery. In one embodiment, the compositions described herein are administered intravenously.

[0074] The term "promoter" refers to a DNA sequence that is recognized by an enzyme / protein in a cell (e.g., a mammalian cell, a hepatic cell) necessary to initiate transcription of a nucleic acid encoding an operably linked coding sequence (e.g., a polypeptide (e.g., any of the exemplary polypeptides described herein)). A promoter typically refers to a nucleotide sequence to which, for example, RNA polymerase and / or any associated factors bind and initiate transcription. A promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter (e.g., a liver-specific promoter).

[0075] The term "enhancer" refers to a nucleotide sequence that can increase transcription of an operably linked nucleic acid (e.g., a nucleic acid encoding a polypeptide (e.g., any of the exemplary polypeptides described herein)). Enhancers can increase transcription levels by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). Unlike promoters, enhancers can act at greater distances from the transcription start site (e.g., compared to promoters).

[0076] The terms "identical" or percent "identity", in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are identical or have a specified percentage, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, of amino acid residues that are identical over a specified region when compared and aligned for maximum matching, e.g., in a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0077] For sequence comparison of polypeptides, typically, one amino acid sequence serves as a reference sequence, and candidate sequences are compared to this reference sequence. Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using public software with known algorithms to achieve maximum alignment. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purpose of this application, for sequence comparison of polypeptide sequences, the BLASTP algorithm standard protein BLAST is used to align two protein sequences with default parameters.

[0078] The term "affinity" refers to the combined strength of all non-covalent interactions between an antigen-binding site and its antigen. Unless otherwise specified, "affinity" refers to the inherent binding affinity, which represents a 1:1 interaction between an antigen-binding domain and an antigen. Affinity can be measured, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Further methods for determining the affinity of an antigen-binding domain and its antigen are known in the art.

[0079] Transferrin receptor binding domains and polypeptides Provided herein is a transferrin receptor binding protein 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). D In some embodiments, the protein binds to the human transferrin receptor 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 and binds to the cynomolgus monkey transferrin receptor.

[0080] Also provided herein is a transferrin receptor binding domain comprising: (a) GX 1 -TX 2 -X 3 -X 4 -X 5 -X 6 -MX 7 (SEQ ID NO:1), wherein X 1 is L or F, and X 2 is S or F, and X 3 is D, S, or E, and X 4 is T or S, and X 5is G or Y, and X 6 is G or A, or X 7 is G or S; (b) AIX 8 -X 9 -X 10 -X 11 -X 12 -X 13 -TX 14 - a second sequence which is YADSVKG (SEQ ID NO: 2), wherein X 8 is T or S, and X 9 is W or S or G or F or Y, and X 10 is S or N, and X 11 is G or A, and X 12 is R, S, or G, and X 13 is H or A, or X 14 is L or Y; and (c) AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 - a third sequence which is DY (SEQ ID NO: 3), wherein X 15 is L or R, and X 16 is V or A, and X 17 is V or A, and X 18 is G or A, and X 19 is I or A, and X 20 is G or A, and X 21 is I or A, and X 22 is E or A, and X 23 is V or A, and X 24 is Q or A, and X 25 is T or A, or X 26 is Y or F.

[0081] In some embodiments, the transferrin receptor binding domain is selected from the group consisting of (a) GLTSDTGGMG (SEQ ID NO: 4), 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), GFTFSTGGMG (SEQ ID NO: 12), GFTFSTGGMS (SEQ ID NO: 13), and GFTFSSGGMS (SEQ ID NO: 14). 4); (b) a first sequence selected from the group consisting of AITWSGRHTLYADSVKG (SEQ ID NO: 15), AISWSGRHTLYADSVKG (SEQ ID NO: 16), AITSSGRHTLYADSVKG (SEQ ID NO: 17), AITGSGRHTLYADSVKG (SEQ ID NO: 18), AITWWNGRHTLYADSVKG (SEQ ID NO: 19), AITWSGSHTLYADSVKG (SEQ ID NO: 20), AITWSGGHTLYADSVKG (SEQ ID NO: 21), AITWSSGRHTYYADSVKG (SEQ ID NO: 22), AITWS A second sequence selected from the group consisting of ARHTLYADSVKG (SEQ ID NO: 23), AITWSGRATLYADSVKG (SEQ ID NO: 24), AITFSGRHTLYADSVKG (SEQ ID NO: 25), and AITYSGRHTLYADSVKG (SEQ ID NO: 26); and (c) ALDVVGIGIEVQTYDY (SEQ ID NO: 27), ARDVVGIGIEVQTYDY (SEQ ID NO: 28), ALDAVGIGIEVQTYDY (SEQ ID NO: 29), ALDVAGIGIEVQTYDY (SEQ ID NO: 30), ALDVVAIGIEV and a third sequence selected from the group consisting of QTYDY (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), and ALDVVGIGIEVQTFDY (SEQ ID NO: 39).

[0082] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain is selected from the group consisting of: (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 ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (c) GLTFDTGGMG (SEQ ID NO: 6), AITWSGR HTLYADSVKG (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); (e) GLTSETGGMG (SEQ ID NO: 8), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (f) GLTSDTYGMG (SEQ ID NO: 9), AITWSGRHTLYADSVKG (SEQ ID NO: 1 5), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (g) GLTSDTGAMG (SEQ ID NO: 10), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (h) GLTSDTGGMS (SEQ ID NO: 11), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (i) GLTSDTGGMG (SEQ ID NO: 4), AISWSGRHTLYADSVKG (SEQ ID NO: 16), and ALDVVGIGI EVQTYDY (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), AITWWNGRHTLYADSVKG (SEQ ID NO:19), and ALDVVGIGIEVQTYDY (SEQ ID NO:27);(m) GLTSDTGGMG (SEQ ID NO: 4), AITWSGSHTLYADSVKG (SEQ ID NO: 20), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (n) GLTSDTGGMG (SEQ ID NO: 4), AITWSGGHTLYADSVKG (SEQ ID NO: 21), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (o) GLTSDTGGMG (SEQ ID NO: 4), AITWSGGHTLYADSVKG (SEQ ID NO: 22), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (p) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ARDVVGIGIEVQTYDY (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 ID NO: 15), and ALDVAGIGIEVQTYDY (SEQ ID NO: 30); (s) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVK G (SEQ ID NO: 15), and ALDVVAIGIEVQTYDY (SEQ ID NO: 31); (t) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGAGIEVQTYDY (SEQ ID NO: 32); (u) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGIAIEVQTYDY (SEQ ID NO: 33); (v) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVG IGAEVQTYDY (SEQ ID NO:34); (w) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIAVQTYDY (SEQ ID NO:35); (x) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEAQTYDY (SEQ ID NO:36); (y) GLTSDTGGMG (SEQ ID NO:4), AITWSGRHTLYADSVKG (SEQ ID NO:15), and ALDVVGIGIEVATYDY (SEQ ID NO:37);(z) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGIGIEVQAYDY (SEQ ID NO: 38); (aa) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRHTLYADSVKG (SEQ ID NO: 15), and ALDVVGIGIEVQTFDY (SEQ ID NO: 39); (bb) GLTSDTGGMG (SEQ ID NO: 4), AITWSARHTLYADSVKG (SEQ ID NO: 23), and ALDVVGIGIEVQTYDY (SEQ ID NO: 27); (cc) GLTSDTGGMG (SEQ ID NO: 4), AITWSGRATLYADSVKG (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) GF TFSTGGMG (SEQ ID NO: 12), AITWSGRHTLYADSVKG (SEQ ID 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) 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).

[0083] Also provided herein is a transferrin receptor binding domain comprising: (a) GX 1 -TLDX 2 -X 3 -AIX 4 (SEQ ID NO: 183), wherein X 1 is S or F, and X 2 is D, H, or Y, and X 3is Y or F, and X 4 is G or A; (b) CISX 5 -X 6 -DGX 7 -TX 8 -YX 9 -DX 10 - a second sequence which is VKG (SEQ ID NO: 184), wherein X 5 is S or R, and X 6 is S or G, and X 7 is I or R, and X 8 is F or Y, and X 9 is A or G, and X 10 is F or S; and (c) AX 11 -X 12 -X 13 -GPNX 14 -CRGWLWX 15 -PX 16 -X 17 - a third sequence which is SGS (SEQ ID NO: 185), wherein X 11 is A, S, or X 12 is K, H, or X 13 is Y or D, X 14 is I, V, X 15 V, E, X 16 P or Q, and X 17 is V, I, or L (SEQ ID NO: 185).

[0084] In some embodiments, the transferrin receptor binding domain comprises: (a) a first sequence selected from the group consisting of GSTLDDYAIG (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).

[0085] 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 AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192); (b) GSTLDHYAIG (SEQ ID NO: 187), CISSRDGITYYADSVKG (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).

[0086] Also provided herein is a transferrin receptor binding domain comprising: (a) GX 1 -TX 2 -X 3 -X 4 -X 5 -X 6 -MX 7 (SEQ ID NO: 195), wherein X 1 is L or F, and X 2 is S or F, and X 3 is D, S, or E, and X 4is T or S, and X 5 is G or Y, and X 6 is G or A, and X 7 is G or S; (b) AIX 8 -X 9 -X 10 -X 11 -X 12 -X 13 -TX 14 - a second sequence which is YADSVKG (SEQ ID NO: 196), wherein X 8 is T or S, and X 9 is W, S, G, F, or Y, and X 10 is S or N, and X 11 is G or A, and X 12 is R, S, or G, and X 13 is H or A, or X 14 is L or Y; and (c) AX 15 -DX 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 - a third sequence which is DY (SEQ ID NO: 197), wherein X 15 is L or R, and X 16 is V or A, and X 17 is V or A, and X 18 is G or A, and X 19 is I or A, and X 20 is G or A, and X 21 is I or A, and X 22 is E or A, and X 23 is V or A, and X 24 is Q or A, and X 25 is T or A, and X 26is Y or F. In some embodiments, the transferrin receptor binding domain comprises GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199), and ALDVVGIGIEVQTYDY (SEQ ID NO: 200).

[0087] Provided herein are transferrin receptor binding domains comprising ESAFSLNAIG (SEQ ID NO: 201), GIGTDGITTYYADFVKD (SEQ ID NO: 202), and NAGSWRTVLSGTHVSRS (SEQ ID NO: 203).

[0088] Provided herein are transferrin receptor binding domains comprising GRDYNHFQRA (SEQ ID NO: 204), RITWSGTITYNESVKG (SEQ ID NO: 205), and ALKTQPPLSQDAGDYTY (SEQ ID NO: 206).

[0089] Provided herein are transferrin receptor binding domains comprising GSTLDDYAIG (SEQ ID NO: 186), CISSSDGITFYGDFVKG (SEQ ID NO: 189), and AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192).

[0090] Provided herein are transferrin receptor binding domains comprising GRSLSTYVMG (SEQ ID NO: 210), ARNGMSTYYTDSVKD (SEQ ID NO: 211), and AGDRSWSRLLRGEYEY (SEQ ID NO: 212).

[0091] Provided herein are transferrin receptor binding domains comprising GSTLDHYAIG (SEQ ID NO: 187), CISRSDGITYYADSVKG (SEQ ID NO: 190), and AAHYGPNVCRGWLWEPPISGS (SEQ ID NO: 193).

[0092] Provided herein are transferrin receptor binding domains comprising GFTLDYFAIA (SEQ ID NO: 188), CISSGDGRTFYADSVKG (SEQ ID NO: 191), and ASHDGPNVCRGWLWVPQLSGS (SEQ ID NO: 194).

[0093] Provided herein are transferrin receptor binding domains comprising GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199), and ALDVVGIGIEVQTYDY (SEQ ID NO: 200).

[0094] In some embodiments of any of the transferrin receptor binding domains described herein, the transferrin receptor binding domain is a humanized transferrin receptor binding domain. In some embodiments of any of the antibodies that comprise a transferrin receptor binding domain described herein, the first sequence, the second sequence, and the third sequence are humanized.

[0095] 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.

[0096] Provided herein is 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 97%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 40-88, 147, and 210-215.

[0097] Also provided herein are polypeptides comprising any of the transferrin receptor binding domains described herein. In some embodiments, the polypeptide also comprises an Fc polypeptide.

[0098] In some embodiments, the polypeptide further comprises a fusion partner, hi 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.

[0099] In some embodiments, the polypeptide comprises a linker between the fusion partner and the transferrin receptor binding domain. In some embodiments, the linker comprises a total of about 1 to about 50 amino acids (e.g., about 1 to about 40 amino acids, about 1 to about 30 amino acids, about 1 to about 20 amino acids, about 1 to about 15 amino acids, about 1 to about 10 amino acids, or about 1 to about 5 amino acids). Non-limiting examples of linkers include (G4S)n (SEQ ID NO: 153), where n is an integer of 1 to 10, such as GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGGS (SEQ ID NO: 156), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 157).

[0100] In some embodiments, the fusion partner is immediately adjacent to the transferrin receptor binding domain.

[0101] In some embodiments, the polypeptide is a single polypeptide. In some embodiments, the single polypeptide comprises 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 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 NOs: 89-138 and 216-221.

[0102] In some embodiments of any of the single polypeptides described herein, the single polypeptide may be or may comprise 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, a VHH-scFv, an IgG-VHH-scFv, or an Fc polypeptide-VHH-scFv.

[0103] In some embodiments of any of the single polypeptides described herein, the transferrin receptor binding domain is fused to an Fc polypeptide.

[0104] In some embodiments of any of the single polypeptides 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 an additional antigen binding domain (e.g., any of the exemplary antigen binding domains described herein that specifically bind to the transferrin receptor, e.g., the human transferrin receptor, or an antigen binding domain that binds to a different antigen). In some embodiments, the fusion partner is glucosylceramidase beta (GBA). In some embodiments, the fusion partner is granulin precursor or progranulin (PGRN).

[0105] In some embodiments of any of the single polypeptides that include GBA as a fusion partner, 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 SEQ ID NO: 179. In some embodiments of any of the single polypeptides that include GBA as a fusion partner, 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 SEQ ID NOs: 139-144.

[0106] In some embodiments of any of the single polypeptides that include PGRN as a fusion partner, 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 SEQ ID NO: 181. In some embodiments of any of the single polypeptides that include PGRN as a fusion partner, 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 SEQ ID NO: 145, 146, or 148. In some embodiments of any of the single polypeptides that include PGRN as a fusion partner, 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 SEQ ID NO: 147.

[0107] In some embodiments, the single polypeptide further comprises a linker (e.g., any of the exemplary linkers described herein) between the fusion partner and the transferrin receptor binding domain. In some embodiments, the fusion partner and the transferrin receptor binding domain are immediately adjacent to one another. In some embodiments, the single polypeptide comprises, from N-terminus to C-terminus, the transferrin receptor binding domain, the linker, and the fusion partner. In some embodiments, the single polypeptide comprises, from N-terminus to C-terminus, the fusion partner, the linker, and the transferrin receptor binding domain. In some embodiments, the linker comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any of the subranges of this range described herein). Non-limiting examples of linkers include (G4S)n (SEQ ID NO: 153), where n is an integer between 1 and 10, such as GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGGS (SEQ ID NO: 156), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 157).

[0108] In some embodiments of any of the single polypeptides described herein, the single polypeptide does not include any other targeting domains.

[0109] In some embodiments, the polypeptide is part of a protein complex. In some embodiments, the protein complex comprises at least a first polypeptide that comprises any of the exemplary transferrin receptor binding domains described herein.

[0110] In some embodiments, the protein complex can include at least a first polypeptide including a transferrin receptor binding domain that includes 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 can include at least a first polypeptide that includes 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.

[0111] In some embodiments of any of the protein complexes 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 an additional antigen binding domain (e.g., any of the exemplary antigen binding domains described herein that specifically bind to a transferrin receptor, e.g., the human transferrin receptor, or an antigen binding domain that binds to a different antigen). In some embodiments, the fusion partner is glucosylceramidase beta (GBA). In some embodiments, the fusion partner is granulin precursor or progranulin (PGRN). In some embodiments of any of the protein complexes described herein, the protein complex comprises a polypeptide comprising a transferrin receptor binding domain and a fusion partner, the polypeptide optionally being fused to an Fc domain. In some embodiments, the polypeptide can comprise a linker (e.g., any of the exemplary linkers described herein) between the polypeptide and the Fc domain.

[0112] In some embodiments of any of the protein complexes having a first and / or second polypeptide that comprises GBA as a fusion partner, the first polypeptide and / or the second polypeptide can 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: 179. In some embodiments, the first polypeptide and / or the second polypeptide can 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: 139-144.

[0113] In some embodiments of any of the protein complexes having a first and / or second polypeptide comprising PGRN as a fusion partner, the first polypeptide and / or the second polypeptide can 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 can 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.

[0114] In some embodiments of any of the protein complexes having a first and / or second polypeptide that includes a fusion partner (e.g., a GBA or PGRN polypeptide), the first and / or second polypeptide can further include a linker (e.g., any of the exemplary linkers described herein) between the fusion partner and the 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 first and / or second polypeptide includes, from N-terminus to C-terminus, the transferrin receptor binding domain, the linker, and the fusion partner (e.g., a GBA or PGRN polypeptide). In some embodiments, the first and / or second polypeptide includes, from N-terminus to C-terminus, the fusion partner (e.g., a GBA or PGRN polypeptide), the linker, and the transferrin receptor binding domain.

[0115] In some embodiments of any of the protein complexes having a first and / or second polypeptide that includes 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 of the subranges of this range described herein). Non-limiting examples of linkers include (G4S)n (SEQ ID NO:153), where n is an integer between 1 and 10, such as GGGGS (SEQ ID NO:154), GGGGSGGGGS (SEQ ID NO:155), GGGGSGGGGSGGGGGS (SEQ ID NO:156), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:157).

[0116] In some embodiments of any of the protein complexes described herein, the protein complex includes 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 comprising one VHH domain and a second functional domain, VHH-Fc, VHH-IgG, IgG-VHH, knob-in-hole assembly, VHH-scFv, IgG-VHH-scFv, or Fc-VHH-scFv.

[0117] 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 include any other targeting domain.

[0118] In some embodiments of any of the protein complexes described herein, the protein complex comprises at least a first polypeptide comprising a transferrin receptor binding domain. Some embodiments of these protein complexes further comprise a second polypeptide comprising a fusion partner (e.g., any of the exemplary fusion partners described herein, e.g., a GBA or PGRN polypeptide). In such embodiments, the first polypeptide and / or the second polypeptide may be fused or linked to an Fc domain.

[0119] In some embodiments of any of the protein complexes described herein, the protein complex may be human or humanized.

[0120] Also provided herein are nucleic acids encoding any of the transferrin receptor binding domains or polypeptides described herein. Also provided herein are expression vectors comprising any of the nucleic acids described herein encoding any of the transferrin receptor binding domains or polypeptides described herein. In some examples, the expression vectors described herein can further comprise a promoter (e.g., any of the exemplary promoters described herein or known in the art) and / or an enhancer (e.g., any of the exemplary enhancers described herein or known in the art), wherein the promoter and / or enhancer are operably linked to the nucleic acid sequence encoding any of the transferrin receptor binding domains or polypeptides described herein.

[0121] Also provided herein are nucleic acids encoding each of the polypeptides in any of the protein complexes described herein. Also provided herein are pairs or sets of nucleic acids that together encode each of the polypeptides in any of the protein complexes described herein.

[0122] Also provided herein is an expression vector comprising any of the nucleic acids described herein. Also provided is a pair or set of expression vectors that together encode each of the polypeptides in any of the protein complexes described herein. In some examples, the expression vector(s) described herein can further comprise a promoter (e.g., any of the exemplary promoters described herein or known in the art) and / or an enhancer (e.g., any of the exemplary enhancers described herein or known in the art), where the promoter and / or enhancer are operably linked to the nucleic acid sequence encoding any of the polypeptides described herein. Further aspects and examples of expression vectors are described herein.

[0123] Antibodies and antibody fragments Provided herein is an antibody or antibody fragment comprising any of the 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.

[0124] In some embodiments, the antibody or antibody fragment can include a transferrin receptor binding domain having 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.

[0125] In some embodiments, the antibody or antibody fragment comprises a VHH domain. In some embodiments, the antibody or antibody fragment can comprise one or more additional single variable domains (e.g., VHH domains) that comprise an antigen binding domain that specifically binds to a transferrin receptor (a transferrin receptor binding domain, e.g., any of the exemplary transferrin receptor binding domains described herein). In some embodiments, the antibody or antibody fragment comprises one or more additional single variable domains that bind to a different antigen. In some embodiments, the antibody or antibody fragment does not comprise any other targeting domain.

[0126] In some embodiments the antibody may be or may comprise 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 comprising 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 may 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 comprising 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.

[0127] In some embodiments, the antibody or antibody fragment comprises an antigen binding domain that specifically binds to a transferrin receptor (e.g., a transferrin receptor binding domain), where the antigen binding domain is fused to an Fc domain. 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 NOs: 89-138 or 216-221.

[0128] In some embodiments, the antibody or antibody fragment may be a human or humanized antibody or antibody fragment.

[0129] In some embodiments, the antibody or antibody fragment further comprises a fusion partner (e.g., a heterologous fusion partner) (e.g., any of the exemplary fusion partners 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 an additional antigen binding domain (e.g., any of the exemplary antigen binding domains described herein that specifically bind to the transferrin receptor, e.g., the human transferrin receptor, or an antigen binding domain that binds to a different antigen). In some embodiments, the fusion partner is a glucosylceramidase beta (GBA) polypeptide. In some embodiments, the fusion partner is a granulin precursor or progranulin (PGRN) polypeptide.

[0130] In some embodiments, the antibody or antibody fragment comprises a GBA polypeptide as a fusion partner, wherein the GBA 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: 179. 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 NOs: 139-144.

[0131] 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 NOs: 145, 146 or 148.

[0132] In some embodiments, the antibody or antibody fragment further comprises a linker (e.g., any of the exemplary linkers described herein) between the fusion partner and the transferrin receptor binding domain. In some embodiments, the fusion partner and the transferrin receptor binding domain are directly adjacent to one another. In some embodiments, the antibody or antibody fragment comprises, from N-terminus to C-terminus, the transferrin receptor binding domain, the linker, and the fusion partner (e.g., a GBA or PGRN polypeptide). In some embodiments, the antibody or antibody fragment comprises, from N-terminus to C-terminus, the fusion partner (e.g., a GBA or PGRN polypeptide), the linker, and the transferrin receptor binding domain.

[0133] In some embodiments, the linker comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any of the subranges of this range described herein). Non-limiting examples of linkers include (G4S)n (SEQ ID NO:153), where n is an integer from 1 to 10, such as GGGGS (SEQ ID NO:154), GGGGSGGGGS (SEQ ID NO:155), GGGGSGGGGSGGGGGS (SEQ ID NO:156), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:157).

[0134] Also provided herein are nucleic acids encoding any of the antibodies or antibody fragments described herein. Also provided herein are expression vectors comprising any of the nucleic acids described herein encoding any of the antibodies or antibody fragments described herein. In some examples, the expression vectors described herein can further comprise a promoter (e.g., any of the exemplary promoters described herein or known in the art) and / or an enhancer (e.g., any of the exemplary enhancers described herein or known in the art), wherein the promoter and / or enhancer are operably linked to the nucleic acid sequence encoding any of the antibodies or antibody fragments described herein. Further aspects and examples of expression vectors are described herein.

[0135] Additional Exemplary Aspects The transferrin receptor binding domains, single polypeptides, protein complexes, antibodies, and antibody fragments described herein comprise at least one transferrin receptor binding domain. Exemplary human wild-type transferrin receptor proteins can include the sequences of SEQ ID NOs: 149, 150, 151, or 152.

[0136] In some embodiments, the transferrin receptor binding domain comprises about 1×10 -5 Less than M, approx. 1x10 -6 Less than M or about 1x10 -7 Less than M (e.g., about 1x10 -8 Less than M, about 1x10 -9 Less than M, about 1x10 -10 Less than M, approx. 1x10 -11 Less than M, about 1x10 -12 Less than M or about 1x10 -13 Dissociation constant (K D ) and 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., performed in phosphate buffered saline).

[0137] In some embodiments where 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 can comprise a linker (e.g., any of the exemplary linkers described herein) between the transferrin receptor binding domain and the Fc region. In some embodiments, the linker comprises a total of about 1 amino acid to about 50 amino acids (e.g., or any of the subranges of this range described herein). Non-limiting examples of linkers include (G4S)n (SEQ ID NO: 153), where n is an integer between 1 and 10, such as GGGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGGSGGGGSGGGGGS (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.

[0138] Non-limiting examples of sequences that specifically bind to the human transferrin receptor include SEQ ID NOs: 40-88, 147, and 89-138.

[0139] 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)).

[0140] Expression vector 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 lentivirus vector, a Sindbis virus vector, an alphavirus-based vector, or an adenovirus vector. AAV vectors are generally described in Asokan et al., Mol. Ther. 20:699-708, 2012, and BJ Carter, "Hand book of Parvoviruses", Ed., P. Tijsser, CRC Press, pp.155-168, 1990. Adenoviral vectors are generally described, for example, in 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, for example, in 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.

[0141] 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 of the exemplary transferrin receptor binding domains described herein), a polypeptide (e.g., any of the exemplary polypeptides described herein), an antibody (e.g., any of the exemplary antibodies described herein), an antibody fragment (e.g., any of the exemplary antibody fragments described herein), or a protein complex (e.g., any of the exemplary protein complexes described herein).

[0142] In some embodiments, the expression vector can be an AAV vector.For example, the AAV vector can be selected from the group 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 of the exemplary AAV vectors described herein, the AAV vector can 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.

[0143] In some embodiments, the nucleic acid further comprises a promoter and / or enhancer operably linked to the sequence encoding any of the transferrin receptor binding domains, antibodies, antibody fragments, single polypeptides, or protein complexes 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 that are constitutive, inducible, and / or tissue-specific are known in the art. Non-limiting examples of promoters include CMV promoter, CAG promoter, human alpha-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 serpin enhancer (e.g., SEQ ID NO: 175). Other promoters and enhancers are described herein.

[0144] In some embodiments, the nucleic acid comprises a promoter operably linked to a nucleic acid sequence encoding a fusion partner (e.g., any of the exemplary fusion partners 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 of the exemplary fusion partners 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 of the exemplary fusion partners described herein).

[0145] In some embodiments, a nucleic acid comprising a promoter and / or enhancer operably linked to a nucleic acid sequence encoding a first polypeptide, a second polypeptide, or both (e.g., any of the exemplary first polypeptides described herein, any of the exemplary second polypeptides described herein) flanked at its 5' and 3' ends by viral inverted terminal repeat (ITR) sequences (e.g., any of the exemplary ITR sequences described herein or known in the art). In some embodiments, a nucleic acid comprising a promoter and / or enhancer operably linked to a nucleic acid sequence encoding a first polypeptide, a second polypeptide, or both (e.g., any of the exemplary first polypeptides described herein, any of the exemplary second polypeptides described herein) flanked at its 5' and 3' ends by viral ITR sequences (e.g., any of the exemplary ITR sequences described herein or known in the art).

[0146] Exemplary ITR sequences are described in BJ Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168, 1990, and U.S. Patent No. 9,150,882 (incorporated herein by reference). Exemplary 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 140 nucleotides, about 100 to about 120 nucleotides, about 100 to about 160 nucleotides, about 100 to about 180 nucleotides, about 100 to about 160 nucleotides, about 100 to about 150 nucleotides, about 100 to about 140 nucleotides, about 100 to about 12 ... 0 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).

[0147] It is within the skill of the artisan to modify the ITR sequence. See, for example, texts such as Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2d Ed., Cold Spring Harbor Laboratory, New York, 1989; and Fischer et al., J. Virol. 70:520-532, 1996). Further examples of ITR sequences may be obtained from any known AAV (AAV ITR). In some examples, the AAV ITR sequence may be an AAV2 ITR sequence, or a functional variant thereof. In some embodiments of any of the AAV vectors described herein, the ITR sequence is a self-complementary ITR (scITR) sequence.

[0148] In some embodiments, a nucleic acid encoding a polypeptide, antibody, antibody fragment, or protein complex comprises a single antigen-binding domain that specifically binds to the transferrin receptor. For example, in some embodiments, a nucleic acid sequence encoding a polypeptide, antibody fragment, antibody, or protein complex encodes a single binding domain that specifically binds to the transferrin receptor and does not encode another binding domain.

[0149] In some embodiments, the expression vector does not include any other targeting domains or amino acid sequences. In some embodiments of any of the expression vectors described herein, the expression vector includes only the first sequence, the linker, and the second sequence.

[0150] In some embodiments, the expression vectors described herein can include 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 a subject's cells or CNS (e.g., hepatocytes) can be detected by a protein binding assay (e.g., Western blot, immunohistochemistry, radioimmunoassay (RIA)), or chemiluminescence).

[0151] In some embodiments, an expression vector described herein includes one or more (e.g., two, three, four, five, or six) promoters (e.g., any of the promoters described herein or known in the art), enhancers (e.g., any of the enhancers described herein or known in the art), Kozak sequences (e.g., any of the Kozak sequences described herein or known in the art), RNA splicing sequences, polyadenylation (poly(A)) signal sequences (e.g., any of the poly(A) signals described herein), and internal ribosome entry site (IRES) sequences (e.g., any of the IRES sequences described herein or known in the art).

[0152] Methods for Producing Transferrin Receptor Binding Domains, Polypeptides, Antibodies, or Antibody Fragments Also provided herein are methods of producing any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments described herein, comprising: (a) culturing a cell (e.g., any of the cells described herein) comprising any of the nucleic acids encoding any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments described herein, or any of the expression vectors described herein comprising a nucleic acid encoding any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments 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 culture medium. 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 (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 isolated transferrin receptor binding domain, polypeptide, antibody, or antibody fragment into a composition (e.g., a pharmaceutical composition).

[0153] Any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments described herein can be produced by any cell, for example, mammalian cells.Non-limiting examples of mammalian cells include human cells, rodent cells (for example, rat cells or mouse cells), rabbit cells, dog cells, cat cells, pig cells, or non-human primate cells.For example, host cells can be CHO cells or HEK cells.

[0154] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor cell proliferation, cell growth, and / or cell differentiation. For example, cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell culture medium that includes supplemental growth factors that aid in cell viability and cell growth.

[0155] Methods for introducing a nucleic acid (e.g., any of the exemplary nucleic acids described herein) and / or an expression vector (e.g., any of the exemplary expression vectors described herein (e.g., AAV vectors)) into a cell (e.g., a mammalian cell) are known in the art. Non-limiting example methods that can be used to introduce a nucleic acid (e.g., any of the exemplary nucleic acids described herein) and / or an expression vector (e.g., any of the 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).

[0156] Also provided herein are methods that further include isolating the transferrin receptor binding domain, polypeptide, antibody, or antibody fragment from the cell culture medium or cells (e.g., mammalian cells) using techniques well known in the art (e.g., ion exchange chromatography (anionic or cationic), metal affinity chromatography, ligand affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and precipitation (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation).

[0157] cell Also provided herein are cells (e.g., mammalian peripheral cells, mammalian hepatocytes, e.g., human hepatocytes) that contain any of the expression vectors, expression vector pairs, nucleic acids, or nucleic acid pairs described herein. Also provided are cells (e.g., mammalian cells, mammalian hepatocytes, e.g., human hepatocytes) that have been transduced with any of the expression vectors or expression vector pairs described herein, edited using lentiviral or CRISPR technology, or otherwise engineered or modified to express any of the polypeptides or protein complexes described herein. One of skill in the art will understand that the expression vectors and nucleic acids described herein can be introduced into any cell (e.g., any mammalian cell, any hepatocyte), and that a variety of techniques are available for modifying the genome of a cell (e.g., a mammalian cell). Non-limiting examples of expression vectors and methods for introducing expression vectors and nucleic acids into a cell (e.g., any mammalian cell, any hepatocyte, e.g., a human hepatocyte) are described herein.

[0158] 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 hepatic cell. 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 ex vivo. In some embodiments, the cell is in vitro.

[0159] Without further elaboration, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the expression vectors of the present invention and practice the methods set forth in the claims. The following examples are intended to specifically point out various aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0160] Compositions and kits Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the transferrin receptor binding domains, polypeptides, antibodies, antibody fragments, expression vectors, nucleic acids, and cells comprising those described herein. Any of the pharmaceutical compositions may comprise any of the transferrin receptor binding domains, polypeptides, antibodies, or antibody fragments described herein and one or more (e.g., one, two, three, four, or five) pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. In some embodiments, any of the pharmaceutical compositions described herein may comprise one or more buffers (e.g., neutral buffered saline, phosphate buffered saline (PBS)), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more proteins, polypeptides, or amino acids (e.g., glycine), one or more antioxidants, one or more chelating agents (e.g., glutathione or EDTA), one or more preservatives, and / or pharma- ceutically acceptable carriers (e.g., PBS, saline, or bacteriostatic water).

[0161] In some embodiments, any of the pharmaceutical compositions described herein can further comprise one or more (e.g., one, two, three, four, or five) agents (e.g., liposomes or cationic lipids) that facilitate entry of any of the expression vectors or nucleic acids described herein into a cell (e.g., a mammalian cell, a hepatic cell).

[0162] In some embodiments, any of the expression vectors or nucleic acids described herein can be formulated with natural and / or synthetic polymers. Non-limiting examples of polymers that may be included in any of the pharmaceutical compositions described herein can include, but are not limited to, poloxamers, chitosans, dendrimers, and poly(lactic-co-glycolic acid) (PLGA) polymers.

[0163] The pharmaceutical compositions provided herein can be formulated, for example, to suit their intended route of administration. In some embodiments, the compositions are formulated for subcutaneous, intramuscular, intravenous, or intrahepatic administration. In some examples, the compositions comprise a therapeutically effective amount of any of the polypeptides, protein complexes, or expression vectors described herein. Single or multiple doses of any of the pharmaceutical compositions described herein can be given (e.g., administered) to a subject, for example, depending on the frequency and dosage required and tolerated by the subject. The dosage of a pharmaceutical composition comprising any of the polypeptides described herein, any of the protein complexes described herein, or any of the expression vectors described herein should provide an amount sufficient to effectively ameliorate or treat a symptom, condition, or disease.

[0164] Also provided are kits comprising any of the compositions (e.g., pharmaceutical compositions) described herein, including any of the nucleic acids, any of the transferrin receptor binding domains, any of the polypeptides, any of the antibodies, any of the antibody fragments, any of the expression vectors, any of the cells described herein, including those described herein. In some embodiments, the kits can include a solid composition (e.g., a lyophilized composition comprising any of the expression vectors, polypeptides, or protein complexes described herein) and a liquid for solubilizing the lyophilized composition.

[0165] In some embodiments, the kit can include at least one dose of any of the compositions (e.g., any of the pharmaceutical compositions) described herein. In some embodiments, the kit can include a pre-loaded syringe containing any of the pharmaceutical compositions described herein. In some embodiments, the kit can include a vial containing any of the pharmaceutical compositions (e.g., formulated as an aqueous pharmaceutical composition) described herein. In some embodiments, the kit can include instructions for carrying out any of the methods described herein. EXAMPLES

[0166] Example 1: Generation of camelid antibodies against the human transferrin receptor A series of experiments were performed to generate camelid antibodies against human transferrin receptor. In these experiments, Creative Biolabs (Shirley, New York) performed the immunization of llamas, isolation of peripheral blood mononuclear cells (PBMCs), and construction of the VHH phage library. Briefly, llamas were immunized with recombinant human transferrin receptor ("TfR")-apical domain every 3 weeks for 3 months. To confirm the immune response, serum titers against the human TfR apical domain were measured. On the last day of the immunization schedule (after the final serum titer check), PBMCs were isolated and total RNA was purified, and a VHH phage library was constructed in pComb3xss phagemid.

[0167] Phage selection for isolating anti-TfR VHH antibodies A series of experiments were performed to isolate anti-TfR VHH antibodies from a VHH phage library. A first round of phage panning using the VHH immune phage library was performed on human TfR (full length), followed by a second round of phage panning on human TfR apical or cynomolgus monkey ("cynomolgus") TfR apical domains. After the second round of phage panning, TG1 E. coli was infected with the phage eluate and plated overnight to isolate individual E. coli colonies. Phage from individual colonies were grown in 96-well plates by adding M13 helper phage to the culture medium and incubated overnight at 30°C. Phage ELISA was performed on human and cynomolgus TfR (full length), as well as on the human TfR apical domain. Counterscreening was also performed in parallel to remove any non-specific binding to streptavidin. Sanger sequencing was performed to sequence VHHs from clones with positive binding to human and cynomolgus TfR.

[0168] Generation of VHH antibodies The variable heavy chain domain of a TfR binder identified from phage panning of an immunollama library was subcloned onto the Fc domain of human IgG1. The expression plasmid contained a CMV promoter to drive expression and a signal peptide for secretion. The heavy chain plasmid was transfected into Expi293 cells. Supernatants were harvested after 5 days and monoclonal antibodies were purified by Protein A chromatography. Affinity and humanized variants of B07 were subcloned onto the Fc domain of human IgG1. These variants were expressed and purified in the same manner. A select set of B07 and humanized B07 variants were expressed as the VHH domain alone. These variants were also expressed and purified in the same manner.

[0169] Example 2: Cellular binding and uptake experiments after treatment with VHH antibodies A series of experiments were performed to evaluate the cellular binding and uptake of a series of VHH antibodies. In these experiments, HEK293T, CHO:cyTfR, and CHO cells were plated in standard growth medium (DMEM (Gibco™ Catalog No. 11995073) + 10% FBS (VWR Catalog No. 89510-188) + 1X pencillin / streptomycin (Gibco Catalog No. 15140122) in 96-well plates at 40,000 cells / well. After approximately 24 hours, molecules were diluted into standard growth medium that had been warmed to 37°C. Old medium was removed from the cells and the diluted molecules were added to the cells. Cells were incubated at 37°C for 45 minutes. Cells were then washed with PBS and then fixed in 4% PFA (Electron Microscopy Sciences Catalog No. 15714-S) for 10 minutes. Cells were washed with PBS and then fixed in 5% BSA, 0.3% Triton® The cells were blocked with PBS containing 1% BSA, 0.3% Triton® X100 for 30 minutes. The cells were stained with anti-human IgG-488 (1:1000; Jackson Immuno Research Catalog No. 109-545-003), cell mask (1:10,000; Thermo Fisher Catalog No. H32721), and DAPI (1:2000; Thermo Fisher Catalog No. D1306) diluted in PBS containing 1% BSA, 0.3% Triton® X100 for at least 30 minutes. The cells were washed with PBS, imaged with Opera Phenix, and the images were analyzed with Harmony software. Figures 2A and 2B show exemplary hTfR binding curves generated from VHH antibodies generated as described in Example 1.

[0170] Example 3: Evaluation of binding affinity of VHH-Fc fusions A series of experiments were performed to evaluate the binding affinity of VHH-Fc fusions to human and cynomolgus TfR apical domains. In these experiments, binding affinity was determined by surface plasmon resonance using a Biacore™ 8K instrument in 1× HBS-EP+ running buffer (GE Healthcare Cat. No. BR100669). A Biacore™ Series S CM5 sensor chip was treated with a human antibody capture kit (GE Healthcare, Cat. No. BR100839). VHH-Fc fusions were captured on each flow cell, and three-fold serial dilutions of human and cynomolgus TfR apical domains (300, 100, 33, 11, 3.7, and 0 nM) were injected at a flow rate of 30 μL / min using a multi-cycle kinetic method. Each sample was analyzed with 270 s association and 10 min dissociation. Some VHH-Fc fusions were measured using 3-fold serial dilutions (100, 33, 11, and 0 nM) of human and cynomolgus TfR apical domains with 300 s association and 10 min dissociation. After each cycle, the chip was regenerated with 3 M magnesium chloride at 50 μL / min for 30 s. Binding responses were corrected by subtracting the RU from the reference flow cell. on and k off A 1:1 Langmuir model of simultaneous fitting of was used for kinetic analysis using the Biacore™ 8K evaluation software. Table 1 shows the binding of the different VHH clones generated as described in Example 1 (huTfR-AD = human transferrin receptor apical domain; cyTfR-AD = cynomolgus transferrin receptor apical domain). [Table 1]

[0171] Example 4: Generation of affinity variants of B07 To generate affinity variants of B07, selected residues in the CDR regions were mutated with the mutations listed in Table 2 with Kabat number. The variable domains containing the mutations were subcloned onto the corresponding constant heavy domain of human IgG1. The heavy chain plasmids were transfected into Expi293 cells. Supernatants were harvested after 5 days and monoclonal antibodies were purified by protein A chromatography. To evaluate the affinity of B07 variants to human and cynomolgus TfR apical domains, binding affinities were determined by surface plasmon resonance using a Biacore™ 8K instrument as described herein. Table 2 shows the effect of each mutation in B07 on binding affinity to human and cynomolgus TfR apical domains. [Table 2]

[0172] Example 5: Humanization of B07 A series of experiments were performed to evaluate the impact of humanization on the B07 VHH antibody. In these experiments, the B07 VHH was humanized by first aligning the heavy chain variable domain to the human antibody germline consensus framework. The human H3 consensus sequence was identified as having the highest homology to the B07 framework, and therefore the H3 sequence was the starting point for humanization. The B07 CDRs were designed to be grafted into the human H3 consensus framework and to include additional variants in which specific amino acid position combinations were back-mutated to the original llama residues (37F, 44E, 45R, 47F, 73K, 78V; Kabat numbering). All variants were generated as fusions with the human IgG1 Fc domain as previously described. The variants were recombinantly expressed and tested for binding to human and cyno TfR. Table 3 shows the impact of humanization on the B07 VHH antibody. [Table 3]

[0173] hB07v17 was further humanized in the CDR H1 region. Additional humanized variants were generated that included reversion of CDR H1 residues to the human VH3 consensus H1 sequence. Mutations at positions 47 and 52a (Kabat numbering) were also tested. Table 4 provides an overview of the mutations made to hB07v17. All variants were generated as fusions with a human IgG1 Fc domain as described herein. Variants were recombinantly expressed and tested for binding to human and cyno TfR. Table 4 shows the effect of humanization in the CDR H1 region on the binding affinity of hB07v17 to human and cyno TfR. [Table 4]

[0174] Example 6: Production of B07-GBA fusion polypeptide A series of experiments were performed to evaluate fusion polypeptides containing the B07 VHH domain or its variants fused to a glucosylceramidase beta (GBA) polypeptide. In these experiments, the B07 VHH domain or the humanized B07 VHH domain was fused to the N-terminus or C-terminus of human GBA. These constructs were used for in vitro GBA activity experiments. Additional combinations of B07 variants fused to GBA with various linker lengths were also generated.

[0175] 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.

[0176] In the in vitro GBA activity experiments, the GBA polypeptide comprising SEQ ID NO:179 was used as a control.

[0177] In a non-limiting example, a B07-GBA fusion polypeptide was generated. The amino acid sequence of an exemplary B07-PGRN fusion polypeptide includes SEQ ID NO:139.

[0178] In a non-limiting example, a B07-GBA fusion polypeptide was generated with a 2X GGGGS linker between the B07 and GBA domains. The amino acid sequence of an exemplary B07-GBA fusion polypeptide includes SEQ ID NO:140.

[0179] In a non-limiting example, a construct was also made with an N-terminal GBA in the GBA-B07 fusion polypeptide. The amino acid sequence of an exemplary GBA-B07 fusion polypeptide includes SEQ ID NO:141.

[0180] 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.

[0181] 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 includes SEQ ID NO:143.

[0182] 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 includes SEQ ID NO:144.

[0183] Example 7: Production of B07-Progranulin Fusion Polypeptides A series of experiments were performed to evaluate fusion polypeptides containing the B07 VHH domain or variants thereof fused to a granulin precursor (progranulin or PGRN) polypeptide. In these experiments, the B07 domain was fused to the N-terminus of human PGRN with two GGGGS linkers.

[0184] A non-limiting example of a nucleic acid sequence encoding a human Progranulin or Granulin Precursor (PRGN) polypeptide includes SEQ ID NO: 180. A non-limiting example of an amino acid sequence of human PGRN includes SEQ ID NO: 181. In a non-limiting example, a B07-PGRN construct was generated. An exemplary B07-PGRN fusion polypeptide amino acid sequence includes SEQ ID NO: 145.

[0185] In a non-limiting example, a 6xHis-Tev-B07-PGRN construct was produced. The addition of a 6xHis tag allows for purification by nickel chromatography. The amino acid sequence of an exemplary 6xHis-Tev-B07-PGRN fusion polypeptide includes SEQ ID NO:146.

[0186] In a non-limiting example, a B07(N68T)-PGRN construct was generated. The N68T mutation in B07 restores the Protein A binding site, allowing purification by Protein A chromatography. The amino acid sequence of an exemplary B07(N68T)-PGRN fusion polypeptide includes SEQ ID NO:148.

[0187] Other embodiments Although the present invention has been described with reference to its detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0188] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0189] [Table 5-1]

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Claims

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

2. K of about 1 nM to about 6 μM Ｄ 2. The transferrin receptor binding protein of claim 1 , which binds to the human transferrin receptor at

3. K of about 1 nM to about 2.5 mM Ｄ 3. The transferrin receptor binding protein of claim 1 or 2, which binds to the cynomolgus monkey transferrin receptor at

4. A transferrin receptor binding domain comprising: (a) G-X １ -T-X ２ -X ３ -X ４ -X ５ -X ６ -M-X ７ (SEQ ID NO:1), wherein X １ is L or F, and X ２ is S or F, and X ３ is D, S or E, and X ４ is T or S, and X ５ is G or Y, and X ６ is G or A, or X ７ is G or S; (b) A-I-X ８ -X ９ -X １０ -X １１ -X １２ -X １３ -T-X １４ -Y-A-D-S-V-K-G (SEQ ID NO:2), ８ is T or S, and X ９ is W, or S, or G, or F, or Y; X １０ is S or N, and X １１ is G or A, and X １２ is R, S, or G; X １３ is H or A, or X １４ is L or Y; and (c) A-X １５ -D-X 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), １５ is L or R, and X １６ is V or A, and X １７ is V or A, and X １８ is G or A, and X １９ is I or A, and X ２０ is G or A, and X ２１ is I or A, and X ２２ is E or A, and X ２３ is V or A, and X ２４ is Q or A, and X ２５ is T or A, or X ２６ is Y or F.

5. 5. The transferrin receptor binding domain of claim 4, comprising: (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), GLTSSTGMG (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) AITWSGRHTLYADSVKG (SEQ ID NO: 15), AISSWSGRHTLYADSVKG (SEQ ID NO: 16), AITSSGRHTLYADSVKG (SEQ ID NO: 17), AITGSGRHTLYADSVKG (SEQ ID NO: 18), AITWNGRHTLYADSVKG (SEQ ID NO: 19), AITWSGSHTLYADSVKG (SEQ ID NO: 20), AITWSGGGHTL A second sequence selected from the group consisting of AITWSGRHTYYADSVKG (SEQ ID NO:21), AITWSARHTLYADSVKG (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) 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: A third sequence selected from the group consisting of 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), and ALDVVGIGIEVQTFDY (SEQ ID NO:39).

6. 6. The transferrin receptor binding domain of claim 4 or 5, comprising: (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 comprising: (a) G-X １ -T-L-D-X ２ -X ３ -A-I-X ４ (SEQ ID NO: 183), wherein X １ is S or F, and X ２ is D, H, or Y; X is ３ is Y or F, and X ４ is G or A; (b) C-I-S-X ５ -X ６ -D-G-X ７ -T-X ８ -Y-X ９ -D-X １０ -V-K-G (SEQ ID NO: 184), ５ is S or R, and X ６ is S or G, and X ７ is I or R, and X ８ is F or Y, and X ９ is A or G, and X １０ is F or S; and (c) A-X １１ -X １２ -X １３ -G-P-N-X １４ -C-R-G-W-L-W-X １５ -P-X １６ -X １７ -S-G-S (SEQ ID NO: 185), １１ is A or S, and X １２ is K or H, and X １３ is Y or D, and X １４ is I or V, X １５ is V or E, and X １６ is P or Q, and X １７ is V, I, or L (SEQ ID NO: 185).

8. 8. The transferrin receptor binding domain of claim 7, comprising: (a) a first sequence selected from the group consisting of GSTLDDYAIG (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).

9. 9. The transferrin receptor binding domain of claim 7 or 8, comprising: (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 comprising: (a) G-X １ -T-X ２ -X ３ -X ４ -X ５ -X ６ -M-X ７ (SEQ ID NO: 195), wherein X １ is L or F, and X ２ is S or F, and X ３ is D, S or E, and X ４ is T or S, and X ５ is G or Y, and X ６ is G or A, and X ７ is G or S; (b) A-I-X ８ -X ９ -X １０ -X １１ -X １２ -X １３ -T-X １４ -Y-A-D-S-V-K-G (SEQ ID NO:196), ８ is T or S, and X ９ is W, S, G, F, or Y, and X １０ is S or N, and X １１ is G or A, and X １２ is R, S, or G; X １３ is H or A, or X １４ is L or Y; and (c) A-X １５ -D-X 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), １５ is L or R, and X １６ is V or A, and X １７ is V or A, and X １８ is G or A, and X １９ is I or A, and X ２０ is G or A, and X ２１ is I or A, and X ２２ is E or A, and X ２３ is V or A, and X ２４ is Q or A, and X ２５ is T or A, and X ２６ is Y or F.

11. 11. The transferrin receptor binding domain of claim 10, comprising GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199), and ALDVVGIGIEVQTYDY (SEQ ID NO: 200).

12. A transferrin receptor binding domain comprising ESAFSLNAIG (SEQ ID NO:201), GIGTDGITTYYADFVKD (SEQ ID NO:202), and NAGSWRTVLSGTHVSRS (SEQ ID NO:203).

13. A transferrin receptor binding domain comprising GRDYNHFQRA (SEQ ID NO:204), RITWSGTITYNESVKG (SEQ ID NO:205), and ALKTQPPLSQDAGDYTY (SEQ ID NO:206).

14. A transferrin receptor binding domain comprising GSTLDDYAIG (SEQ ID NO: 186), CISSSDGITFYGDFVKG (SEQ ID NO: 189), and AAKYGPNICRGWLWVPPVSGS (SEQ ID NO: 192).

15. Transferrin receptor binding domains comprising GRSLSTYVMG (SEQ ID NO:210), ARNGMSTYYTDSVKD (SEQ ID NO:211), and AGDRSWSRLLRGEYEY (SEQ ID NO:212).

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

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

18. A transferrin receptor binding domain comprising: GLTSDTGGMG (SEQ ID NO: 198), AITWSGRHTLYADSVKG (SEQ ID NO: 199), and ALDVVGIGIEVQTYDY (SEQ ID NO: 200).

19. The transferrin receptor binding domain according to any one of claims 1 to 18, which 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. 22. The polypeptide of claim 21, further comprising a fusion partner.

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

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

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

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

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

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

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

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

32. 32. The antibody of claim 31, which is humanized.

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

34. 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. 35. The antibody of claim 33 or 34, wherein the fusion partner is immediately 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. 38. The antibody fragment of claim 37 comprising a VHH domain.

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

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

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

44. 1. A composition comprising: (i) a transferrin receptor binding domain according to any one of claims 1 to 20; a polypeptide according to any one of claims 21 to 30; an antibody according to any one of claims 31 to 36; or an antibody fragment according to any one of claims 37 to 43; and (ii) a pharma- ceutically acceptable carrier.

45. 45. A kit comprising the composition of claim 44.

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

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

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

49. 1. A method for producing a transferrin receptor binding domain, polypeptide, antibody, or antibody fragment, 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; (b) harvesting the transferrin receptor binding domain, the polypeptide, the antibody, or the antibody fragment from the host cell or the culture medium.

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

51. 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 that is at least 80% identical to any one of SEQ ID NOs: 40-88, 147, and 210-215.

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

54. 54. The transferrin receptor binding domain of claim 53, comprising a sequence which is any one of SEQ ID NOs: 40-88, 147, and 210-215.

55. A transferrin receptor binding domain according to any one of claims 52 to 54, which is a VHH domain.

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

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

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

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

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

62. 62. The polypeptide of any one of claims 57 to 61 which is a single polypeptide.

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

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

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

66. 62. A polypeptide according to any one of claims 57 to 61 which is part of a protein complex.

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

68. 68. The antibody of claim 67, which is humanized.

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

70. 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. 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. 71. The antibody of claim 69 or 70, wherein the fusion partner is immediately 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. 74. An antibody fragment according to claim 73, comprising a VHH domain.

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

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

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

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

81. 81. A kit comprising the composition of claim 80.

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

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

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

85. 1. A method for producing a transferrin receptor binding domain, polypeptide, antibody, or antibody fragment, 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; (b) harvesting the transferrin receptor binding domain, the polypeptide, the antibody, or the antibody fragment from the host cell or the culture medium.

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

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

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