Anti-transferrin receptor antibodies and uses thereof

Abstract

The present disclosure provides anti-transferrin receptor antibodies, compositions comprising same, and methods of using them. The present disclosure also provides polynucleotides and vectors encoding the anti-transferrin receptor antibodies, as well as cells comprising them, methods of making the antibodies, and molecules comprising the antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 239,630, filed September 1, 2021, and U.S. Provisional Application No. 63 / 388,088, filed July 11, 2022. The contents of the foregoing applications are incorporated herein by reference in their entireties.

[0002] The present disclosure relates to anti-transferrin receptor antibodies, compositions comprising same and uses thereof. The present disclosure also provides related polynucleotides and vectors encoding the anti-transferrin receptor antibodies, and cells containing them. [Background technology]

[0003] Transferrin receptor (TfR) is a cell membrane-bound glycoprotein that is involved in the regulation of intracellular iron uptake and cell proliferation. Iron uptake occurs through internalization of iron-loaded transferrin (Tf), which is mediated by interaction with TfR. Transferrin receptor 1 (TfR1), also known as CD71 or p90, is a ubiquitously expressed high-affinity receptor that plays an important role in muscle development and denervation-induced muscle atrophy (Li Y, et al. Neural Regen Res 2021;16:1308-16). The widespread presence of TfR in muscle tissues may allow drug delivery to muscle in vivo by targeting TfR (Schnyder A, et al. Biochem J. 2004 Jan 1;377(Pt 1):61-7). TfR can be targeted by monoclonal antibodies specific for the extracellular domain of the receptor (Daniels TR et al., Clin Immunol. 2006 Nov;121(2):144-58). Targeting muscle brings some unique challenges, both for uptake in muscle and limited uptake in other tissues: muscle tissue is considered a difficult tissue for drug delivery because the vascular endothelium in muscle acts as a barrier to drug uptake (Ebner DC et al., Current pharmaceutical design. 21.10.2174 / 1381612820666140929095755 and US20190240346). Therefore, there is a need to develop more efficient drug delivery methods to muscle, such as new and improved anti-TfR antibodies that can be used to treat various muscle-related diseases, including muscular dystrophies and lysosomal storage diseases. In addition, such antibodies can also be used in gene therapy for non-muscle disorders. Due to the widespread expression of TfR1 in non-muscle tissues, such as liver cells, tumor cells, immune cells, and endothelial cells of the blood-brain barrier, there is also a need to develop antibodies that target these tissues for drug delivery and / or therapeutic purposes. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Publication No. 20190240346 [Non-patent literature]

[0005] [Non-Patent Document 1] Li Y,et al.Neural Regen Res 2021;16:1308-16 [Non-Patent Document 2] Schnyder A,et al.Biochem J.2004 Jan 1;377(Pt 1):61-7 [Non-Patent Document 3] Daniels TR et al., Clin Immunol.2006 Nov;121(2):144-58 [Non-Patent Document 4] Ebner DC et al.,Current pharmaceutical design.21.10.2174 / 1381612820666140929095755 Summary of the Invention

[0006] The present disclosure relates to anti-transferrin receptor antibodies and methods of use thereof for treating muscular disorders, arteriosclerotic diseases, cardiac-related diseases, and lysosomal storage diseases. The antibodies of the present disclosure may also be used to target brain tissue, liver tissue, tumor cells, and immune cells.

[0007] In a first aspect, the disclosure features an antibody that binds to the human transferrin receptor, comprising a heavy chain variable region (VH) comprising a VH complementarity Determining Region (CDR) 1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence GIDFSSSGYMX (SEQ ID NO: 149), where X is any amino acid, VH CDR2 comprises the amino acid sequence XIYTYSSNTYYAXXXKG (SEQ ID NO: 151), where X is any amino acid, and VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106); and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), and VL CDR2 comprises the amino acid sequence RASXLXS (SEQ ID NO: 153), where X is any amino acid, and CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO:110).

[0008] In some embodiments, the VH CDR1 comprises the amino acid sequence GIDFSSSGYMX 1 (SEQ ID NO: 150), where X 1 is C, A, or H, and VH CDR2 is the amino acid sequence X 2 IYTYSSNTYYAX 3 X 4 X 5 KG (SEQ ID NO: 152), where X 2 is C or A, and X3 is S or A, and X 4 is W or S, and X 5 is A or V, VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), and VL CDR2 comprises the amino acid sequence RASX 6 LX 7 S (SEQ ID NO: 154), where X 6 is T or S, and X 7 is A or E, and VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110). In some embodiments, VH-CDR1, VH-CDR2, and VH-CDR3 each correspond to the VH CDRs of a single VH clone shown in Figure 1A, and VL-CDR1, VL-CDR2, and VL-CDR3 each correspond to the VL CDRs of a single VL clone shown in Figure 1C. In some embodiments, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 each correspond to the VH CDRs and VL CDRs of a single clone shown in Table 1A.

[0009] In some embodiments, (a) the VH CDR1 comprises the amino acid sequence GIDFSSSGYMC (SEQ ID NO: 101), the VH CDR2 comprises the amino acid sequence CIYTYSSNTYYASWAKG (SEQ ID NO: 104), the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASTLAS (SEQ ID NO: 109), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110); (b) the VH CDR1 comprises the amino acid sequence GIDFSSSGYMH (SEQ ID NO: 102), the VH CDR2 comprises the amino acid sequence AIYTYSSNTYYASWAKG (SEQ ID NO: 105), the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), and the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASTLAS (SEQ ID NO: 109), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110). (c) the VH CDR1 comprises the amino acid sequence GIDFSSSGYMC (SEQ ID NO: 101), the VH CDR2 comprises the amino acid sequence CIYTYSSNTYYAASVKG (SEQ ID NO: 103), and the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASSLES (SEQ ID NO: 108), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110), or (d) the VH CDR1 comprises the amino acid sequence GIDFSSSGYMC (SEQ ID NO: 101), VH CDR2 comprises the amino acid sequence CIYTYSSNTYYASWAKG (SEQ ID NO: 104), VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), VL CDR2 comprises the amino acid sequence RASSLES (SEQ ID NO: 108), and VLCDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO:110).

[0010] In some embodiments, (i) the VH is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 12, or 15-18, and (ii) the VL is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 35-37. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 4, 12, or 15-18, and the VL comprises the amino acid sequence of SEQ ID NOs: 34-37.

[0011] In some embodiments, (a) the VH comprises the amino acid sequence of SEQ ID NO:4, and the VL comprises the amino acid sequence of SEQ ID NO:34; (b) the VH comprises the amino acid sequence of SEQ ID NO:12, and the VL comprises the amino acid sequence of SEQ ID NO:35; (c) the VH comprises the amino acid sequence of SEQ ID NO:15, and the VL comprises the amino acid sequence of SEQ ID NO:35; (d) the VH comprises the amino acid sequence of SEQ ID NO:16, and the VL comprises the amino acid sequence of SEQ ID NO:35; (e) the VH comprises the amino acid sequence of SEQ ID NO:17, and the VL comprises the amino acid sequence of SEQ ID NO:35; or (f) the VH comprises the amino acid sequence of SEQ ID NO:18, and the VL comprises the amino acid sequence of SEQ ID NO:35.

[0012] In a second aspect, the disclosure features an antibody that binds to the human transferrin receptor and competes for binding to the human transferrin receptor with an antibody comprising: (a) a VH comprising the amino acid sequence of SEQ ID NO:4 and a VL comprising the amino acid sequence of SEQ ID NO:34; (b) a VH comprising the amino acid sequence of SEQ ID NO:12 and a VL comprising the amino acid sequence of SEQ ID NO:35; (c) a VH comprising the amino acid sequence of SEQ ID NO:15 and a VL comprising the amino acid sequence of SEQ ID NO:35; (d) a VH comprising the amino acid sequence of SEQ ID NO:16 and a VL comprising the amino acid sequence of SEQ ID NO:35; (e) a VH comprising the amino acid sequence of SEQ ID NO:17 and a VL comprising the amino acid sequence of SEQ ID NO:35; or (f) a VH comprising the amino acid sequence of SEQ ID NO:18 and a VL comprising the amino acid sequence of SEQ ID NO:35.

[0013] In a third aspect, the disclosure features an antibody that binds to the human transferrin receptor, comprising a heavy chain variable region (VH) comprising a VH complementarity Determining Region (CDR) 1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence GFSFSNSYWIX (SEQ ID NO: 155), where X is any amino acid, VH CDR2 comprises the amino acid sequence XINTDADSTNYAXXXXG (SEQ ID NO: 157), where X is any amino acid, and VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119); the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence XASQNIGSNLA (SEQ ID NO: 159), and VL CDR2 comprises the amino acid sequence DASKLXS (SEQ ID NO: 161), where X is any amino acid, CDR3 comprises the amino acid sequence QXTVRGGAYGXA (SEQ ID NO: 163), where X is any amino acid.

[0014] In some embodiments of the third aspect, the VH CDR1 has the amino acid sequence GFSFSNSYWIX 1 (SEQ ID NO: 156), where X 1 is C, A, or H, and VH CDR2 is the amino acid sequence X 2 INTDADSTNYAX 3 X 4 X 5 X 6 G (SEQ ID NO: 158), where X 2 is C or A, and X 3 is S or D, and X 4 is W or S, and X 5 is A or V, and X 6 is R or K, VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), and VL CDR1 comprises the amino acid sequence X 7 ASQNIGSNLA (SEQ ID NO: 160), where X 7 is Q or R, and VL CDR2 has the amino acid sequence DASKLX 8 S (SEQ ID NO: 162), where X8 is A or E, and VL CDR3 has the amino acid sequence QX 9 TVRGGAYGX 10 A (SEQ ID NO: 164), where X 9 is C, Q, A, S, T, or V, and X 10 is N or L.

[0015] In some embodiments of the third aspect, VH-CDR1, VH-CDR2, and VH-CDR3 each correspond to the VH CDRs of a single VH clone shown in Figure 1B, and VL-CDR1, VL-CDR2, and VL-CDR3 each correspond to the VL CDRs of a single VL clone shown in Figure ID. In some embodiments of the third aspect, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 each correspond to the VH CDRs and VL CDRs of a single clone shown in Table 2A.

[0016] In some embodiments of the third aspect, (a) VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), VL CDR1 comprises the amino acid sequence QASQNIGSNLA (SEQ ID NO: 121), VL CDR2 comprises the amino acid sequence DASKLAS (SEQ ID NO: 123), and VL CDR3 comprises the amino acid sequence QCTVRGGAYGNA (SEQ ID NO: 124); (b) VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), VH CDR2 comprises the amino acid sequence CINTDADSTNYADSVKG (SEQ ID NO: 118), VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), and VL CDR1 comprises the amino acid sequence QASQNIGSNLA (SEQ ID NO: 121), (c) VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), and VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and VL CDR3 comprises the amino acid sequence QCTVRGGAYGNA (SEQ ID NO: 124), (d) VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), and VH (e) VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and VL CDR3 comprises the amino acid sequence QSTVRGGAYGNA (SEQ ID NO: 125);wherein CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and VL CDR3 comprises the amino acid sequence QTTVRGGAYGNA (SEQ ID NO: 126); or (f) VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), and VL CDR3 comprises the amino acid sequence QTTVRGGAYGNA (SEQ ID NO: 126). CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122) and VL CDR3 comprises the amino acid sequence QVTVRGGAYGNA (SEQ ID NO: 127).

[0017] In some embodiments of the third aspect, (i) the VH is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 30-33, and (ii) the VL is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 41-44. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 30-33, and the VL comprises the amino acid sequence of any one of SEQ ID NOs: 41-44.

[0018] In some embodiments of the third aspect, (a) the VH comprises the amino acid sequence of SEQ ID NO: 19, and the VL comprises the amino acid sequence of SEQ ID NO: 38, (b) the VH comprises the amino acid sequence of SEQ ID NO: 30, and the VL comprises the amino acid sequence of SEQ ID NO: 41, (c) the VH comprises the amino acid sequence of SEQ ID NO: 31 and the VL comprises the amino acid sequence of SEQ ID NO: 41, (d) the VH comprises the amino acid sequence of SEQ ID NO: 32 and the VL comprises the amino acid sequence of SEQ ID NO: 41, (e) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 41, (f) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 42, (g) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 43, or (h) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 44.

[0019] In a fourth aspect, the disclosure features an antibody that binds to the human transferrin receptor and competes for binding to the human transferrin receptor with an antibody comprising: (a) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 38; (b) a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (c) a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (d) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (e) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (f) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 42; (g) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (h) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 44.

[0020] In some embodiments of any of the above aspects, the antibody is a Fab fragment or a Fab' fragment. In some embodiments, the antibody is a bispecific antibody, a single chain antibody, a Fab fragment, a F(ab') 2Fragment, Fab' fragment, Fsc fragment, Fv fragment, scFv, sc(Fv) 2 , or a diabody.

[0021] In some embodiments, the antibody comprises a constant heavy chain (CH) domain and a constant light chain (CL) domain. In some embodiments, the CH domain comprises a CH1 domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 45-52. In some embodiments, the CH1 domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 45, 47, and 49. In some embodiments, the CH1 domain is fused to a hinge comprising the amino acid sequence set forth in any one of SEQ ID NOs: 53-55 and 57. In some embodiments, the CH1 domain comprises the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the CH1 domain is fused to a hinge comprising the amino acid sequence ES or the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the CH1 domain is fused to a hinge comprising the amino acid sequence ES, ESK, or the amino acid sequence set forth in any one of SEQ ID NOs: 53-90. In some embodiments, the CL domain comprises the amino acid sequence set forth in SEQ ID NO: 91 or 92.

[0022] In some embodiments, the antibody comprises (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 93 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 94; (ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 95 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96; (iii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 97 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96; (iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96; or (v) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0023] In another aspect, the disclosure features an antibody that binds to the human transferrin receptor at an epitope located in whole or in part within amino acids K231-E369 of SEQ ID NO: 1. In some embodiments, the antibody binds to one or more residues selected from the group consisting of residues K231, D245, L246, Y247, T248, P249, E350, G351, D352, C353, P354, S355, D356, K358, T359, D360, S361, R364, M365, V366, T367, and E369 of SEQ ID NO:1. In some embodiments, the antibody binds to one or more amino acids of SEQ ID NO:1 selected from the group consisting of K231, D245 to P249, E244 to V250, E350 to S361, M349 to T362, R364 to E369, and C363 to S370.

[0024] In another aspect, the disclosure features an antibody that binds to the human transferrin receptor at an epitope located in whole or in part within amino acids D139 to K145 and G490 to E582 of SEQ ID NO: 1. In some embodiments, the antibody binds to one or more residues selected from the group consisting of residues D139, T141, K145, G490, T491, V517, T518, Y573, K574, I577, E578, R579, I580, P581, and E582 of SEQ ID NO:1. In some embodiments, the antibody binds to one or more amino acids of any of the following sequences: D139-E369, D139-E582, T141-E369, T141-E582, K145-E369, K145-E582, K231-E369, K231-E582, D245-E369, D245-E582, E350-E369, E350-E582, K358-E369, K358-E582, R364-E369, R364-E582, G490-E582, V517-E582, K573-E582, I577-E582, or Y573-E582 of SEQ ID NO:1. In some embodiments, the antibody binds to one or more amino acids of any of the following sequences: D139 to T141, D139 to K145, T141 to K145, T138 to K145, T138 to L146, G490 to T491, L489 to T491, L489 to S492, V517 to T518, P516 to T518, P516 to G519, Y573 to E582, Y573 to L583, T572 to E582, or T572 to L583 of SEQ ID NO:1.

[0025] In another aspect, the disclosure features a nucleic acid(s) encoding the antibody of any one of the above aspects.In yet another aspect, the disclosure features an expression vector(s) comprising the nucleic acid(s) operably linked to a promoter.In another aspect, the disclosure features an isolated cell comprising the nucleic acid(s) or expression vector(s).

[0026] In another aspect, the disclosure features an isolated cell comprising a first expression vector comprising a first nucleic acid encoding a first polypeptide comprising a VH of an antibody of the disclosure operably linked to a promoter, and a second expression vector comprising a second nucleic acid encoding a second polypeptide comprising a VL of an antibody of the disclosure operably linked to a promoter.

[0027] In yet another aspect, the disclosure features a method of making an antibody of the disclosure, comprising culturing a cell of the disclosure and isolating the antibody. In another aspect, the disclosure features a pharmaceutical composition comprising an antibody of the disclosure and a pharma- ceutically acceptable carrier. In another aspect, the disclosure features a conjugate comprising an antibody of the disclosure and an agent.

[0028] In some embodiments, the agent is a nucleic acid. In some embodiments, the nucleic acid is an mRNA, an siRNA, an antisense oligonucleotide, a microRNA (miRNA), a guide RNA (gRNA), or a phosphoramidate morpholino oligomer (PMO). In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiments, the agent is linked to the antibody via a linker.

[0029] In another aspect, the disclosure features an in vivo drug delivery method, the method comprising administering to a human subject a conjugate of the disclosure. In some embodiments, (a) the human subject has a muscle disease, muscle atrophy, arteriosclerotic disease, heart-related disease, or lysosomal storage disease, and the method delivers the drug to muscle tissue, (b) the human subject has a neurological disorder (e.g., brain disease), and the method delivers the drug to brain tissue, (c) the human subject has a liver disease, and the method delivers the drug to liver cells, (d) the human subject has cancer, and the method delivers the drug to tumor cells, or (e) the human subject has an immune disorder, and the method delivers the drug to immune cells. [Brief description of the drawings]

[0030] [Figure 1A] This is an alignment of VH sequences of the ANTIBODY-A anti-TfR1 antibody family with union CDRs (underlined). [Figure 1B] This is an alignment of VH sequences of the ANTIBODY-B anti-TfR1 antibody family with union CDRs (underlined). [Figure 1C] This is an alignment of VL sequences of the ANTIBODY-A anti-TfR1 antibody family with union CDRs (underlined). [Figure 1D] This is an alignment of VL sequences of the ANTIBODY-B anti-TfR1 antibody family with union CDRs (underlined). [Figure 2A] VH sequence alignment of the ANTIBODY-A anti-TfR1 antibody family with Chothia CDRs (underlined). [Figure 2B] VH sequence alignment of the ANTIBODY-B anti-TfR1 antibody family with Chothia CDRs (underlined). [Figure 2C] VL sequence alignment of the ANTIBODY-A anti-TfR1 antibody family with Chothia CDRs (underlined). [Figure 2D] VL sequence alignment of the ANTIBODY-B anti-TfR1 antibody family with Chothia CDRs (underlined). [Diagram 3] Atomic model of humanized ANTIBODY-A Fab bound to the human transferrin receptor in the presence of the endogenous ligand, transferrin, integrated into cryo-EM density at 3.4 Å resolution. [Figure 4]Partial alignment of the transferrin receptor apical domain sequence with the ANTIBODY-A epitope. Residues within 5 Å of the ANTIBODY-A Fab observed in the cryo-EM structure of ANTIBODY-A bound to the human transferrin receptor ectodomain are highlighted in grey. Epitope residues that differ from the human transferrin receptor are in bold. Strictly conserved residues are indicated by asterisks, strongly conserved residues by colons, and moderately conserved residues by periods. Sequence source: human (Homo sapiens, Uniprot P02786.2), cynomolgus monkey (Macaca fascicularis, Uniprot G8F602), mouse (Mus musculus, GenBank NP_035768.1). [Diagram 5] Atomic model of humanized ANTIBODY-B Fab bound to the human transferrin receptor in the presence of the endogenous ligand, transferrin, integrated into cryo-EM density at 3.96 Å resolution. [Figure 6] Partial alignment of the transferrin receptor protease-like domain sequence with the ANTIBODY-B epitope. Residues within 5 Å of ANTIBODY-B, as observed in the cryo-EM structure of ANTIBODY-B bound to the cyno transferrin receptor ectodomain, are highlighted in grey. Epitope residues that differ from the human transferrin receptor are in bold. Strictly conserved residues are indicated by asterisks, strongly conserved residues by colons, and moderately conserved residues by periods. Sequence source: human (Homo sapiens, Uniprot P02786.2), cynomolgus monkey (Macaca fascicularis, Uniprot G8F602), mouse (Mus musculus, GenBank NP_035768.1). [Figure 7A] 1 depicts antibody structures corresponding to Protein Index 1 ("A"), Protein Index 2 ("B"), Protein Index 3 ("C"), and Protein Index 4 ("D"). [Figure 7B]1 depicts the structures of antibodies corresponding to protein indexes 5, 6, 7, 8, 9, 10, 11, and 12. [Figure 7C] 1 depicts the structures of antibodies corresponding to protein indices 13, 14, 15, 16, 17, 18, 19, and 20. [Figure 7D] 1 depicts the structure of the antibody corresponding to protein index 21. [Figure 7E] 1 depicts the structure of the antibody corresponding to protein index 22. [Figure 8A] 1 is a graph depicting antibodies identified by protein indexes 1, 2, 3, and 4 binding to the cynomolgus monkey transferrin receptor (cyTfR). [Figure 8B] 1 is a graph depicting antibodies identified by protein indexes 1, 2, 3, and 4 binding to the human transferrin receptor (huTfR). [Figure 9A] 1 is a graph depicting the exclusion of transferrin by antibodies identified by protein indexes 1 and 2 that bind to the human transferrin receptor. [Figure 9B] 1 is a graph depicting the exclusion of transferrin by antibodies identified by protein indexes 3 and 4 that bind to the human transferrin receptor. [Figure 10] 1 is a graph depicting the exclusion of transferrin by antibodies identified by protein index 3 ("x") and protein index 14 (triangles) binding to the human transferrin receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present disclosure provides antibodies that specifically bind to transferrin receptor 1 (TfR1). Related polypeptides, polynucleotides, vectors, cells, compositions and conjugates that include the antibodies, methods of making the antibodies, and methods of delivering the compositions and conjugates are also provided. The present disclosure also provides methods of using the novel anti-TfR antibodies.

[0032] definition Unless otherwise defined herein, technical and scientific terms used in this description have the meanings commonly understood by those skilled in the art. For the interpretation of this specification, the following terminology explanations apply, and whenever appropriate, terms used in the singular form also include the plural form and vice versa. If any explanation of the described terms contradicts any document incorporated herein by reference, the explanation of the terms below shall prevail.

[0033] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and binds to a target through at least one antigen-binding site. "Antibody" is used in the broadest sense herein and encompasses various antibody structures, including "antibody fragments" and "antigen-binding fragments." Thus, the term "antibody" includes, but is not limited to, recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single-chain Fv (scFv) antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0034] The term "intact antibody" or "full length antibody" refers to an antibody having a structure substantially similar to a native antibody structure, including, for example, an antibody that contains two light chains (each containing a variable region and a light chain constant region (CL)) and two heavy chains (each containing a variable region and at least the heavy chain constant regions CH1, CH2, and CH3 and a hinge region between the CH1 and CH2 regions).

[0035] As used herein, the term "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an antibody and an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single chain antibody molecules (e.g., scFv, sc(Fv) 2), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antibody fragments.

[0036] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous antibody population involved in highly specific recognition and binding to a single antigenic determinant or epitope. The term "monoclonal antibody" includes intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')). 2 , Fv), single chain antibodies (e.g., scFv), fusion proteins comprising antibody fragments, and any other modified immunoglobulin molecule that comprises at least one antigen binding site. Furthermore, "monoclonal antibody" refers to such antibodies produced by any number of techniques, including, but not limited to, hybridoma production, phage display libraries, recombinant expression, and transgenic animals.

[0037] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a first source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0038] As used herein, the term "humanized antibody" refers to an antibody comprising a human heavy chain variable region and a light chain variable region, in which naturally occurring CDR amino acid residues have been replaced by corresponding CDR residues from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), which non-human antibody has a desired specificity, affinity, and / or activity. In some embodiments, one or more framework region amino acid residues of the human heavy chain variable region or the human light chain variable region have been replaced by the corresponding residue from the non-human antibody. Furthermore, a humanized antibody may contain amino acid residues that are not found in a human antibody or a non-human antibody. In some embodiments, these modifications are made to further refine and / or optimize antibody properties. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (e.g., CH1, hinge, CH2, CH3, Fc), typically that of a human immunoglobulin.

[0039] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence that corresponds to an antibody produced by a human and / or an antibody that has been made using any of the techniques for making human antibodies known to those of skill in the art, including, but not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma techniques.

[0040] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen or target capable of being recognized and bound by a particular antibody. When the antigen or target is a polypeptide, epitopes can be formed from both adjacent amino acids and non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids (also called linear epitopes) are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding (also called conformational epitopes) are usually lost upon protein denaturation. Epitopes usually comprise at least three, and more commonly at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a number of software bioinformatics tools available on the internet. X-ray crystallography or electron microscopy (e.g., cryo-electron microscopy) may be used to characterize epitopes on target proteins by analyzing amino acid residue interactions of antigen / antibody complexes.

[0041] As used herein, the term "specifically binds" or "binds" refers to an antibody that interacts with a particular antigen, epitope, protein, or target molecule more frequently, more rapidly, more persistently, with higher affinity, or some combination of the above, than with another substance. An antibody that specifically binds to an antigen can be identified, for example, by immunoassay, ELISA, surface plasmon resonance (SPR), or other techniques known to those skilled in the art. In some embodiments, an antibody that specifically binds to an antigen (e.g., human TfR1) can bind to a related antigen (e.g., cyno TfR1). An antibody that specifically binds to an antigen can bind to a target antigen with a higher affinity than its affinity for a different antigen. The different antigen can be a related antigen. In some embodiments, an antibody that specifically binds to an antigen can bind to a target antigen with an affinity that is at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, or at least 100 times higher than its affinity for a different antigen. In some embodiments, an antibody that specifically binds to a particular antigen binds to a different antigen with such low affinity that the binding cannot be detected using the assays described herein or other methods known in the art. In some embodiments, affinity is measured using SPR technology in a Biacore system as described herein or known to those skilled in the art.

[0042] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that are modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, or any other manipulation or modification). Also included in this definition are polypeptides that contain one or more amino acid analogs, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies, the term "polypeptide" is understood to include polypeptides as a single chain as well as polypeptides of two or more associated chains.

[0043] The terms "polynucleotide" as well as "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0044] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are identical or have a certain percentage of identical nucleotides or amino acid residues when compared and aligned (optionally introducing gaps) to maximize correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the disclosure are substantially identical, meaning that they, when compared and aligned for maximum correspondence, have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a sequence region that is at least about 10, at least about 20, at least about 20-40, at least about 40-60 nucleotides or amino acid residues, at least about 60-80 nucleotides or amino acid residues in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 nucleotides or amino acid residues, e.g., over at least about 80-100 nucleotides or amino acid residues, and in some embodiments the sequences are substantially identical over the entire length of the sequences being compared, e.g., (i) the coding region of a nucleotide sequence or (ii) the entire length of an amino acid sequence.

[0045] As used herein, the phrase "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue with a similar side chain. A family of amino acid residues with similar side chains is generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of phenylalanine with tyrosine is considered to be a conservative substitution. Generally, conservative substitutions in a polypeptide and / or antibody sequence do not abrogate binding of the polypeptide or antibody to its target binding site, and methods for identifying conservative nucleotide and amino acid substitutions that do not abolish binding are well known in the art.

[0046] As used herein, the term "vector" refers to a construct capable of delivering and typically expressing one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors embedded in liposomes.

[0047] As used herein, the term "isolated" refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An "isolated" antibody is substantially free of material from the donor cell from which it is derived. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is one that has been purified to the extent that it is no longer in the form in which it is found in nature. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure. A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition may be isolated from a natural source (e.g., tissue) or from a source such as an engineered cell line.

[0048] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0049] As used herein, the term "pharmaceutical acceptable" refers to a substance that is approved or approvable by a regulatory agency, or that is listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, including humans.

[0050] As used herein, the term "pharmaceutically acceptable excipient, carrier, or adjuvant" refers to an excipient, carrier, or adjuvant that may be administered to a subject together with at least one antibody of the present disclosure and that is generally safe, non-toxic, and does not affect the pharmacological activity of the therapeutic agent. Generally, those skilled in the art and the US FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.

[0051] As used herein, the term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of an antibody to be effective. A pharmaceutical formulation or composition generally includes additional components, such as, for example, pharma- ceutical acceptable excipients, carriers, adjuvants, buffers, and the like.

[0052] As used herein, the term "conjugate" refers to a combination in which two substances are covalently linked (e.g., an antibody of the present disclosure connected to a therapeutic agent). In a conjugate, the two substances may be directly linked or linked via a linker. In the present disclosure, one of the two substances is an antibody of the present disclosure, and the other is a drug (e.g., a bioactive agent). The linker may be a cleavable linker or a non-cleavable linker.

[0053] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an antibody of the disclosure required to reach a target tissue, or the amount of a conjugate, fusion protein or fusion polypeptide, or complex comprising an antibody of the disclosure and a therapeutic agent sufficient to (i) reduce and / or ameliorate a disease, disorder, or condition in a subject, and / or (ii) the severity and / or duration of symptoms in a subject. The term also encompasses the amount of a conjugate required to (i) reduce or ameliorate the development or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, onset, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic effect(s) or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the conjugates provided herein).

[0054] As used herein, the term "therapeutic effect" refers to the effect and / or ability of an agent (e.g., an antibody, conjugate, fusion protein or polypeptide of the disclosure, or a complex comprising an antibody) to (i) reduce and / or ameliorate a disease, disorder, or condition in a subject, and / or (ii) the severity and / or duration of symptoms in a subject. The term also encompasses the ability of an agent (e.g., a conjugate) to (i) reduce or ameliorate the development or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, onset, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic effect(s) or therapeutic effect(s) of another agent or therapy (e.g., an agent other than a conjugate provided herein).

[0055] As used herein, reference to a value or parameter with "about" or "approximately" includes (and describes) embodiments directed to that value or parameter. For example, a reference to "about X" includes the reference to "X." "About X" means + / - 10% of X. Thus, "about 10" means a value between 9 and 11.

[0056] TfR1 and anti-TfR1 antibodies The transferrin receptor, also known as CD71, is a transmembrane glycoprotein expressed at different levels in various sites of the human body, whose function is to mediate the uptake of iron from the plasma glycoprotein, transferrin, into cells. Iron uptake from transferrin requires binding of transferrin to the transferrin receptor, internalization of transferrin in endocytic vesicles by receptor-mediated endocytosis, and release of iron from the protein by lowering the pH of the endosome. Ponka P, Lok CN.. Int J Biochem Cell Biol.1999 Oct;31(10):1111-37 and Xiaopeng Mo,in Brain Targeted Drug Delivery System,2019. TfR is highly expressed in muscle (e.g., cardiac muscle and gastrocnemius). Apotransferrin (i.e., non-iron conjugate) binds to TfR when it binds to two iron 3+ ions to form holotransferrin (i.e., iron conjugate). The complex of TfR and holotransferrin is transported into cells by receptor-mediated endocytosis. CD71 and transferrin dissociate in the endosomal environment, transferrin is transported into cells, and CD71 is recycled to the cell membrane. Therefore, it is believed that transferrin is transported into cells by appropriately binding to TfR and dissociating appropriately. The transferrin receptor system has been utilized to deliver anticancer drugs, proteins, and therapeutic genes into malignant cells, and to deliver other therapeutic agents to the brain through the blood-brain barrier.

[0057] Two transferrin receptors, TfR1 and TfR2, have been characterized in humans and cynomolgus monkeys. TfR1 is a ubiquitously expressed high affinity receptor, whereas TfR2 expression is restricted to certain cell types and is not affected by intracellular iron concentration. TfR2 binds to transferrin with 25-30 times lower affinity than TfR1. The antibodies of the present disclosure bind to TfR1.

[0058] Representative amino acid (aa) sequences of human TfR1 (UniProt No. P02786.2), cyno TfR1 ("cyno" TfR1 (Genbank No. XP_005545315.1), and mouse TfR1 (Genbank No. NP_035768.1) are set forth herein as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. As used herein, references to amino acid positions in TfR1 refer to the numbering of the amino acid sequence including the signal sequence. The sequences of human TfR1, cyno TfR1, and mouse TfR1 are as follows: Human TfR1 (UniProt No. P02786.2, SEQ ID NO: 1) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF Cyno TfR1 (UniProt No. G8F602, Accession No. 2) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDDEENADNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF Mouse TfR1 (Genbank No. NP_035768.1, SEQ ID NO: 3) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPQTISRAAAEKLFGKMEGSCPARWNIDSSCLELSQNQNVKLIVKN VLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF

[0059] The present disclosure provides antibodies that bind to TfR1. In some embodiments, the anti-TfR1 antibodies bind to a TfR1 fragment. In some embodiments, the anti-TfR1 antibodies bind to the extracellular domain of TfR1. In some embodiments, the anti-TfR1 antibodies bind to the protease-like domain, the helical domain, and / or the apical domain(s) of TfR1. In some embodiments, the anti-TfR1 antibodies bind to a conformational epitope on TfR1. In some embodiments, the anti-TfR1 antibodies bind to a conformational epitope located in the apical domain of TfR1. In some embodiments, the anti-TfR1 antibodies bind to a conformational epitope located in the protease-like domain of TfR1. In some embodiments, the anti-TfR1 antibodies bind to a conformational epitope located in the helical domain of TfR1. In some embodiments, the anti-TfR1 antibodies bind to a conformational epitope located in the helical domain of TfR1. In some embodiments, the anti-TfR1 antibodies bind to human TfR1. In some embodiments, the anti-TfR1 antibody binds to cyno TfR1. In some embodiments, the anti-TfR1 antibody binds to human TfR1 and to cyno TfR1.

[0060] In some embodiments, the anti-TfR1 antibody binds to human TfR1 wholly or partially within amino acids K231-E369 of SEQ ID NO: 1. In some embodiments, the anti-TfR1 antibody binds to human TfR1 at an epitope that includes amino acids within K231-E369 of SEQ ID NO: 1. In some embodiments, the anti-TfR1 antibody binds to at least one of the amino acids within K231-E369 of SEQ ID NO: 1.

[0061] In some embodiments, the anti-TfR1 antibody binds to human TfR1 wholly or partially within amino acids D139 to K145 and G490 to E582 of SEQ ID NO: 1. In some embodiments, the anti-TfR1 antibody binds to human TfR1 at an epitope that includes amino acids within D139 to K145 and G490 to E582 of SEQ ID NO: 1. In some embodiments, the anti-TfR1 antibody binds to at least one amino acid within D139 to K145 and G490 to E582 of SEQ ID NO: 1.

[0062] In some embodiments, the anti-TfR1 antibody specifically binds to a discontinuous epitope of human TfR1 or cynomolgus monkey TfR1. In one example, the anti-TfR1 antibody specifically binds to human TfR1, and the antibody binds to one or more residues selected from the group consisting of residues K231, D245, L246, Y247, T248, P249, E350, G351, D352, C353, P354, S355, D356, K358, T359, D360, S361, R364, M365, V366, T367, and E369. In another example, an anti-TfR1 antibody specifically binds to cynomolgus monkey TfR1, wherein the antibody binds to one or more residues selected from the group consisting of residues K231, D245, L246, D247, S248, P249, E350, G351, D352, C353, P354, S355, D356, K358, T359, D360, S361, K364, M365, V366, T367, and E369. In one example, an anti-TfR1 antibody specifically binds to human TfR1, where the antibody binds to one or more residues selected from the group consisting of residues D139, T141, K145, G490, T491, V517, T518, Y573, K574, 1577, E578, R579, 1580, P581, and E582. In another example, an anti-TfR1 antibody specifically binds to cynomolgus monkey TfR1, where the antibody binds to one or more residues selected from the group consisting of residues D139, T141, K145, G490, T491, V517, T518, Y573, K574, V577, E578, R579, 1580, P581, and E582.

[0063] In some embodiments, the anti-TfR1 antibody binds to human TfR1 at one or more amino acids within amino acids D139 to E582 of SEQ ID NO:1, wherein the one or more amino acids are selected from the following sequences: D139 to E369, D139 to E582, T141 to E369, T141 to E582, K145 to E369, K145 to E582, K231 to E36 9, K231-E582, D245-E369, D245-E582, E350-E369, E350-E582, K358-E369, K358-E582, R364-E369, R364-E582, G490-E582, V517-E582, K573-E582, I577-E582, and / or Y573-E582.

[0064] In some embodiments, the anti-TfR1 antibody binds to human TfR1 at one or more of the following amino acids: K231, D245 to P249, E244 to V250, E350 to S361, M349 to T362, R364 to E369, and / or C363 to S370 of SEQ ID NO:1.

[0065] In some embodiments, the anti-TfR1 antibody binds to one or more amino acids within the range of amino acids K231 to S370 of SEQ ID NO:1.

[0066] In some embodiments, the anti-TfR1 antibody binds to human TfR1 at one or more amino acids within amino acids D139 to L583 of SEQ ID NO:1, wherein the one or more amino acids are within any of the following sequences: D139 to T141, D139 to K145, T141 to K145, T138 to K145, T138 to L146, G490 to T491, L489 to T491, L489 to S492, V517 to T518, P516 to T518, P516 to G519, Y573 to E582, Y573 to L583, T572 to E582, and / or T572 to L583.

[0067] In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region spanning amino acids 607-760 of human TfR1 (SEQ ID NO:1), for example, the antibody binds to one or more residues within a region spanning amino acids 607-730, 607-700, 607-670, 640-760, 640-730, 640-700, 670-760, 670-730, 700-760, or 730-760 of human TfR1.

[0068] In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region spanning amino acids 189-383 of human TfR1 (SEQ ID NO: 1), e.g., the antibody binds to one or more residues within a region spanning amino acids 189-350, 189-320, 189-290, 189-260, 189-230, 220-383, 220-350, 220-320, 220-290, 220-260, 230-383, 230-350, 230-320, 230-383, In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region spanning amino acids 208-348 of human TfR1. In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region of human TfR1 spanning amino acids 340 to 370. In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region of human TfR1 spanning amino acids 190 to 230.

[0069] In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region spanning amino acids 122-188 of human TfR1 (SEQ ID NO:1), for example, the antibody binds to one or more residues within a region spanning amino acids 122-170, 122-150, 130-188, 130-170, 130-150, 140-188, 140-170, 150-188, or 150-170 of human TfR1.

[0070] In some embodiments, the anti-TfR1 antibody binds to one or more residues within a region spanning amino acids 384-606 of human TfR1 (SEQ ID NO: 1), e.g., the antibody binds to one or more residues within the region spanning amino acids 384-570, 384-540, 384-510, 384-480, 384-450, 384-420, 410-606, 410-570, 410-540, 410-510, 410-480, 410-450, 440-606, 440-570, , 440-540, 440-510, 440-480, 470-606, 470-590, 470-570, 470-540, 470-510, 490-606, 490-590, 490-570, 490-540, 490-510, 510-606, 510-590, 510-570, 510-540, 540-606, 540-590, 540-570, 570-590, or 570-606.

[0071] In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to the extracellular domain of TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to the protease-like domain, the helical domain, and / or the apical domain(s) of TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to a conformational epitope on TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to a conformational epitope located in the apical domain of TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to a conformational epitope located in the protease-like domain of TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to a conformational epitope located in the helical domain of TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to human TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to cyno TfR1. In some embodiments, the anti-TfR1 Ab (e.g., ANTIBODY A or ANTIBODY B described herein) binds to human TfR1 and cyno TfR1.

[0072] In some embodiments, the anti-TfR1 antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In some examples, the antibody comprises a human kappa light chain constant region. In other embodiments, the antibody comprises a human lambda light chain constant region. In some embodiments, the antibody is an antibody fragment comprising an antigen-binding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide-linked scFv. In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bivalent antibody.

[0073] In some instances, the antibody is a Fab, Fab', F(ab) 2 , scFv, sc(Fv) 2 In some embodiments, the antibody or antigen-binding fragment (e.g., Fab) has an interchain disulfide that is eliminated. The Fab or Fab' contains a variable heavy domain (VH) and a variable light domain (VL).

[0074] In some embodiments, the antibody is isolated, hi some embodiments, the antibody is substantially pure.

[0075] In some embodiments, the anti-TfR1 antibody is a monoclonal antibody. The monoclonal antibody may be prepared by any method known to those skilled in the art. In some embodiments, the monoclonal antibody is prepared using hybridoma methods known to those skilled in the art. For example, a mouse, rat, rabbit, hamster, or other suitable host animal is immunized as described above using hybridoma methods. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a cyno protein or a fragment thereof.

[0076] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol. Hybridoma cells are selected using special media, as known in the art, and unfused lymphocytes and myeloma cells are eliminated by the selection process. Hybridomas producing monoclonal antibodies directed against a selected antigen can be identified by a variety of methods, including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution or other techniques. Hybridomas can be grown in in vitro culture using standard methods, or grown in vivo as ascites tumors in animals. Monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art, including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0077] In some embodiments, monoclonal antibodies are produced using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding the antibodies are isolated from mature B cells or hybridoma cells (e.g., by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody) and their sequences are determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into an appropriate expression vector. The appropriate expression vector produces the monoclonal antibody when transfected into a host cell (E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins).

[0078] In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of the desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

[0079] In some embodiments, monoclonal antibodies are modified by the use of recombinant DNA technology to generate surrogate antibodies. In some embodiments, the light and heavy chain constant domains of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate chimeric antibodies. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. In some embodiments, the variable region(s) are subjected to site-directed or high-density mutagenesis to optimize the specificity and / or affinity of the monoclonal antibody.

[0080] In some embodiments, the anti-TfR1 antibody is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, a humanized antibody contains one or more amino acid residues from a non-human source introduced into its sequence. In some embodiments, humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody. In some embodiments, a humanized antibody is constructed by substituting all six CDRs of a non-human antibody (e.g., a mouse antibody) for the corresponding CDRs of a human antibody.

[0081] The selection of which human heavy and / or light chain variable regions to use to generate a humanized antibody can be made by various methods known in the art based on various factors. In some embodiments, a "best-fit" method is used where the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to the sequence of the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody. In some embodiments, a particular variable region framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from the consensus sequence of the most abundant human subclass. In some embodiments, human germline genes are used as the source of variable region framework sequences.

[0082] Other humanization methods include, but are not limited to, (i) a method termed "superhumanization", which is described as the direct introduction of CDRs into human germline frameworks, (ii) a method termed Human String Content (HSC), which is based on a metric of "antibody humanness", (iii) methods based on the generation of large libraries of humanized variants (including phage, ribosome, and yeast display libraries), and (iv) methods based on shuffling of framework regions.

[0083] In some embodiments, the anti-TfR1 antibody is a "human antibody." Human antibodies can be prepared using a variety of techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In either case, cells producing antibodies directed against a target antigen can be generated and isolated. In some embodiments, human antibodies are selected from phage libraries, which express human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires derived from unimmunized donors. Techniques for generating and using antibody phage libraries are well known in the art. After antibodies are identified, affinity maturation strategies known in the art can be employed to generate high affinity human antibodies, including but not limited to chain shuffling and site-directed mutagenesis. In some embodiments, human antibodies are generated in transgenic mice containing human immunoglobulin loci. These mice are capable of producing the full repertoire of human antibodies upon immunization, in the absence of endogenous immunoglobulin production.

[0084] In some embodiments, the anti-TfR1 antibody is a bispecific antibody. A bispecific antibody can recognize and bind to at least two different antigens or epitopes. The different epitopes can be in the same molecule (e.g., two epitopes on TfR1) or on different molecules (e.g., one epitope on TfR1 and one epitope on a different target). In some embodiments, a bispecific antibody has increased efficacy compared to an individual antibody or a combination of two or more antibodies. In some embodiments, a bispecific antibody has reduced toxicity compared to an individual antibody or a combination of two or more antibodies. It is known to those skilled in the art that any therapeutic agent can have unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents. In this case, the two individual binding agents have different PK profiles. In some embodiments, a bispecific antibody has the ability to focus the action of two agents to a common area (e.g., tissue) of a subject. In some embodiments, bispecific antibodies have the ability to focus the action of two agents on a common target (e.g., a specific cell type). In some embodiments, bispecific antibodies have the ability to direct the action of two agents to more than one biological pathway or function. In some embodiments, bispecific antibodies have the ability to target two different cells and bring them into close proximity to each other.

[0085] In some embodiments, bispecific antibodies have reduced toxicity and / or side effects. In some embodiments, bispecific antibodies have reduced toxicity and / or side effects compared to a mixture of two individual antibodies or an antibody as a single agent. In some embodiments, bispecific antibodies have an increased therapeutic index. In some embodiments, bispecific antibodies have an increased therapeutic index compared to a mixture of two individual antibodies or an antibody as a single agent.

[0086] Several techniques for making bispecific antibodies are known to those skilled in the art. In some embodiments, bispecific antibodies comprise heavy chain constant regions with modifications to amino acids that are part of the interface between the two heavy chains. Such modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation. In some embodiments, bispecific antibodies are generated using the knobs-into-holes (KIH) strategy. See, for example, Ridgway et al. Protein Eng. 1996; 9(7): 617-21 and Klein et al. MAbs. 2012; 4(6): 653-663.

[0087] In some embodiments, the bispecific antibody comprises a light chain constant region having modifications in amino acids that are part of the interface between the two light chains. Such modifications are made to reduce or eliminate mispairing of the light chains. See, e.g., Lewis et al. Nat Biotech 2014;32(2):191-98. In some embodiments, the bispecific antibody comprises an scFv in which the VH and VL are covalently linked and the CH1 and CL are removed. In some embodiments, the bispecific antibody comprises an scFab or Fcab (see, e.g., Wozniak-Knopp et al. PEDS 2010;23(4):289-97), a single domain antibody (e.g., with a VHH derived from a camelid species or shark), or a Duet Mab (see, e.g., Mazor et al. Mabs 2015;7(2):377-89).

[0088] Bispecific antibodies can be intact antibodies or antibody fragments that contain the antigen-binding site.

[0089] The present disclosure contemplates anti-TfR1 antibodies with more than one specificity. In some embodiments, tri- or tetra-specific antibodies are generated. Anti-TfR1 antibodies with more than two valencies are contemplated. In some embodiments, tri- or tetra-valent antibodies are generated.

[0090] The CDRs of an antibody are defined by those skilled in the art using various methods / systems. Such systems and / or definitions have been developed and improved over the years, including Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is the one commonly used. The Chothia definition is based on the location of the loop regions on the structure. The IMGT system is based on sequence variability and the location in the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs (e.g., abYsis) are available for analyzing antibody sequences and determining CDRs, and such software programs are known to those skilled in the art.

[0091] The CDR sequences depicted in Figures 1A to 1D include the union of all positions in the CDR definition of Kabat (Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit. National Institutes of Health, Bethesda, MD) and the CDR definition of Chothia (Chothia, C. & Lesk, A. M. J. Mol. Biol (1987) 196, 901-917) (Chothia, C. et al. Nature (1989) 342, 877-883) (Al-Lazikani, B., Lesk, A. M. & Chothia, C. J. Mol. Biol (1997) 273, 927-948). This "union" definition of CDR is also known as the "Wolfguy" definition by Bujotzek et al. (Bujotzek A1, Dunbar J, Lipsmeier F, Schafer W, Antes I, Deane CM, Georges G. (2015) "Prediction of VH-VL domain orientation for antibody variable domain modeling." Proteins Apr;83(4):681-95. doi:10.1002 / prot.24756). In some embodiments, the CDR definition is based on a combination of the Kabat and Chothia definitions (exemplary system). However, it will be understood that reference to the heavy chain CDR(s) and / or light chain CDR(s) of a particular antibody encompasses all CDR definitions known to those skilled in the art. In one example, an anti-TfR1 antibody used in the methods described herein comprises six CDRs of any ANTIBODY-A clone disclosed herein or any ANTIBODY-B clone disclosed herein based on the Wolfguy definition or the Union definition.In one example, an anti-TfR1 antibody used in any of the methods described herein comprises six CDRs of any ANTIBODY-A clone disclosed herein or any ANTIBODY-B clone disclosed herein based on the Chothia definition. In one example, an anti-TfR1 antibody used in the methods described herein comprises six CDRs of any ANTIBODY-A clone disclosed herein or any ANTIBODY-B clone disclosed herein based on the Kabat definition. In one example, an anti-TfR1 antibody used in the methods described herein comprises six CDRs of any ANTIBODY-A clone disclosed herein or any ANTIBODY-B clone disclosed herein based on the AbM definition. In one example, an anti-TfR1 antibody used in the methods described herein comprises six CDRs of any ANTIBODY-A clone disclosed herein or any ANTIBODY-B clone disclosed herein based on the IMGT definition. In one example, an anti-TfR1 antibody used in the methods described herein comprises six CDRs of any ANTIBODY-A clone disclosed herein or any ANTIBODY-B clone disclosed herein, based on the Contact definition.

[0092] In some embodiments, the anti-TfR1 antibody is an anti-TfR1 antibody that comprises one, two, three, four, five, and / or six CDRs of any one of the antibodies described herein. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 1A, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 1A. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 2A, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 2A.

[0093] In some embodiments, the anti-TfR1 antibody is an anti-TfR1 antibody that comprises one, two, three, four, five, and / or six CDRs of any one of the antibodies described herein. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 1B, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 1B. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 2B, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 2B.

[0094] In some embodiments, the anti-TfR1 antibody comprises the three heavy chain CDRs and the three light chain CDRs of any one of the clones shown in Table 1 A. In some embodiments, the anti-TfR1 antibody comprises the three heavy chain CDRs and the three light chain CDRs of any one of the clones shown in Table 2A.

[0095] In some embodiments, the anti-TfR1 antibody comprises the three heavy chain CDRs and the three light chain CDRs of any one of the clones shown in Table 1 B. In some embodiments, the anti-TfR1 antibody comprises the three heavy chain CDRs and the three light chain CDRs of any one of the clones shown in Table 2B.

[0096] In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 3A, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 3B. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 4A, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 4B.

[0097] In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 3C, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 3D. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Table 4C, and / or (ii) one, two, and / or three light chain CDRs of any one of the clones shown in Table 4D.

[0098] In some embodiments, the anti-TfR1 antibody comprises (i) the three heavy chain CDRs of any one of the clones shown in Table 3A, and (ii) the three light chain CDRs of any one of the clones shown in Table 3B. In some embodiments, the anti-TfR1 antibody comprises (i) the three heavy chain CDRs of any one of the clones shown in Table 4A, and (ii) the three light chain CDRs of any one of the clones shown in Table 4B.

[0099] In some embodiments, the anti-TfR1 antibody comprises (i) the three heavy chain CDRs of any one of the clones shown in Table 3C, and (ii) the three light chain CDRs of any one of the clones shown in Table 3D. In some embodiments, the anti-TfR1 antibody comprises (i) the three heavy chain CDRs of any one of the clones shown in Table 4C, and (ii) the three light chain CDRs of any one of the clones shown in Table 4D. [Table 1A] [Table 1B] [Table 2A] [Table 2B-1] [Table 2B-2]

Table 3A

Table 3B

Table 3C-1

Table 3C-2

Table 3D

Table 4A-1

Table 4A-2

Table 4B

Table 4C-1

Table 4C-2

Table 4D-1

Table 4D-2

[0100] In some embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, CDR2, and CDR3, and / or the light chain variable region CDR1, CDR2, and CDR3 of an antibody described herein. In some embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of an antibody described herein. In some embodiments, the anti-TfR1 antibody includes a humanized version or variant of an antibody described herein.

[0101] In some embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, CDR2, and CDR3, and / or the light chain variable region CDR1, CDR2, and CDR3 of any ANTIBODY-A clone disclosed herein, a humanized version thereof, or a variant thereof. In some embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, the heavy chain variable region CDR2, and the heavy chain variable region CDR3 of any ANTIBODY-A clone disclosed herein. In other embodiments, the anti-TfR1 antibody comprises the light chain variable region CDR1, the light chain variable region CDR2, and the light chain variable region CDR3 of any ANTIBODY-A clone disclosed herein. In certain embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, the heavy chain variable region CDR2, the heavy chain variable region CDR3, the light chain variable region CDR1, the light chain variable region CDR2, and the light chain variable region CDR3 of any antibody ANTIBODY-A clone disclosed herein. In some embodiments, the anti-TfR1 antibody is a humanized version of any of the ANTIBODY-A clones disclosed herein. In some embodiments, the anti-TfR1 antibody is a variant of any of the ANTIBODY-A clones disclosed herein.

[0102] In some embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, CDR2, and CDR3, and / or the light chain variable region CDR1, CDR2, and CDR3 of any ANTIBODY-B clone disclosed herein, a humanized version thereof, or a variant thereof. In some embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, the heavy chain variable region CDR2, and the heavy chain variable region CDR3 of any ANTIBODY-B clone disclosed herein. In other embodiments, the anti-TfR1 antibody comprises the light chain variable region CDR1, the light chain variable region CDR2, and the light chain variable region CDR3 of any ANTIBODY-B clone disclosed herein. In certain embodiments, the anti-TfR1 antibody comprises the heavy chain CDR1, the heavy chain variable region CDR2, the heavy chain variable region CDR3, the light chain variable region CDR1, the light chain variable region CDR2, and the light chain variable region CDR3 of any antibody ANTIBODY-B clone disclosed herein. In some embodiments, the anti-TfR1 antibody is a humanized version of ANTIBODY-B. In some embodiments, the anti-TfR1 antibody is a variant of ANTIBODY-B.

[0103] In some embodiments, the anti-TfR1 antibody is a variant of an anti-TfR1 antibody described herein comprising 1-30 conservative amino acid substitutions. In some embodiments, the anti-TfR1 antibody variant comprises 1-25 conservative amino acid substitutions. In some embodiments, the anti-TfR1 antibody variant comprises 1-20 conservative amino acid substitutions. In some embodiments, the anti-TfR1 antibody variant comprises 1-15 conservative amino acid substitutions. In some embodiments, the anti-TfR1 antibody variant comprises 1-10 conservative amino acid substitution(s). In some embodiments, the anti-TfR1 antibody variant comprises 1-5 conservative amino acid substitution(s). In some embodiments, the anti-TfR1 antibody variant comprises 1-3 conservative amino acid substitution(s). In some embodiments, the conservative amino acid substitution(s) are in the CDRs of the antibody. In some embodiments, the conservative amino acid substitution(s) are not in the CDRs of the antibody. In some embodiments, the conservative amino acid substitution(s) are in framework regions of the antibody.

[0104] In some embodiments, the anti-TfR1 antibody comprises (a) a heavy chain variable region CDR1 comprising GIDFSSSGYMC (SEQ ID NO: 101), a heavy chain variable region CDR2 comprising CIYTYSSNTYYAASVKG (SEQ ID NO: 103), and a heavy chain variable region CDR3 comprising GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), and / or (b) a light chain variable region CDR1 comprising QASQNINSYLA (SEQ ID NO: 107), a light chain variable region CDR2 comprising RASSLES (SEQ ID NO: 108), and a light chain variable region CDR3 comprising QSYYYSGSSNYNA (SEQ ID NO: 110). In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region CDR1 comprising GIDFSSSGYMC (SEQ ID NO: 101), a heavy chain variable region CDR2 comprising CIYTYSSNTYYAASVKG (SEQ ID NO: 103), and a heavy chain variable region CDR3 comprising GTYGYTGYTYTMGYFSL (SEQ ID NO: 106). In some embodiments, the anti-TfR1 antibody comprises a light chain variable region CDR1 comprising QASQNINSYLA (SEQ ID NO: 107), a light chain variable region CDR2 comprising RASSLES (SEQ ID NO: 108), and a light chain variable region CDR3 comprising QSYYYSGSSNYNA (SEQ ID NO: 110). In some embodiments, the anti-TfR1 antibody comprises (a) a heavy chain variable region CDR1 comprising GIDFSSSGYMC (SEQ ID NO: 101), a heavy chain variable region CDR2 comprising CIYTYSSNTYYAASVKG (SEQ ID NO: 103), and a heavy chain variable region CDR3 comprising GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), and (b) a light chain variable region CDR1 comprising QASQNINSYLA (SEQ ID NO: 107), a light chain variable region CDR2 comprising RASSLES (SEQ ID NO: 108), and a light chain variable region CDR3 comprising QSYYYSGSSNYNA (SEQ ID NO: 110).In some embodiments, the anti-TfR1 antibody comprises (a) a heavy chain variable region comprising a heavy chain CDR1 comprising GIDFSSSGYMC (SEQ ID NO: 101), a heavy chain CDR2 comprising CIYTYSSNTYYAASVKG (SEQ ID NO: 103), and a heavy chain CDR3 comprising GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), or (b) a light chain variable region comprising a light chain CDR1 comprising QASQNINSYLA (SEQ ID NO: 107), a light chain CDR2 comprising RASSLES (SEQ ID NO: 108), and a light chain CDR3 comprising QSYYYSGSSNYNA (SEQ ID NO: 110).

[0105] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region CDR1 comprising GIDFSSSGYMC (SEQ ID NO: 101), or a variant thereof comprising one, two, three, or four amino acid substitutions; a heavy chain variable region CDR2 comprising CIYTYSSNTYYAASVKG (SEQ ID NO: 103), or a variant thereof comprising one, two, three, or four amino acid substitutions; a heavy chain variable region CDR3 comprising GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), or a variant thereof comprising one, two, three, or four amino acid substitutions; and variants thereof comprising one, two, three, or four amino acid substitutions, a light chain variable region CDR1 comprising QASQNINSYLA (SEQ ID NO: 107) or variants thereof comprising one, two, three, or four amino acid substitutions, a light chain variable region CDR2 comprising RASSLES (SEQ ID NO: 108) or variants thereof comprising one, two, three, or four amino acid substitutions, and a light chain variable region CDR3 comprising QSYYYSGSSNYNA (SEQ ID NO: 110) or variants thereof comprising one, two, three, or four amino acid substitutions. In some embodiments, the CDR comprises one amino acid substitution. In some embodiments, the CDR comprises two amino acid substitutions. In some embodiments, the CDR comprises three amino acid substitutions. In some embodiments, the CDR comprises four amino acid substitutions. In some embodiments, the one or more amino acid substitutions are conservative substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain variable region CDR2. In some embodiments, the CDR is a heavy chain variable region CDR3. In some embodiments, the CDR is a light chain variable region CDR1. In some embodiments, the CDR is a light chain variable region CDR2. In some embodiments, the CDR is a light chain variable region CDR3. In some embodiments, the one or more substitutions are made as part of a humanization process. In some embodiments, the one or more substitutions are made as part of a germline humanization process. In some embodiments, the one or more substitutions are made as part of an affinity maturation process. In some embodiments, the one or more substitutions are made as part of an optimization process.

[0106] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence GIDFSSSGYMX (SEQ ID NO: 149), where X is any amino acid, VH CDR2 comprises the amino acid sequence XIYTYSSNTYYAXXXKG (SEQ ID NO: 151), where X is any amino acid, and VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), and VL CDR2 comprises the amino acid sequence RASXLXS (SEQ ID NO: 153), where X is any amino acid, and VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110). In some embodiments, the VH CDR1 comprises the amino acid sequence GIDFSSSGYMX 1 (SEQ ID NO: 150), where X 1 is C, A, or H, and VH CDR2 is the amino acid sequence X 2 IYTYSSNTYYAX 3 X 4 X 5 KG (SEQ ID NO: 152), where X 2 is C or A, and X 3 is S or A, and X 4 is W or S, and X 5 is A or V, VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), and VL CDR2 comprises the amino acid sequence RASX 6 LX 7 S (SEQ ID NO: 154), where X 6 is T or S, and X 7 is A or E, and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110).

[0107] In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region having the three VH CDRs of any ANTIBODY-A clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 16, and a light chain variable region having the three VL CDRs of any ANTIBODY-A clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 35.

[0108] In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 16, and a light chain variable region that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 35. In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region having one or more (e.g., one, two, or three) substitutions, deletions, or insertions in the sequence set forth in SEQ ID NO: 16, and a light chain variable region having one or more substitutions, deletions, or insertions in the sequence set forth in SEQ ID NO: 35. In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region having the three VH CDRs of any of the ANTIBODY-A clones disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VH sequences shown in FIG. 1A, and a light chain variable region having the three VL CDRs of any of the ANTIBODY-B clones disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences shown in FIG. 1C.

[0109] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 16 and / or a light chain variable region having at least 80% sequence identity to SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 16. In some embodiments, the anti-TfR1 antibody comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:35.

[0110] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 16 and / or a light chain variable region having at least about 80% sequence identity to SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 16 and a light chain variable region having at least about 80% sequence identity to SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 90% sequence identity to SEQ ID NO: 16 and / or a light chain variable region having at least about 90% sequence identity to SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 90% sequence identity to SEQ ID NO: 16 and a light chain variable region having at least about 90% sequence identity to SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 16 and / or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 16 and a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 35.

[0111] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 16. In some embodiments, the anti-TfR1 antibody comprises a light chain variable region comprising SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 16 and a light chain variable region comprising SEQ ID NO: 35.

[0112] In some embodiments, the anti-TfR1 antibody comprises CDR1, CDR2, and CDR3 of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16, and CDR1, CDR2, and CDR3 of a light chain variable region having the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-TfR1 antibody comprises (i) a heavy chain variable region comprising CDR1, CDR2, and CDR3 of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16, and (ii) a light chain variable region comprising CDR1, CDR2, and CDR3 of a light chain variable region having the amino acid sequence of SEQ ID NO: 35.

[0113] In some embodiments, the anti-TfR1 antibody comprises (a) a heavy chain variable region CDR1 comprising GFSFSNSYWIC (SEQ ID NO: 116), a heavy chain variable region CDR2 comprising CINTDADSTNYASWARG (SEQ ID NO: 117), and a heavy chain variable region CDR3 comprising QNNVFDPGYNL (SEQ ID NO: 119), and / or (b) a light chain variable region CDR1 comprising RASQNIGSNLA (SEQ ID NO: 120), a light chain variable region CDR2 comprising DASKLES (SEQ ID NO: 122), and a light chain variable region CDR3 comprising QCTVRGGAYGNA (SEQ ID NO: 124). In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region CDR1 comprising GFSFSNSYWIC (SEQ ID NO: 116), a heavy chain variable region CDR2 comprising CINTDADSTNYASWARG (SEQ ID NO: 117), and a heavy chain variable region CDR3 comprising QNNVFDPGYNL (SEQ ID NO: 119). In some embodiments, the anti-TfR1 antibody comprises a light chain variable region CDR1 comprising RASQNIGSNLA (SEQ ID NO: 120), a light chain variable region CDR2 comprising DASKLES (SEQ ID NO: 122), and a light chain variable region CDR3 comprising QCTVRGGAYGNA (SEQ ID NO: 124). In some embodiments, the anti-TfR1 antibody comprises (a) a heavy chain variable region CDR1 comprising GFSFSNSYWIC (SEQ ID NO: 116), a heavy chain variable region CDR2 comprising CINTDADSTNYASWARG (SEQ ID NO: 117), and a heavy chain variable region CDR3 comprising QNNVFDPGYNL (SEQ ID NO: 119), and (b) a light chain variable region CDR1 comprising RASQNIGSNLA (SEQ ID NO: 120), a light chain variable region CDR2 comprising DASKLES (SEQ ID NO: 122), and a light chain variable region CDR3 comprising QCTVRGGAYGNA (SEQ ID NO: 124).In some embodiments, the anti-TfR1 antibody comprises (a) a heavy chain variable region comprising a heavy chain CDR1 comprising GFSFSNSYWIC (SEQ ID NO: 116), a heavy chain CDR2 comprising CINTDADSTNYASWARG (SEQ ID NO: 117), and a heavy chain CDR3 comprising QNNVFDPGYNL (SEQ ID NO: 119), or (b) a light chain variable region comprising a light chain CDR1 comprising RASQNIGSNLA (SEQ ID NO: 120), a light chain CDR2 comprising DASKLES (SEQ ID NO: 122), and a light chain CDR3 comprising QCTVRGGAYGNA (SEQ ID NO: 124).

[0114] In some embodiments, the anti-TfR1 antibody comprises: (a) a heavy chain variable region CDR1 comprising GFSFSNSYWIC (SEQ ID NO: 116), or a variant thereof comprising one, two, three, or four amino acid substitutions; a heavy chain variable region CDR2 comprising CINTDADSTNYASWARG (SEQ ID NO: 117), or a variant thereof comprising one, two, three, or four amino acid substitutions; a heavy chain variable region CDR3 comprising QNNVFDPGYNL (SEQ ID NO: 119), or a variant thereof comprising one, two, three, or four amino acid substitutions; and variants thereof comprising one, two, three, or four amino acid substitutions, a light chain variable region CDR1 comprising RASQNIGSNLA (SEQ ID NO: 120) or variants thereof comprising one, two, three, or four amino acid substitutions, a light chain variable region CDR2 comprising DASKLES (SEQ ID NO: 122) or variants thereof comprising one, two, three, or four amino acid substitutions, and a light chain variable region CDR3 comprising QCTVRGGAYGNA (SEQ ID NO: 124) or variants thereof comprising one, two, three, or four amino acid substitutions. In some embodiments, the CDR comprises one amino acid substitution. In some embodiments, the CDR comprises two amino acid substitutions. In some embodiments, the CDR comprises three amino acid substitutions. In some embodiments, the CDR comprises four amino acid substitutions. In some embodiments, the one or more amino acid substitutions are conservative substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain variable region CDR2. In some embodiments, the CDR is a heavy chain variable region CDR3. In some embodiments, the CDR is a light chain variable region CDR1. In some embodiments, the CDR is a light chain variable region CDR2. In some embodiments, the CDR is a light chain variable region CDR3. In some embodiments, the one or more substitutions are made as part of a humanization process. In some embodiments, the one or more substitutions are made as part of a germline humanization process. In some embodiments, the one or more substitutions are made as part of an affinity maturation process. In some embodiments, the one or more substitutions are made as part of an optimization process.

[0115] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence GFSFSNSYWIX (SEQ ID NO: 155), where X is any amino acid, VH CDR2 comprises the amino acid sequence XINTDADSTNYAXXXXG (SEQ ID NO: 157), where X is any amino acid, and VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119); and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence XASQNIGSNLA (SEQ ID NO: 159), VL CDR2 comprises the amino acid sequence DASKLXS (SEQ ID NO: 161), where X is any amino acid, and VL CDR3 comprises the amino acid sequence QXTVRGGAYGXA (SEQ ID NO: 163), where X is any amino acid. In some embodiments, the VH CDR1 comprises the amino acid sequence GFSFSNSYWIX 1 (SEQ ID NO: 156), where X 1 is C, A, or H, and VH CDR2 is the amino acid sequence X 2 INTDADSTNYAX 3 X 4 X 5 X 6 G (SEQ ID NO: 158), where X 2 is C or A, and X 3 is S or D, and X 4 is W or S, and X 5 is A or V, and X 6 is R or K, VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), and VL CDR1 comprises the amino acid sequence X 7 ASQNIGSNLA (SEQ ID NO: 160), where X 7 is Q or R, and VL CDR2 has the amino acid sequence DASKLX 8 S (SEQ ID NO: 162), where X 8 is A or E, and VL CDR3 has the amino acid sequence QX 9 TVRGGAYGX 10A (SEQ ID NO: 164), where X 9 is C, Q, A, S, T, or V, and X 10 is N or L.

[0116] In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region having the three VH CDRs of any ANTIBODY-B clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO:33, and a light chain variable region having the three VL CDRs of any ANTIBODY-B clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO:41.

[0117] In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:33, and a light chain variable region that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:41.

[0118] In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region having one or more substitutions, deletions, or insertions in the sequence set forth in SEQ ID NO:33, and a light chain variable region having one or more substitutions, deletions, or insertions in the sequence set forth in SEQ ID NO:41.

[0119] In some embodiments of the methods described herein, the anti-TfR1 antibody comprises a heavy chain variable region having the three VH CDRs of any ANTIBODY-B clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VH sequences shown in FIG. 1B, and a light chain variable region having the three VL CDRs of any ANTIBODY-B clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences shown in FIG. 1D.

[0120] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 33 and / or a light chain variable region having at least 80% sequence identity to SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 33. In some embodiments, the anti-TfR1 antibody comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 41.

[0121] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 33 and / or a light chain variable region having at least about 80% sequence identity to SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 33 and a light chain variable region having at least about 80% sequence identity to SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 90% sequence identity to SEQ ID NO: 33 and / or a light chain variable region having at least about 90% sequence identity to SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 90% sequence identity to SEQ ID NO: 33 and a light chain variable region having at least about 90% sequence identity to SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 33 and / or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 33 and a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 41.

[0122] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 33. In some embodiments, the anti-TfR1 antibody comprises a light chain variable region comprising SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 33 and a light chain variable region comprising SEQ ID NO: 41.

[0123] In some embodiments, the anti-TfR1 antibody comprises CDR1, CDR2, and CDR3 of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 33, and CDR1, CDR2, and CDR3 of a light chain variable region having the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-TfR1 antibody comprises (i) a heavy chain variable region comprising CDR1, CDR2, and CDR3 of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 33, and (ii) a light chain variable region comprising CDR1, CDR2, and CDR3 of a light chain variable region having the amino acid sequence of SEQ ID NO: 41.

[0124] Humanized VH / VL constructs of the present disclosure include, but are not limited to, those SEQ ID NOs: set forth in Figures 1A-1D.

[0125] Anti-TfR1 antibody constant region In some embodiments, the variable regions of the anti-TfR1 antibodies described herein are fused to a constant region. The constant region has a constant heavy chain (CH) domain (e.g., CH1, hinge, CH2, and / or CH3 domain(s) or any combination thereof) and a constant light chain (CL) domain. In some embodiments, the CH domain is from an IgG1 or IgG4 molecule. In some embodiments, the CH domain is from an IgG2, IgG3, or IgG molecule. The VH of the anti-TfR1 antibodies described herein may be fused to any one of the following constant heavy chain (CH) constructs shown in Table 5 below. The VL of the anti-TfR1 antibodies described herein may be fused to any one of the following constant light chain (CL) constructs shown in Table 5 below. In some embodiments, the VH / CH1 (CH1 from IgG1) construct is further fused to a hinge region (e.g., ES) shown in Table 5 below. In some embodiments, the VH / CH1 (CHI from IgG1) construct is further fused to a hinge region comprising the amino acid sequence E, EP, EPK, or any one of SEQ ID NOs: 58-74. In some embodiments, the VH / CH1 (CHI from IgG4) construct is further fused to a hinge region (e.g., ES) as shown in Table 5 below. In some embodiments, the VH / CH1 (CHI from IgG4) construct is further fused to a hinge region comprising the amino acid sequence E, ES, ESK, or any one of SEQ ID NOs: 75-90. In some embodiments, the VH / VL-CH1 / CL construct does not contain a hinge. In some embodiments, the hinge region is any hinge region known in the art. In some embodiments, the hinge region is naturally occurring, e.g., derived from a naturally occurring IgG1, IgG2, IgG3, or IgG4 molecule. In other embodiments, the hinge region contains modification(s) relative to a naturally occurring hinge.

[0126] In some embodiments, the anti-TfR1 antibodies of the present disclosure are those in which at least one or more of the constant regions have been modified or deleted. In some embodiments, the antibodies may include one or more modifications to the heavy chain constant domain (CH1, CH2 or CH3) and / or the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes two or more human constant regions. In some embodiments, the VH is fused to any CH1 construct known in the art and the VL is fused to any CL known in the art. In some embodiments, the constant light chain (CL) of the construct is a naturally occurring human kappa constant region. In some embodiments, the modifications to the constant region include the addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 and CH3 domains are removed from the antibody. In some embodiments, the deleted constant region is replaced with a short amino acid spacer, which provides some of the flexibility of the molecule normally conferred by the absent constant region. In some embodiments, the modified antibody comprises a CH1 domain fused directly to the hinge region of the antibody. In some embodiments, the modified antibody comprises a Fab fused to a bottom Fc.

[0127] In some embodiments, an anti-TfR1 antibody of the present disclosure contains a linker (e.g., a linker shown in Table 5 below). In some embodiments, the linker is disposed between the Fc region and the Fab region of an anti-TfR1 antibody of the present disclosure. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0128] Other exemplary constant regions (e.g., hinge regions) that can be combined with the antibody variable regions described herein include, but are not limited to, the hinge regions described in Peters SJ, et al. J Biol Chem. 2012 Jul 13;287(29):24525-33, and Heads JT, et al. Protein Sci. 2012 Sep;21(9):1315-22, which are incorporated by reference in their entireties.

[0129] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1796 (SEQ ID NO: 192) and a second polypeptide comprising TOC1775 (SEQ ID NO: 94).

[0130] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1797 (SEQ ID NO: 193), a second polypeptide comprising TOC1801 (SEQ ID NO: 195), and a third polypeptide comprising TOC1775 (SEQ ID NO: 94).

[0131] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1798 (SEQ ID NO: 180) and a second polypeptide comprising TOC1775 (SEQ ID NO: 94).

[0132] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1799 (SEQ ID NO: 194), a second polypeptide comprising TOC1801 (SEQ ID NO: 195), and a third polypeptide comprising TOC1775 (SEQ ID NO: 94).

[0133] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1785 (SEQ ID NO: 174) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0134] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1786 (SEQ ID NO: 175) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0135] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1787 (SEQ ID NO: 176) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0136] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1710 (SEQ ID NO: 177) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0137] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1711 (SEQ ID NO: 178) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0138] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1712 (SEQ ID NO: 179) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0139] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1713 (SEQ ID NO: 180) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0140] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1714 (SEQ ID NO: 181) and a second polypeptide comprising TOC1715 (SEQ ID NO: 196).

[0141] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1788 (SEQ ID NO: 182) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0142] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1789 (SEQ ID NO: 183) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0143] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1790 (SEQ ID NO: 184) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0144] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1791 (SEQ ID NO: 185) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0145] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1792 (SEQ ID NO: 186) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0146] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1793 (SEQ ID NO: 187) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0147] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1794 (SEQ ID NO: 188) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0148] In some embodiments, the anti-TfR1 antibody comprises a first polypeptide comprising TOC1795 (SEQ ID NO: 189) and a second polypeptide comprising TOC1721 (SEQ ID NO: 197).

[0149] In some embodiments, the anti-TfR1 antibody comprises a polypeptide comprising TOC1728 (SEQ ID NO: 190).

[0150] In some embodiments, the anti-TfR1 antibody comprises a polypeptide comprising TOC1729 (SEQ ID NO: 191).

[0151] Also provided herein is an antibody that competes with one or more of the antibodies described herein for binding to human TfR1. In some embodiments, the antibody of the present disclosure binds to the same epitope as one of the anti-TfR1 antibodies described herein. In some embodiments, the antibody binds to an epitope that overlaps with the epitope bound by one of the anti-TfR1 antibodies described herein. In some embodiments, the antibody that competes with one or more of the antibodies described herein for binding to TfR1 is identified using the epitope binning method described herein.

[0152] In some embodiments, the antibody competes with an anti-TfR1 antibody described herein for binding to TfR1. In some embodiments, the antibody competes with an anti-TfR1 antibody described herein for binding to human TfR1. In some embodiments, the antibody competes with a reference antibody for binding to TfR1 (e.g., human TfR1), the reference antibody comprising: (a) a heavy chain variable region comprising: a heavy chain variable region CDR1 comprising: GIDFSSSGYMC (SEQ ID NO: 101); a heavy chain variable region CDR2 comprising: CIYTYSSNTYYAASVKG (SEQ ID NO: 103); and a heavy chain variable region CDR3 comprising: GTYGYTGYTYTMGYFSL (SEQ ID NO: 106); and (b) a light chain variable region CDR1 comprising: QASQNINSYLA (SEQ ID NO: 107); a light chain variable region CDR2 comprising: RASSLES (SEQ ID NO: 108); and a light chain variable region CDR3 comprising: QSYYYSGSSNYNA (SEQ ID NO: 110). In some embodiments, the antibody competes with a reference antibody for binding to TfR1 (e.g., human TfR1), where the reference antibody comprises a heavy chain variable region comprising SEQ ID NO:16 and a light chain variable region comprising SEQ ID NO:35.

[0153] In some embodiments, the antibody competes with a reference antibody for binding to TfR1 (e.g., human TfR1), the reference antibody comprising (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising GFSFSNSYWIC (SEQ ID NO: 116), a heavy chain variable region CDR2 comprising CINTDADSTNYASWARG (SEQ ID NO: 117), and a heavy chain variable region CDR3 comprising QNNVFDPGYNL (SEQ ID NO: 119), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising RASQNIGSNLA (SEQ ID NO: 120), a light chain variable region CDR2 comprising DASKLES (SEQ ID NO: 122), and a light chain variable region CDR3 comprising QCTVRGGAYGNA (SEQ ID NO: 124). In some embodiments, the antibody competes with a reference antibody for binding to TfR1 (e.g., human TfR1), the reference antibody comprising a heavy chain variable region comprising SEQ ID NO: 33 and a light chain variable region comprising SEQ ID NO: 41.

[0154] In some embodiments, the anti-TfR1 antibodies described herein include antibodies in which at least one or more of the constant regions have been modified or deleted. In some embodiments, the antibodies may include one or more modifications to one or more of the heavy chain constant regions (CH1, hinge, CH2, or CH3) and / or to the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes two or more human constant regions. In some embodiments, the modifications to the constant region include the addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant region or hinge region of the modified antibody. In some embodiments, the entire CH2 domain is removed from the antibody. In some embodiments, the deleted constant region is replaced with a short amino acid spacer, which provides some of the flexibility of the molecule that is normally imparted by the absent constant region. In some embodiments, the modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody, hi some embodiments, the modified antibody comprises a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domain.

[0155] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:93. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:93 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:93. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 90% sequence identity to SEQ ID NO:94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 93 and a light chain having at least 90% sequence identity to SEQ ID NO: 94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the anti-TfR1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 93 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 93 and / or a light chain of SEQ ID NO: 94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 93. In some embodiments, the anti-TfR1 antibody is an antibody comprising a light chain of SEQ ID NO: 94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 93 and a light chain of SEQ ID NO: 94.

[0156] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 93. In some embodiments, the anti-TfR1 antibody comprises a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 94. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 93 and a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 94.

[0157] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:95. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:95 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:95. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 90% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 95 and a light chain having at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the anti-TfR1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 95 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 95 and / or a light chain of SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 95. In some embodiments, the anti-TfR1 antibody is an antibody comprising a light chain of SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 95 and a light chain of SEQ ID NO: 96.

[0158] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 95. In some embodiments, the anti-TfR1 antibody comprises a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 95 and a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 96.

[0159] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:97. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:97 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:97. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 90% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 97 and a light chain having at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the anti-TfR1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 97 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 97 and / or a light chain of SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 97. In some embodiments, the anti-TfR1 antibody is an antibody comprising a light chain of SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 97 and a light chain of SEQ ID NO: 96.

[0160] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 97. In some embodiments, the anti-TfR1 antibody comprises a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 97 and a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 96.

[0161] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:98. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:98 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:98. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 90% sequence identity to SEQ ID NO:96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 98 and a light chain having at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the anti-TfR1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 98 and / or a light chain of SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 98. In some embodiments, the anti-TfR1 antibody is an antibody comprising a light chain of SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 98 and a light chain of SEQ ID NO: 96.

[0162] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 98. In some embodiments, the anti-TfR1 antibody comprises a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 98 and a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) relative to SEQ ID NO: 96.

[0163] In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 99. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 99 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 99 and a light chain having at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the anti-TfR1 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 99 and / or a light chain of SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 99. In some embodiments, the anti-TfR1 antibody is an antibody comprising a light chain of SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a heavy chain of SEQ ID NO: 99 and a light chain of SEQ ID NO: 100.

[0164] The present disclosure further encompasses additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies described herein, or antibody fragments thereof. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including, but not limited to, specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, and / or solubility. Those skilled in the art will recognize that amino acid changes may alter post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0165] Antibody effector functions can be modulated by amino acid mutations and / or domain substitutions (e.g., including but not limited to those described in Dumet et al. MABS 2019;11(8):1341-50). Additional properties such as pharmacokinetics (e.g., Dall'acqua et al J of Immunology 2002;169(9)5171-80), glycosylation, immunogenicity, solubility, and stability can be engineered by modification of the Fc by mutation or substitution. In addition, novel antigen specificities can be engineered into the constant domain to create new paratopes (e.g., Wozniak-Knopp et al. PEDS 2010;23(4):289-97). The affinity or avidity of the Fab can be modulated by altering the linkage between the antibody domains (e.g., removing the Fab from the top of the antibody and linking the Fab to the Fc C-terminus with a linker of any length from 0-40 amino acids and fusing it to the N-terminus of either the VH or VL domain of the Fab), thereby creating an "upside-down" antibody, potentially with modulated affinity or avidity for binding to antigen and modulated effector function (e.g., Weber et al. Cell Reports 2018;22:149-62).

[0166] A variation may be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide, resulting in a change in the amino acid sequence compared to the sequence of the native antibody or polypeptide. In some embodiments, the amino acid substitution is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties (e.g., a conservative amino acid substitution, such as replacing leucine with serine). The insertion or deletion may optionally range from about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the parent molecule. In some embodiments, biologically useful and / or relevant amino acid sequence variations may be determined by systematically making insertions, deletions, or substitutions in the sequence and examining the activity of the resulting variant proteins compared to the parent protein.

[0167] In some embodiments, variants may include amino acid residues added to the amino and / or carboxyl termini of an antibody or polypeptide. The length of added amino acid residues may range from 1 to 100 or more residues. In some embodiments, variants include an N-terminal methionyl residue. In some embodiments, variants include additional polypeptides / proteins (e.g., Fc regions) to create fusion proteins. In some embodiments, variants may be engineered to be detectable and include detectable labels and / or proteins (e.g., fluorescent tags or enzymes).

[0168] In some embodiments, cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody are substituted or deleted to modulate the properties of the antibody (e.g., to improve oxidative stability and / or prevent aberrant disulfide bridges). Conversely, in some embodiments, one or more cysteine ​​residues are added to create disulfide bond(s) to improve stability.

[0169] In some embodiments, the antibodies of the present disclosure comprise hinge region variants that are unable to form disulfide bonds between identical heavy chains (e.g., reducing homodimer formation). In some embodiments, the antibodies comprise heavy chains with amino acid changes that alter electrostatic interactions. In some embodiments, the antibodies comprise heavy chains with amino acid changes that alter hydrophobic / hydrophilic interactions.

[0170] In some embodiments, the antibodies of the present disclosure are "deimmunized." Deimmunization of an antibody generally consists of the introduction of specific amino acid mutations (e.g., substitutions, deletions, additions) that result in the elimination of predicted T-cell epitopes without significantly reducing the binding affinity or other desired properties of the antibody.

[0171] Variant antibodies or polypeptides described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0172] In some embodiments, the anti-TfR1 antibodies described herein are chemically modified. In some embodiments, the anti-TfR1 antibodies are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linkage to a cellular ligand or other protein. Any of a number of chemical modifications can be performed by known techniques.

[0173] Generally speaking, antigen-antibody interactions are non-covalent and reversible, and are formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. When describing the strength of an antigen-antibody complex, the terms affinity and / or avidity are often used. The binding of an antibody to its antigen is a reversible process, and the affinity of the binding is usually measured using the equilibrium dissociation constant (K D ) is reported as K D is the ratio of the antibody's dissociation rate (koff) (how quickly the antibody dissociates from its antigen) to the antibody's association rate (kon) (how quickly the antibody binds to its antigen). In some embodiments, K D The k value is calculated by measuring the k and k rates of a particular antibody / antigen interaction and then using the ratio of these values ​​to determine the K D In some embodiments, the K D The K value is used to assess and rank the strength of individual antibody / antigen interactions. D The lower the avidity, the higher the affinity of the antibody for its target. In some embodiments, affinity is measured using SPR technology in the Biacore system. Avidity gives an indication of the overall strength of the antibody-antigen complex. It depends on three main parameters: (i) the affinity of the antibody for the target, (ii) the valency of both the antibody and the antigen, and (iii) the structural arrangement of the interacting moieties.

[0174] Binding characteristics of anti-TfR1 antibodies In some embodiments, the anti-TfR1 antibody has a K D In some embodiments, the anti-TfR1 antibody binds to TfR1 within a range (monovalent affinity). In some embodiments, the anti-TfR1 antibody has a K of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. D In some embodiments, the anti-TfR1 antibody binds to TfR1 with a K DIn some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K of about 0.1 nM or less. D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D (monovalent affinity). In some embodiments, the dissociation constant of an antibody to TfR1 is determined using TfR1 protein or a fragment thereof immobilized on a Biacore chip and flowing the binding agent across the chip. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to TfR1 is determined using the binding agent captured on a Biacore chip and flowing soluble TfR1 across the chip.

[0175] In some embodiments, the anti-TfR1 antibody has a K of about 5 nM or less. D In some embodiments, the anti-TfR1 antibody binds to TfR1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K of about 0.01 nM to about 2.5 nM. DIn some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K of about 0.1 nM to about 5 nM. D In some embodiments, the anti-TfR1 antibody binds to Tfr1 with a K of about 1 nM to about 5 nM. D It binds to Tfr1 with monovalent affinity.

[0176] In some embodiments, the anti-TfR1 antibody has a half maximal effective concentration (EC 50 ) (monovalent affinity). In some embodiments, the anti-TfR1 antibody binds to TfR1 with an EC 50 In some embodiments, the anti-TfR1 antibody binds to Tfr1 with an EC of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. 50 In some embodiments, the anti-TfR1 antibody binds to TfR1 with an EC 50 (monovalent affinity). In some embodiments, the anti-TfR1 antibody binds to cyno TfR1 and / or human TfR1 with an EC 50 It binds to TfR1 with monovalent affinity.

[0177] In some embodiments, EC 50 The value is determined by binding of an anti-TfR1 antibody to cells expressing TfR1 using fluorescence activated cell sorting (FACS). 50Values ​​are determined by assays on plates coated with the huTfR1 ectodomain, including but not limited to enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD).

[0178] Method for producing anti-TfR1 antibody The anti-TfR1 antibodies described herein may be produced by any suitable method known in the art. Such methods range from direct protein synthesis to constructing a DNA sequence encoding a polypeptide sequence and expressing the sequence in a suitable host. In some embodiments, the DNA sequence is constructed using recombinant techniques by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence may be mutagenized by site-directed mutagenesis to obtain a functional variant thereof. In some embodiments, the DNA sequence encoding the polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides may be designed based on the amino acid sequence of the desired polypeptide and on the codon selection that favors the host cell that will produce the recombinant polypeptide of interest. Standard methods may be applied to synthesize a polynucleotide sequence encoding the isolated polypeptide of interest. For example, the complete amino acid sequence may be used to construct a reverse-translated gene. Additionally, DNA oligomers may be synthesized that contain a nucleotide sequence encoding the particular polypeptide to be isolated. For example, several small oligonucleotides encoding portions of the desired polypeptide may be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0179] Once assembled (by synthesis, site-directed mutagenesis, or otherwise), a polynucleotide sequence encoding a particular polypeptide of interest may be inserted into an expression vector and operatively linked to appropriate expression control sequences for protein expression in a desired host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and / or expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order for a transfected gene to be expressed at high levels in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0180] In some embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies against human TfR1. For example, a recombinant expression vector can be a replicable DNA construct that includes a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an anti-TfR1 antibody operatively linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally includes an assembly consisting of (1) a genetic element(s) that has a regulatory role in gene expression (e.g., a transcriptional promoter or enhancer), (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Regulatory elements may include operator sequences to control transcription. Also included may be the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. DNA regions are "operatively linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide. A promoter is operatively linked to a coding sequence if it controls the transcription of the coding sequence. Or a ribosome binding site is operatively linked to a coding sequence if it is positioned to allow translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence that allows extracellular secretion of the translated protein by the host cell. In some embodiments, in situations where the recombinant protein is expressed without a leader or transport sequence, the polypeptide may include an N-terminal methionine residue. Optionally, this residue can then be cleaved from the expressed recombinant protein to obtain the final product.

[0181] The choice of expression control sequences and expression vectors generally depends on the choice of host. A wide variety of expression host / vector combinations can be employed. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids (such as plasmids derived from E. coli, including pCR1, pBR322, pMB9, and their derivatives), as well as plasmids with broader host ranges (such as M13 and other filamentous single-stranded DNA phages).

[0182] In some embodiments, the anti-TfR1 antibodies of the disclosure are expressed from one or more vectors. In some embodiments, the heavy chain polypeptide is expressed by one vector and the light chain polypeptide is expressed by a second vector. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed by one vector. Thus, the disclosure provides a vector encoding an anti-TfR1 antibody described herein. In one embodiment, the vector encodes a heavy chain polypeptide of an anti-TfR1 antibody described herein. In one embodiment, the vector encodes a light chain polypeptide of an anti-TfR1 antibody described herein. In one embodiment, the vector encodes a heavy chain polypeptide and a light chain polypeptide of an anti-TfR1 antibody described herein.

[0183] Suitable host cells for expressing anti-TfR1 antibodies or TfR1 proteins or fragments thereof for use as antigens or immunogens include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of suitable promoters. Prokaryotes include gram-negative or gram-positive bacteria (e.g., E. coli or Bacillus). Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems can also be employed. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts, as well as methods of protein production, including antibody production, are well known in the art.

[0184] A variety of mammalian culture systems can be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (from monkey kidney), L-929 (from mouse fibroblast), C127 (from mouse mammary tumor), 3T3 (from mouse fibroblast), CHO (from Chinese hamster ovary), HeLa (from human cervical carcinoma), BHK (from hamster kidney fibroblast), HEK-293 (from human embryonic kidney) cell lines and their variants. Mammalian expression vectors can include nontranscribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, as well as 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, and transcription termination sequences.

[0185] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those of skill in the art.

[0186] Thus, the present disclosure provides a cell comprising an anti-TfR1 antibody described herein. The present disclosure also provides a cell comprising one or more polynucleotides encoding an anti-TfR1 antibody described herein, or one or more vectors encoding an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a polynucleotide encoding an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a first polynucleotide encoding a heavy chain of an anti-TfR1 antibody described herein, and a second polynucleotide encoding a light chain of an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a polynucleotide encoding a heavy chain and a light chain of an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a vector encoding an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a first vector encoding a heavy chain of an anti-TfR1 antibody described herein, and a second vector encoding a light chain of an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a vector encoding a heavy chain and a light chain of an anti-TfR1 antibody described herein. In some embodiments, the cells produce an anti-TfR1 antibody described herein. In some embodiments, the cells produce an antibody. In some embodiments, the cells produce an antibody that binds to human TfR1. In some embodiments, the cells produce an antibody that binds to cyno TfR1. In some embodiments, the cells produce an antibody that binds to human TfR1 and cyno TfR1. In some embodiments, the cells produce an antibody designated ANTIBODY-A. In some embodiments, the cells produce a humanized version of ANTIBODY-A. In some embodiments, the cells produce an antibody designated ANTIBODY-B. In some embodiments, the cells produce a humanized version of ANTIBODY-B. In some embodiments, the cells are prokaryotic cells (e.g., E. coli). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are hybridoma cells.

[0187] The protein produced by the host cell can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Purification can be facilitated by attaching affinity tags (such as hexahistidine (SEQ ID NO: 209), maltose binding domain, influenza envelope sequence, and glutathione-S-transferase) to the protein and passing it through a suitable affinity column. Affinity chromatography used to purify immunoglobulins includes, but is not limited to, protein A, protein G, and protein L chromatography. The isolated protein can be physically characterized using techniques known to those skilled in the art, including, but not limited to, proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography. The purity of the isolated proteins can be measured using techniques known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).

[0188] In some embodiments, supernatants from expression systems that secrete recombinant protein into culture medium are first concentrated using commercially available protein concentration filters (e.g., Amicon® or Millipore Pellicon® ultrafiltration units). Following the concentration step, the concentrate may be applied to a suitable purification matrix. In some embodiments, an anion exchange resin (e.g., a matrix or substrate with pendant diethylaminoethyl (DEAE) groups) is employed. The matrix may be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. In some embodiments, a cation exchange step is employed. Suitable cation exchangers include a variety of insoluble matrices that contain sulfopropyl or carboxymethyl groups. In some embodiments, hydroxyapatite supports are employed, including but not limited to ceramic hydroxyapatite (CHT). In some embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC supports (e.g., silica gel with pendant methyl or other aliphatic groups) are employed to further purify the recombinant protein. In some embodiments, hydrophobic interaction chromatography (HIC) is used to separate recombinant proteins based on their hydrophobicity. HIC uses conditions and matrices that function under less denaturing conditions than some other techniques, making it a useful separation technique for purifying proteins while maintaining biological activity. Some or all of the above purification steps can be used in various combinations to obtain homogeneous recombinant proteins.

[0189] In some embodiments, the antibodies of the disclosure are Fabs, which can be prepared by first producing intact monoclonal Abs, followed by chemical or enzymatic cleavage (e.g., pepsin, papain, or ficin digestion) of the monoclonal antibodies to produce F(ab')s. 2Fragments can be obtained by obtaining a Fab fragment, followed by reducing the fragment to obtain a Fab fragment. Such techniques are known in the art. See, e.g., Victor CG et al., Biosensors and Bioelectronics, 2016(85):32-45. Alternatively, the antibodies of the present disclosure can be produced as F(ab') 2 It is produced by recombinantly synthesizing an antibody fragment, followed by chemical reduction of the fragment to provide the Fab units.

[0190] Polynucleotides In some embodiments, the disclosure encompasses polynucleotides that include polynucleotides that encode a polypeptide (e.g., an anti-TfR1 antibody) described herein. The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of a polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotides include polynucleotides (e.g., nucleotide sequences) that encode the heavy chain of an anti-TfR1 antibody described herein. In some embodiments, the polynucleotides include polynucleotides (e.g., nucleotide sequences) that encode the light chain of an anti-TfR1 antibody described herein. In some embodiments, the polynucleotides include polynucleotides (e.g., nucleotide sequences) that encode the heavy chain of an anti-TfR1 antibody described herein, and polynucleotides (e.g., nucleotide sequences) that encode the light chain of an anti-TfR1 antibody described herein.

[0191] In some embodiments, the polynucleotide comprises a polynucleotide (eg, a nucleotide sequence) that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 4-164. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) that encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100.

[0192] In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising two or more amino acid sequences selected from the group consisting of any one of SEQ ID NOs: 4-164. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 93 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 95 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 97 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 98 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 99 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 100.

[0193] In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:4 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:34. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:12 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:15 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:16 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:17 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:18 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:35.

[0194] In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:19 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:30 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:41. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:31 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:41. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:32 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:41. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:33 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:33 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:43. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising the amino acid sequence of SEQ ID NO:33 and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:44.

[0195] The disclosure also provides variants of the polynucleotides described herein, such as those encoding fragments, analogs, and / or derivatives of the polypeptides. In some embodiments, the disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide described herein.

[0196] In some embodiments, the polynucleotide comprises a polynucleotide having a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-164. Also provided are polynucleotides comprising a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-164. In some embodiments, the hybridization is under high stringency conditions, as known to those of skill in the art.

[0197] As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% identical to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or some nucleotides may be inserted into the reference sequence up to 5% of the total nucleotides in the reference sequence.These mutations of the reference sequence may occur at the 5' or 3' end position of the reference nucleotide sequence, or at any position between these end positions, interspersed among the nucleotides in the reference sequence individually, or interspersed in one or more consecutive groups within the reference sequence.

[0198] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not change the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). In some embodiments, polynucleotide variants contain one or more mutated codons, including one or more (e.g., one, two, or three) substitutions for a codon that change the amino acid encoded by that codon. Methods for introducing one or more substitutions into a codon are known in the art, such as PCR mutagenesis and site-directed mutagenesis. Polynucleotide variants may be produced for a variety of reasons, such as to optimize codon expression for a particular host (e.g., to change the codons of human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0199] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0200] In some embodiments, a polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a polynucleotide that aids in the expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control trafficking of the polypeptide). The polypeptide may have a leader sequence that is cleaved by the host cell to form a "mature" form of the polypeptide.

[0201] In some embodiments, a polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame with a marker or tag sequence. For example, in some embodiments, the marker sequence is a hexahistidine (SEQ ID NO: 209) tag (HIS tag), which allows for efficient purification of the polypeptide fused to the marker. In some embodiments, when a mammalian host (e.g., COS-7 cells) is used, the marker sequence is a hemagglutinin (HA) tag, derived from the influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG™ tag. In some embodiments, the marker is used in combination with other markers or tags.

[0202] In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure.

[0203] Vectors and cells Also provided are vectors and cells comprising any one of the polynucleotides described herein. In some embodiments, an expression vector comprises a polynucleotide molecule encoding an anti-TfR1 antibody described herein. In some embodiments, an expression vector comprises a polynucleotide molecule encoding a polypeptide that is a part of an anti-TfR1 antibody described herein. In some embodiments, an expression vector comprises a polynucleotide molecule encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, an expression vector comprises a polynucleotide molecule encoding a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, an expression vector comprises a polynucleotide molecule encoding a heavy chain polypeptide and a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, a host cell comprises an expression vector comprising a polynucleotide molecule encoding an anti-TfR1 antibody described herein. In some embodiments, a host cell comprises an expression vector comprising a polynucleotide molecule encoding a polypeptide that is a part of an anti-TfR1 antibody described herein. In some embodiments, a host cell comprises a polynucleotide molecule encoding an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule encoding a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a first polynucleotide encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein and a second polynucleotide encoding a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises (ii) a first expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein, and (ii) a second expression vector comprising a polynucleotide molecule encoding a light chain polypeptide of an anti-TfR1 antibody described herein.

[0204] Analysis of the physical / chemical properties of anti-TfR1 antibodies The anti-TfR1 antibodies of the present disclosure may be analyzed for their physical / chemical properties and / or biological activity by various methods known in the art. In some embodiments, the anti-TfR1 antibodies are tested for their ability to bind to TfR1 (e.g., human TfR1 and / or cyno TfR1). Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS. In some embodiments, the anti-TfR1 antibodies are tested for their ability to inhibit, reduce, or block transferrin binding to the TfR1 receptor. In some embodiments, the anti-TfR1 antibodies are tested for their ability to inhibit, reduce, or block TfR1 activity. In some embodiments, the anti-TfR1 antibodies are tested for their ability to co-internalize with TFR1 and induce increased TfR1 internalization. In addition, the antibodies may be evaluated for solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency.

[0205] In some embodiments, monoclonal antibodies generated against TfR1 are grouped based on the epitope each antibody recognizes, in a process known as "epitope binning." Generally, antibodies are tested in a pairwise combinatorial fashion, and antibodies that compete with each other are grouped into bins. For example, in a premix binning assay, a primary antibody is immobilized on a surface, and a premix solution of a secondary antibody and antigen is flowed over the immobilized primary antibody. In parallel, a target protein is immobilized on a surface, and two antibodies are flowed over the immobilized antigen, and the two antibodies compete to bind to the target. From this technique, antibodies that block each other can be identified. A competitive blocking profile is created for each antibody compared to the other antibodies. The results determine which bin each antibody falls into. High-throughput methods of epitope binning are known in the art, which allow for the screening and characterization of a large number of antibodies. Antibodies that bind to similar epitopes often share similar functions. Conversely, antibodies that bind to different epitopes may have different functional activities.

[0206] In some embodiments, the epitope bin comprises at least one antibody from the group consisting of ANTIBODY-A and ANTIBODY-B.

[0207] Epitope mapping is a method to identify the binding site, region, or epitope on a target protein to which an antibody (or other binding agent) binds. A variety of methods for mapping epitopes on a target protein are known in the art. These methods include mutagenesis (including but not limited to shotgun mutagenesis, site-directed mutagenesis, and alanine scanning), domain or fragment scanning, peptide scanning (e.g., Pepscan technology), display (e.g., phage display, microbial display, and ribosome / mRNA display), methods involving proteolysis and mass spectrometry, and structure determination (e.g., X-ray crystallography and NMR). Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ).

[0208] In some embodiments, the anti-TfR1 antibodies described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

[0209] In some embodiments, an assay is provided to identify anti-TfR1 antibodies that affect TfR1 activity. In some embodiments, SPR, ELISA, or FACS assays are used to evaluate the ability of anti-TfR1 antibodies to block TfR1 binding. In some embodiments, cytotoxicity assays are used to evaluate the ability of anti-TfR1 antibodies to affect natural killer (NK) cell activity. In some embodiments, proliferation assays are used to evaluate the ability of anti-TfR1 antibodies to affect T cell activity.

[0210] In some embodiments, the anti-TfR1 antibodies described herein are antagonists of human TfR1. In some examples, the terms "inhibit", "induce", "reduce", "increase", "enhance" are compared to the level / activity before treatment with a conjugate comprising an anti-TfR1 antibody. In some examples, the terms "inhibit", "induce", "reduce", "increase", "enhance" are compared to the level / activity before treatment with a conjugate comprising an anti-TfR1 antibody.

[0211] Anti-TfR1 antibody conjugates, fusions, and complexes The present disclosure also provides conjugates, fusion proteins, and complexes comprising the anti-TfR1 antibodies described herein. In some embodiments, the anti-TfR1 antibodies of the present disclosure can be conjugated to a second molecule. In some embodiments, the anti-TfR1 antibodies are conjugated to a cytotoxic agent or cytotoxic moiety. In some embodiments, the anti-TfR1 antibodies are conjugated to a cytotoxic agent to form an ADC (antibody drug conjugate).

[0212] Conjugates comprising the anti-TfR1 antibodies described herein can be prepared using any suitable method known in the art. In some embodiments, the components of the conjugate are linked by covalent interactions. In some embodiments, the conjugates are prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0213] In some embodiments, the anti-TfR1 antibodies described herein are conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and / or detection. Detectable substances include, but are not limited to, enzymes (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase), prosthetic groups (such as biotin and flavin(s)), fluorescent substances (such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin), bioluminescent substances (such as luciferase), radioactive substances (such as riboflavin, ... 212 Bi, 14 C. 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H, 166 Ho, 131 I, 125 I, 123 I, 121 I, 115 In, 113 In, 112 In, 111 In, 140 La, 177 Lu, 54 Mn, 99 Mo, 32 P, 103 Pd, 149 Pm, 142 Pr, 186 Re, 188 Re, 105 Rh, 97 Ru, 35 S, 47 Sc, 75 Se, 153 Sm, 113 Sn,117 Sn, 85 Sr, 99m Tc, 201 Ti, 133 Xe, 90 Y, 69 Yb, 175 Yb, 65 Zn, positron emitting metals, and magnetic metal ions.

[0214] The anti-TfR1 antibodies described herein can also be conjugated to a second antibody to form an antibody heteroconjugate.

[0215] The anti-TfR1 antibody described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized anti-TfR1 antibody is used in immunoassays. In some embodiments, the immobilized anti-TfR1 antibody is used in the purification of target antigen (e.g., human TfR1 or cyno TfR1).

[0216] In some embodiments, the anti-TfR1 antibody of the present disclosure may be conjugated to a molecule or drug, such as a nucleic acid (e.g., an antisense oligonucleotide, a short interfering RNA (siRNA), an RNA (messenger RNA (mRNA), microRNA (miRNA), guide RNA (gRNA), etc.), a phosphoramidate morpholino oligomer, or an aptamer, etc.). In some embodiments, the anti-TfR1 antibody is conjugated to a particle (e.g., a lipid particle or a nanoparticle) that can contain a therapeutic agent, such as those described herein. In some embodiments, the anti-TfR1 antibody is conjugated to a viral particle (e.g., a viral particle containing a therapeutic nucleic acid and / or protein, e.g., a viral particle for gene therapy). The anti-TfR1 antibody may be linked to the drug by a linker. Methods for preparing antibody-nucleic acid conjugates, such as the conjugates contemplated in the present disclosure, are well known in the art. See, for example, U.S. Patent Application Publication No. US20190240346, as well as U.S. Patent Nos. US10881743 and US10550188, and International Patent Application Publication No. WO1991004753, the disclosures of which are incorporated by reference in their entireties.

[0217] In some embodiments, fusion proteins comprising the anti-TfR1 antibodies described herein may be produced using any suitable method known in the art. Such fusion proteins may include fusions of the anti-TfR1 antibodies of the present disclosure described herein (including bispecific, multispecific, or multivalent anti-TfR1 antibodies) with a therapeutic polypeptide or protein. In one example, avidin may be added to the heavy chain C-terminus to produce a fusion protein, as described in Candelaria PV et al. Front Immunol. 2021; 12: 607692. The fusion protein may be further conjugated or complexed with a second molecule or drug, such as a biotinylated drug, as described in Daniels TR, et al. Biochim Biophys Acta. 2012; 1820 (3): 291-317.

[0218] In some embodiments, a complex comprising the anti-TfR1 Ab described herein can be produced using any suitable method known in the art. In some embodiments, the components of the complex are linked by non-covalent interactions. Such compounds include an anti-TfR1 antibody conjugated to another agent (e.g., a therapeutic agent), or an anti-TfR1 antibody conjugated to a lipid or nanoparticle (bearing a therapeutic polypeptide or protein).

[0219] Tissue targeting and uses of anti-TfR1 antibodies In some embodiments, the antibodies of the present disclosure can be used to target muscle tissue (e.g., skeletal or voluntary muscles, cardiac muscles, smooth muscles, etc.). In some embodiments, described herein is a method of treating muscle atrophy or myotonic dystrophy in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate / fusion / complex comprising an anti-TfR1 antibody described herein. In some examples, the muscle atrophy is associated with and / or induced by cachexia (e.g., cancer cachexia), denervation, myopathy, motor neuron disease, diabetes, chronic obstructive pulmonary disease, liver disease, congestive heart failure, chronic renal failure, chronic infection, sepsis, fasting, sarcopenia, glucocorticoid-induced atrophy, disuse, or space flight. In some cases, the myotonic dystrophy is DM1. Examples of muscle diseases that can be treated with conjugates comprising the anti-TfR1 antibodies of the present disclosure include, but are not limited to, muscle diseases, muscle atrophy, arteriosclerotic diseases, heart-related diseases, and lysosomal storage diseases.

[0220] Examples of muscle diseases include muscular dystrophies (e.g., Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (Becker MD), Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, limb-girdle muscular dystrophy, Fukuyama congenital muscular dystrophy, myotonic dystrophy, periodic paralysis, diaphragmatic paralysis, diaphragmatic atony, distal myopathy, myotonic syndrome, mitochondrial disease, and muscle wasting diseases.

[0221] Examples of muscle atrophy include sarcopenia (age-related muscle atrophy), disuse muscle atrophy, cachexia, amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy.

[0222] Examples of diseases associated with muscle wasting include DMD, Becker MD, Limb-girdle MD, Myotonic MD and Facioscapulohumeral Muscular Dystrophy (FSHD), myositis, myopathies (including hereditary and acquired myopathies), motor neuron diseases (such as Lou Gehrig's disease or ALS), and neurodegenerative diseases (such as Parkinson's disease, Huntington's disease and Alzheimer's disease).

[0223] An example of an arteriosclerotic disease is peripheral arterial occlusive disease.

[0224] Examples of heart-related diseases include angina pectoris (including exertional angina and variant angina), acute coronary syndrome (including unstable angina, acute myocardial infarction, and post-myocardial infarction heart failure), heart failure (including HFrEF, HFpEF, acute heart failure, chronic heart failure, and decompensated heart failure), cardiomyopathies (including dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy), cor pulmonale, asymptomatic myocardial ischemia, arrhythmias (conduction disorders, sinus node dysfunction, ectopic supraventricular rhythms, atrioventricular block, atrial fibrillation, atrial flutter, reentrant supraventricular tachycardia (SVT, PSVT), Wolff-Parkinson-White (WPW) syndrome, leg block, and pulmonary artery disease. These include the maintenance of cardiac function and prevention of cardiac events during non-pharmacological therapy for severe heart failure (e.g., intra-aortic balloon pumping, ventricular assist device, Batista surgery, cell transplantation, gene therapy, heart transplantation), valvular abnormalities (including aortic regurgitation, aortic stenosis, mitral valve prolapse (MVP), mitral regurgitation, mitral stenosis, pulmonary regurgitation, pulmonary artery stenosis, tricuspid regurgitation, tricuspid stenosis), endocarditis, cardiac tumors, decreased cardiac function after cardiopulmonary bypass surgery, and prevention of cardiac events during non-pharmacological therapy for severe heart failure (e.g., intra-aortic balloon pumping, ventricular assist device, Batista surgery, cell transplantation, gene therapy, heart transplantation). For a list of diseases that may be treated with conjugates comprising the anti-TfR1 antibodies of the present disclosure, see, e.g., U.S. Patent Application Publication No. US20190240346, as well as U.S. Pat. Nos. US10881743 and US10550188, and International Patent Application Publication No. WO1991004753, the disclosures of which are incorporated by reference in their entireties.

[0225] In addition to muscle, the antibodies of the present disclosure may be used to target other tissues in which TfR1 is expressed. Examples of such tissues include, but are not limited to, hepatocytes, immune cells, tumors, and brain tissue. Transferrin receptors are involved in many types of diseases, including anemia, neurodegenerative diseases, and cancer. TfR is known to be expressed in liver tissue and is involved in liver iron overload in alcoholic liver disease (ALD) (Suzuki Y, et al., Alcohol Clin Exp Res. 2002 Aug; 26 (8 Suppl): 26S-31S). TfR is also expressed in endothelial cells of the blood-brain barrier (Johnsen KB, et al. Progress in Neurobiology. 181: 101665). Furthermore, TfR1 is known to be abnormally expressed in various cancers (Shen Y, et al. Am J Cancer Res. 2018; 8 (6): 916-931). Expression of TfR also increases in response to immune cell activation (Harel E, et al. PLoS ONE 2011;6(9):e24202). TfR is expressed on proliferating cells, is required for their proliferation, and can be detected after mitogenic stimulation of normal peripheral blood T and B cells (Neckers, LM et al., J Immunol November 1, 1984,133(5)2437-2441).

[0226] Pharmaceutical Compositions The present disclosure provides compositions comprising the anti-TfR1 antibodies described herein. The present disclosure also provides pharmaceutical compositions comprising the anti-TfR1 antibodies described herein and a pharma- ceutically acceptable vehicle.

[0227] The formulation is prepared by combining the anti-TfR1 antibody of the present disclosure with a pharma- ceutically acceptable vehicle (e.g., a carrier or excipient) for storage and / or use. Generally, pharma- ceutically acceptable carriers, excipients, and / or stabilizers are considered by those skilled in the art to be inactive ingredients of a formulation or pharmaceutical composition.

[0228] Suitable pharma- ceutically acceptable vehicles include, but are not limited to, non-toxic buffers (such as phosphate, citric acid, and other organic acids), salts (such as sodium chloride), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol), alkyl parabens (such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides (e.g., less than about 10 amino acid residues), protein Examples of suitable surfactants include proteins (such as serum albumin, gelatin, or immunoglobulin), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), carbohydrates (such as monosaccharides, disaccharides, glucose, mannose, or dextrin), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (such as Zn-protein complexes), and nonionic surfactants (such as TWEEN (registered trademark) or polyethylene glycol (PEG)) (Remington: The Science and Practice of Pharmacy, 22 nd Edition, 2012, Pharmaceutical Press, London. In some embodiments, the formulation is in the form of an aqueous solution. In some embodiments, the formulation is in lyophilized or alternatively dried form.

[0229] Therapeutic preparations may be in unit dosage form. Such preparations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form solid preformulation compositions containing a homogeneous mixture of the compounds of the present disclosure, or pharma-ceutically acceptable non-toxic salts thereof. The solid preformulation compositions are then subdivided into unit dosage forms of the type described above. Tablets, pills, etc. of the preparation or composition can be coated or otherwise compounded to obtain a dosage form that offers the advantage of prolonged action. For example, a tablet or pill can include an inner composition covered with an outer component. Moreover, the two components can be separated by an enteric layer which serves to resist disintegration and permit the inner component to pass intact through the stomach or to be delayed in release of the inner component. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0230] The binding agent of the present disclosure may be formulated in any form suitable for delivery to target cells / tissues. In some embodiments, the anti-TfR1 antibody may be formulated as a liposome, a microparticle, a microcapsule, an albumin microsphere, a microemulsion, a nanoparticle, a nanocapsule, or a macroemulsion. In some embodiments, the pharmaceutical formulation comprises an anti-TfR1 antibody of the present disclosure complexed with a liposome. Methods for obtaining liposomes are known to those skilled in the art. For example, some liposomes may be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).

[0231] In some embodiments, anti-TfR1 antibody is formulated as a sustained release preparation. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the drug, the matrices being in the form of shaped articles (e.g., films or microcapsules). Sustained release matrices include, but are not limited to, polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate)), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0232] The pharmaceutical compositions or formulations of the present disclosure can be administered in many ways, either for local or systemic treatment. In some embodiments, administration is topical, by epidermal or transdermal patch, ointment, lotion, cream, gel, drop, suppository, spray, liquid, and powder. In some embodiments, administration is intrapulmonary, intratracheal, and intranasal, by inhalation or insufflation of powder or aerosol, including by nebulizer. In some embodiments, administration is oral. In some embodiments, administration is parenteral, including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular) administration. In some embodiments, administration is by intravenous injection or infusion. In some embodiments, administration is by intramuscular injection.

[0233] Various delivery systems are known and can be used to administer the anti-TfR1 antibodies described herein. In some embodiments, the anti-TfR1 antibodies or compositions described herein are delivered in a controlled or sustained release system. In some embodiments, a pump is used to achieve controlled or sustained release. In some embodiments, a polymeric material is used to achieve controlled or sustained release of the anti-TfR1 antibodies herein. Examples of polymers used in sustained release formulations include, but are not limited to, poly 2-hydroxyethyl methacrylate, polymethyl methacrylate, polyacrylic acid, polyethylene-co-vinyl acetate, polymethacrylic acid, polyglycolide (PLG), polyanhydrides, poly N-vinylpyrrolidone, polyvinyl alcohol (PVA), polyacrylamide, polyethylene glycol (PEG), polylactide (PLA), polylactide-co-glycolide (PLGA), and polyorthoesters. Any polymer used in a sustained release formulation must be inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

[0234] Additional delivery systems can be used to administer the anti-TfR1 antibodies described herein, including, but not limited to, injectable drug delivery devices and osmotic pumps. Injectable drug delivery devices include, for example, handheld devices (e.g., autoinjectors) or wearable devices. Various types of osmotic pump systems can include single compartment systems, dual compartment systems, and multi-compartment systems.

[0235] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that a specific material is mentioned, it is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. EXAMPLES

[0236] Example 1: Production of rabbit anti-transferrin receptor 1 (TfR1) monoclonal antibody Rabbit anti-TfR1 monoclonal antibody was produced by the following process. New Zealand white rabbits were immunized twice with cyno transferrin receptor as an immunogen, and then boosted with human transferrin receptor. Serum responses to human and cyno transferrin receptor were confirmed by binding to Ba / F3 cell lines expressing human and cyno transferrin receptor. Whole blood was collected 2 weeks after the last immunization, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque.

[0237] To generate monoclonal antibodies, cyno-transferrin receptor was immobilized on plates, incubated with PBMCs, and used to capture B cells with appropriate specificity. The attached B cells were incubated for 1 week in cell culture conditions that promote differentiation and proliferation of antibody-secreting cells. Supernatants from individual wells were assessed for human and cyno-transferrin receptor reactivity using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The ELISA assay measured binding to transferrin receptor complexed with holo-transferrin. The format used for the ELISA entailed coating human or cyno-transferrin receptor on a 96-well plate and then adding holo-transferrin to each well. After 1.5 hours of incubation, diluted supernatants were added to the plate for 1 hour. Plate-bound antibodies were detected with anti-rabbit IgG HRP reagent. The FACS assay used the Baf / 3 cell line expressing human and cyno-transferrin receptor. B cells from wells that were positive for both human transferrin receptor and cyno transferrin receptor were collected, and the VH and VL antibody sequences were examined.

[0238] RNA was isolated from B cells and used for cDNA synthesis. Heavy and light chains (Vkappa) were PCR amplified and cloned into pCR4 vector by TOPO / TA cloning. The cloned products were transformed into E. coli and resistant colonies were sequenced using Sanger sequencing.

[0239] Example 2: Selection of chimeric antibodies RabANTIBODY-A and RabANTIBODY-B Consensus VH and VL sequences were identified and used to express the resulting rabbit antibodies with human IgG1 frameworks. Chimeric rabbit antibody candidates were transiently expressed in Chinese Hamster Ovary S (CHO-S) cells and purified. Binding to human and cyno TfR1 was confirmed using FACs binding to Ba / F3 cells overexpressing human or cyno TfR1, and affinity to recombinant extracellular domains of human or cyno TfR1 was then measured using SPR analysis.

[0240] Two antibodies (RabANTIBODY-A and RabANTIBODY-B) were selected for humanization based on the following properties: 1) Single-digit nM monovalent affinity for human TfR1; 2) Cross-reactivity to cyno TfR1, 3) The difference in affinity between cyno TfR1 and human TfR1 is less than 10x; 4) No cross-reactivity to TfR2; and 5) The epitope does not overlap with the transferrin-binding region of TfR1.

[0241] The amino acid sequences of the complementarity determining regions (CDRs) and mature heavy and light chain variable regions of RabANTIBODY-A and RabANTIBODY-B are shown below. The CDRs described below and herein include the union of all positions in the CDR definition of Kabat (Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS & Foeller, C. (1991) and the CDR definition of Chothia (Chothia, C. & Lesk, AMJ Mol. Biol (1987) 196, 901-917) (Chothia, C. et al. Nature (1989) 342, 877-883) (Al-Lazikani, B., Lesk, AM & Chothia, CJ Mol. Biol (1997) 273, 927-948). This "union" definition of CDR is also known as the "Wolfguy" definition by Bujotzek et al. (Bujotzek A,. (2015) Proteins Apr;83(4):681-95), shown herein in Table 6 and as the underlined positions in the sequence alignments of Figures 1A-1D. The Chothia definitions of CDRs are shown herein in Table 7 and as the underlined positions in the sequence alignments of Figures 2A-2D. [Table 6-1] [Table 6-2] [Table 7] [ka] [ka]

[0242] Example 3: Humanization of anti-TfR1 antibodies The RabANTIBODY-A and RabANTIBODY-B antibodies were humanized using the method described in U.S. Patent No. 8,961,976 (incorporated herein in its entirety by reference). Briefly, the method entails grafting mature non-human CDRs into a selected human framework, followed by the introduction of "back mutations" of human framework residues into the amino acid sequence of the mature non-human sequence to preserve stability and binding affinity. Furthermore, in order to minimize the risk of immunogenicity, where it is predicted that the resulting more human-like sequence should not significantly compromise binding affinity, attempts have been made to (i) use human sequences in some sections of the CDRs that may not affect affinity, and (ii) select "forward mutations" of some non-human CDR residues to the amino acid sequence of the human antibody. Such a method is described in U.S. Patent No. 8,349,324 (incorporated herein in its entirety by reference).

[0243] Step 1: Select the human acceptor framework: Selection methods included: (a) A combined approach to sequence comparison that scores all human germline candidate sequences and places greater weight on matches at specific structurally important positions; (b) evaluating each mismatch position with the aid of a 3D structural homology model; and (c) A slight preference for humVH3 over humVH1 due to the typically greater stability and SpA binding potential of humVH3, and a slight preference for humVK1 over humVK3 due to its longer history of use in humanizing rabbit antibodies.

[0244] For ANTIBODY-A, the human germline humIGHV3-72*1+humIGHJ1*1 and humIGKV1-5*1+humIGKJ4*1 were selected as the human acceptor frameworks. For ANTIBODY-B, the human germline humIGHV3-30*1+humIGHJ4*1 and humIGKV1-5*1+humIGKJ4*1 were selected as the human acceptor frameworks.

[0245] Step 2: Design the most human-like designs, H0 and L0: The most human-like designs were named "H0" and "L0", which are CDR-grafted sequences, potentially adding some human CDR residues by using sections of human CDR sequences and / or by using "forward mutations", as described above. CDRs are defined to include the union of all positions in the CDR definition of Kabat (Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit. National Institutes of Health, Bethesda, MD) and the CDR definition of Chothia (Chothia, C. & Lesk, AMJ Mol. Biol (1987) 196, 901-917) (Chothia, C. et al. Nature (1989) 342, 877-883) (Al-Lazikani, B., Lesk, AM & Chothia, CJ Mol. Biol (1997) 273, 927-948). This "union" definition of CDRs is also known as the "Wolfguy" definition by Bujotzek et al. (Bujotzek A et al., Proteins Apr;83(4):681-95). This "union" definition of CDRs is shown herein as the underlined positions in the sequence alignments of Figures 1A-1D. AHo position numbering is used throughout this humanization section. See, for example, Honegger A & Plueckthun A, JMB 2001 309:657-670. For each of ANTIBODY-A and ANTIBODY-B, design H0 included AHo# C42A C57A (removing an extra disulfide bond from the mature rabbit CDRs). For ANTIBODY-A, design L0 included human CDR-L2, excluding rabbit R58. For ANTIBODY-B, design L0 contained mature rabbit unpaired cysteine ​​Q108 instead of 108C in CDR-L3, L137 in CDR-L3, R24 in CDR-L1, and E71 in CDR-L2.

[0246] Step 3: Study the predicted structure and construct a list of suggested mutations: For each rabbit / human mismatch, and for many of their neighboring structures, rational design (and in some cases computational energy calculations) was used to predict how mutations to mature rabbit amino acids, or in some cases other newly occurring amino acids, might improve or risk stability, might be required to preserve binding affinity, or might increase the risk of immunogenicity.

[0247] For ANTIBODY-A VH, the mutations used included the set {E1-(deletion)V2Q Q3S}, Q141P, A24K, the set {A42C A57C}, the set {ASV 72-74 SWA} in the "CDR-H2 Kabat extension," and the set {RDDSKNSL 82-89 KT-SSTTV} ("RDDSKNSL" disclosed as SEQ ID NO:210 and "KT-SSTTV" disclosed as SEQ ID NO:211) or the set {R82K D84- L89V} in the so-called "CDR-H4" framework loop, which is shorter than that of the mature rabbit sequence.

[0248] For ANTIBODY-B VH, the mutations used included the set {Q1-(deletion)V2Q Q3S}, Q141P, the set {A42C A57C}, A56G which may affect the 42C 57C disulfide bond, the set {DSVK 72-75 SWAR} in the "CDR-H2 Kabat extension" ("DSVK" disclosed as SEQ ID NO:212 and "SWAR" disclosed as SEQ ID NO:213), the set {A24K N87T} and / or D83T and / or N84-(deletion) and / or the set {N87T A24K} and / or L89V in the so-called "CDR-H4" framework loop which is shorter than that of the mature rabbit sequence.

[0249] For the ANTIBODY-A VK, the mutations used include C98P, the set DIQ 1-3 ELD, and the set S69T E71A.

[0250] For ANTIBODY-B VK, mutations used included C98P, Q108C to reintroduce the unpaired cysteine ​​in the mature rabbit sequence, the set {DIQM 1-4 ELVL} ("DIQM" disclosed as SEQ ID NO:214, and "ELVL" disclosed as SEQ ID NO:215), Q108A to try amino acids as small as 108C but without the potential for inappropriate disulfide bonding, and L137N.

[0251] Step 4: Classify the mutations into several designs: To triage the predicted risks, the mutations of interest were grouped into some VH designs and some VK designs. Immunogenicity risks were predicted by comparison with human germline and CD4 episcore methods. See, for example, Dhanda et.al., Frontiers in Immunology, 2018, 9, 1369. For each of ANTIBODY-A and ANTIBODY-B, the number of designs was doubled to test rabbit extra disulfide VH 42C 57C (in the "C" design), as well as human VH A42 A57.

[0252] For ANTIBODY-A, 10 VH designs (H0, H1, H2, H3, H4, H1C, H2C, H3C, and H4C) and 3 VL designs (L0, L1, and L2) were generated.

[0253] For ANTIBODY-B, ten VH designs and three VL designs were generated for similar, but not identical, reasons as for ANTIBODY-A.

[0254] The humanized variants are shown in Figures 1A-1D with Union CDRs. In Figures 2A-2D, the humanized variants are shown with Chothia CDRs. Various combinations of humanized heavy and light chain plasmids were transiently expressed in CHO-S cells. The titers in the supernatants were estimated using Octet anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor.

[0255] FACS binding assays were performed with Ba / F3 cells overexpressing either human TfR1 or cyno TfR1. Cells were dispensed into U-bottom 96-well plates at 50,000 cells / well / 50 μl FACS buffer and placed on ice. Serially diluted Fab supernatants or purified Fabs were then added to the cells followed by incubation on ice. Cells were washed twice with FACS buffer and secondary antibodies were added to the wells at a dilution of 1:400 followed by incubation on ice for 30 min. Cells were washed twice with FACS buffer and fixed with 2% PFA prepared in PBS. Finally, flow cytometry analysis was performed by measuring PE fluorescence.

[0256] Tables 8 and 9 show the expression titers and ECs, as measured by Octet, of the humanized versions of ANTIBODY-A (Table 8) and ANTIBODY-B (Table 9) derived from supernatants from transient transfections. 50 Octet titers were estimated using anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor. FACS binding assays were performed with serially diluted supernatants. EC 50 Values ​​were determined from best-fit curves using GraphPad Prism. ~=approximate EC estimated by Prism. 50 >=EC greater than the value estimated by GraphPad Prism 50 . NA=EC 50 Fabs that showed strong affinity to both human and cyno TfR1 were purified and the FACS binding assay was repeated with the purified material. [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2]

[0257] Tables 10 and 11 show the ECs of the humanized versions of ANTIBODY-A and ANTIBODY-B selected from the purified products. 50 The FACS binding assay was performed using serially diluted purified products. EC 50 Values ​​were determined from best-fit curves using GraphPad Prism. [Table 10] [Table 11] The complete heavy and light chain sequences of the above exemplary constructs are shown in Table 12 below. [Table 12-1] [Table 12-2]

[0258] Example 4: Continued anti-TfR1 antibody design and selection process to remove excess disulfides and unpaired cysteines In the initial design, the redundant rabbit VH disulfide bond was removed by mutating C35 C50 (AHo # C42 C57) from C,C to A,A. Additionally, the unpaired cysteine ​​C90 (AHo # C108) of ANTIBODY-B VL was removed by mutating it to Q or A. These attempts were found to result in weaker binding affinity in the constructs tested, and therefore subsequent rounds of design were performed. For ANTIBODY-A H3 / L0 and for ANTIBODY-B H1 / L2, the second disulfide C,C was mutated to S,V or S,R or S,Y or H,A, respectively, borrowed from various similar human germline sequences, and then validated by molecular modeling, energy theory calculations, and / or publicly available structures. For ANTIBODY-B H1C / L2, and for ANTIBODY-B H4C / L2, the unpaired cysteine ​​was mutated to S or T or V. The subsequent designs, which removed the extra disulfide and the unpaired cysteine, are shown in Figures 1A-1D.

[0259] Table 13 shows the expression titers and EC2s measured by Octet for humanized version variants of anti-TfR1 antibodies ANTIBODY-A and ANTIBODY-B derived from supernatants from transient transfections. 50 Octet titers were estimated using anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor. FACS binding assays were performed with serially diluted supernatants. EC 50 Values ​​were determined from best-fit curves using GraphPad Prism. [Table 13]

[0260] Table 14 shows the Octet titers and EC2s of humanized version mutants of ANTIBODY-A and ANTIBODY-B from transient transfection supernatants in which the extra rabbit disulfide pairs have been removed. 50Octet titers were estimated using anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor. FACS binding assays were performed with serially diluted supernatants. EC 50 Values ​​were determined from best-fit curves using GraphPad Prism. [Table 14-1] [Table 14-2] Table 15 shows the EC values ​​of ANTIBODY-B mutants selected from the purified products. 50 In these mutants, the unpaired cysteine ​​was mutated on the light chain. FACS binding assays were performed with serially diluted purified Fab. EC 50 Values ​​were determined from best-fit curves using GraphPad Prism. [Table 15]

[0261] Example 5: Affinity and kinetics for human and cynomolgus TfR1 binding Binding of anti-TfR1 Fab fragments in their chimeric rabbit and humanized forms to recombinant human and cyno transferrin receptor extracellular domains was assessed using SPR to obtain kinetic and affinity values.

[0262] SPR results were obtained using a Biacore 8K+ (Cytiva) with a CM5 sensor chip (Series S, Cytiva) prepared for capture of histidine-tagged ligands according to the manufacturer's protocol (His Capture Kit, Cytiva). The running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% bovine serum albumin, 0.005% surfactant P20, pH 7.4, and flowed at 30 ul / min. To determine kinetics and affinity, recombinant human or cynomolgus TfR1 ECD-His was captured at 15–20 sensor response units (RU) and Fab fragments were injected sequentially at 0.78, 3.1, 12.5, 50, and 200 nM for 4 min each (single cycle kinetics mode), followed by a 15 min buffer flow to monitor dissociation. For Tf competitive binding analysis, human or cyno TfR1 ECD-His was captured at 25–40 RU, followed by a 4 min injection of either buffer or 1 mM holo-human transferrin (Sigma T0665), followed by sequential injections of 50 nM and 500 nM Fab with or without 1 mM holo-human transferrin, respectively, for 3 min, and dissociation was monitored for 10 min. After each cycle, the capture surface was regenerated with 2x injections of 10 mM glycine (pH 1.5) for 1 min. SPR responses were reference subtracted against the signal from the sensor surface without TfR1 and against the signal from cycles in which buffer was injected instead of Fab. Kinetics and affinity parameters were determined by fitting the data to a 1:1 binding model using Biacore Insight Evaluation Software (Cytiva).

[0263] The TfR1 ECD protein was captured on the SPR sensor surface. The association rate (k a ), dissociation rate (k d ), and the corresponding equilibrium dissociation constant (K D) was determined by fitting the SPR binding responses obtained from sequential injections of Fab (0.78-200 nM) to a 1:1 binding model. Table 16 shows the affinity and kinetic parameters of each Fab tested. [Table 16-1] [Table 16-2]

[0264] Example 6: Competition with transferrin for binding to human and cynomolgus monkey TfR1 The effect of holo-human transferrin on the binding of anti-TfR1 Fab fragments to human and cyno TfR1 was evaluated by SPR. For chimeric rabbit Fab fragments (used at 50 nM and 500 nM) that bind to surface-captured human and cyno TfR1, binding kinetics (k a , k d ), affinity (K D ), and response level (R max ) parameters were determined (Table 17). [Table 17]

[0265] These assays show that binding of rabbit ANTIBODY-A chi Fab or rabbit ANTIBODY-B chi Fab to either human TfR1 extracellular domain (ECD) or cyno TfR1 ECD was not significantly altered by the presence of holo-huTf in competition assays, suggesting that neither antibody competes with transferrin.

[0266] Example 7: Anti-TfR1 antibodies do not bind to TfR2 The cross-reactivity of anti-TfR1 antibodies with TfR2 was evaluated during FACS. Briefly, FACS binding assays were performed with BaF3 cells overexpressing human TfR2. Cells were dispensed into U-bottom 96-well plates at 50,000 cells / well / 50 μl FACS buffer and placed on ice. Then, 250 nM of bivalent huIgG1 antibody was added to the cells, followed by incubation on ice. Cells were washed twice with FACS buffer and secondary antibodies were added to the wells at a dilution of 1:400, followed by incubation on ice for 30 min. Cells were washed twice with FACS buffer and fixed with 2% PFA prepared in PBS. Finally, flow cytometry analysis was performed by measuring PE fluorescence.

[0267] Cells incubated with chimeric ANTIBODY-A mAb or chimeric ANTIBODY B mAb did not show any change in PE signal compared to cells incubated with secondary antibody alone, indicating a lack of binding to the cell surface TfR2 receptor (data not shown).

[0268] Example 8: Exemplary anti-TfR1 antibodies The following exemplary anti-TfR1 antibodies were constructed and shown to bind to their target (TfR1) with high affinity: (1)(i) a heavy chain of SEQ ID NO: 93 + a light chain of SEQ ID NO: 94; (2)(ii) a heavy chain of SEQ ID NO: 95 + a light chain of SEQ ID NO: 96; (3) (iii) a heavy chain of SEQ ID NO: 97 + a light chain of SEQ ID NO: 96; (4) (iv) a heavy chain of SEQ ID NO: 98 + a light chain of SEQ ID NO: 96, and Any of these antibodies can be linked to an agent (such as a nucleic acid (e.g., an antisense oligonucleotide, a short interfering RNA (siRNA), a messenger RNA (mRNA), a microRNA (miRNA), a guide RNA (gRNA), a phosphoramidate morpholino oligomer, or an aptamer)) to create a conjugate. In some embodiments, the anti-TfR1 antibody is conjugated to a particle (e.g., a lipid particle or a nanoparticle) that can contain a therapeutic agent such as those described herein.

[0269] Example 9: Cryo-EM structure of ANTIBODY-A To investigate the molecular basis of antigen recognition, a cryo-EM structure of humanized ANTIBODY-A Fab (containing the heavy chain sequence shown in SEQ ID NO: 95 and the light chain sequence shown in SEQ ID NO: 96) in complex with human (hu) transferrin receptor (TfR) and hu transferrin (Tf) was determined at 3.4 Å resolution. An atomic model of the complex was obtained by fitting a homology model of ANTIBODY-A and available crystal structures of TfR and Tf to the cryo-EM density map ( FIG. 3 ). The final model reveals that ANTIBODY-A binds to a conformational epitope located in the apical domain of TfR. Within 5 Å of the binding partner are 22 residues of TfR and 18 residues of ANTIBODY-A Fab. The discontinuous epitope is formed by residues K231, D245, L246, Y247, T248, P249, E350, G351, D352, C353, P354, S355, D356, K358, T359, D360, S361, R364, M365, V366, T367, and E369 of hu TfR and contains a combination of hydrophobic, hydrophilic, and charged residues. The antigen-binding site of ANTIBODY-A is formed by four CDRs (H1, H2, H3, L3) and consists of S29, S30, S31, Y33, Y52, Y54, S55, N57, Y59, Y100, Y102, T103, G104, Y105, T106, and Y107 of the heavy chain and Y93 and G95 of the light chain (Table 18). [Table 18]

[0270] ANTIBODY-A recognizes human and cynomolgus TfR but does not cross-react with mouse TfR. These structural studies provided detailed insight into the binding properties of ANTIBODY-A. The binding epitope in the TfR apical domain is largely conserved between human and cynomolgus but significantly diverges from mouse, consistent with the lack of binding of ANTIBODY-A derived from this species (Figure 4).

[0271] Example 10: Cryo-EM structure of ANTIBODY-B To investigate the molecular basis of antigen recognition, a cryo-EM structure of humanized ANTIBODY-B Fab (containing the heavy chain sequence shown in SEQ ID NO: 99 and the light chain sequence shown in SEQ ID NO: 100) in complex with hu TfR and hu Tf was determined at 3.96 Å resolution. An atomic model of the complex was obtained by fitting a homology model of ANTIBODY-B and available crystal structures of TfR and Tf to the cryo-EM density map ( FIG. 5 ). The final model revealed that ANTIBODY-B binds to a conformational epitope located in the protease-like domain of TfR. Within 5 Å of the binding partner are 15 residues of TfR and 18 residues of ANTIBODY-B Fab. The discontinuous epitope is formed by residues D139, T141, K145, G490, T491, V517, T518, Y573, K574, I577, E578, R579, I580, P581, and E582 of hu TfR and contains a combination of hydrophobic, hydrophilic, and charged residues. The antigen-binding site of ANTIBODY-B is formed by five CDRs (H1, H2, H3, L1, and L3) and consists of N30, W33, N52, D54, A55, D56, S57, T58, N59, N100, and V101 of the heavy chain and N28, I29, G30, V92, R93, G94, and G95 of the light chain (Table 19). [Table 19-1] [Table 19-2]

[0272] ANTIBODY-B recognizes human and cynomolgus TfR but does not cross-react with mouse TfR. These structural studies have provided detailed insight into the binding properties of ANTIBODY-B. The binding epitope in the TfR protease-like domain is largely conserved between human and cynomolgus but significantly diverges from mouse, consistent with the lack of binding of ANTIBODY-B derived from this species (Figure 6).

[0273] Example 11: Format variants of the humanized version of ANTIBODY-A Twenty-two different format variants of the humanized version of ANTIBODY-A were prepared. Table 20 describes each of the antibody variants by an index number (used to identify the antibody used in the experiment), the name of the individual proteins (Fc chain and / or non-Fc chain) that make up the antibody, an identification of a diagram showing the general structure of the antibody (selected from Figures 7A-7E), and a description of the antibody. The amino acid sequences of each of the individual proteins (Fc chain and / or non-Fc chain) that make up each antibody are provided in Table 21. [Table 20-1] [Table 20-2] [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5] [Table 21-6] [Table 21-7] [Table 21-8]

[0274] Hamster TfR-deficient CHO cells were transduced with lentiviral plasmids expressing either the full-length human TfR or the full-length cynomolgus TfR. Flow cytometry was performed by incubating CHO cells with either the cynomolgus TfR or the human TfR at 50,000 cells per well (100 microliters) with titrated test articles (antibodies identified by protein index 1, 2, 3, or 4) for 1 hour on ice, followed by two washes, incubation with a secondary anti-human Fc PE-labeled conjugate (Jackson Immunoresearch), two washes, and fixation with 1% paraformaldehyde before analysis on a flow cytometer.

[0275] All four antibody formats tested (identified by protein indexes 1, 2, 3, and 4) bound well to both cynomolgus and human TfR, with similar binding to the two species (Figures 8A-8B and Table 22). The bivalent constructs showed slightly higher binding to cells due to their binding avidity. [Table 22]

[0276] The clearance of transferrin by the humanized version of ANTIBODY-A was evaluated in various formats: CHO cells lacking hamster TfR and overexpressing human TfR were incubated with 3 nM AlexaFluor647-conjugated transferrin and titrated required concentrations of test articles (antibodies identified by protein index 1, 2, 3, or 4) for 1 hour on ice, washed three times, fixed with 1% paraformaldehyde, and analyzed by flow cytometry for clearance of fluorescent transferrin.

[0277] Both right side up ("RSU") constructs (protein indexes 1 and 2) showed minimal Tf exclusion (Figure 9A). The bivalent RSU (protein index 1) showed approximately 10% Tf exclusion at concentrations >2 nM.

[0278] Both inverted ("USD") constructs (protein indexes 3 and 4) showed approximately 40% Tf exclusion (Figure 9B), likely due to a steric clash between the Fc of these constructs and transferrin.

[0279] Binding of anti-TfR humanized ANTIBODY-A monovalent Fc fusions (protein index 2 and 4) to TfR in the presence or absence of human holo-transferrin was examined by surface plasmon resonance at 25 degrees Celsius on a Biacore 8K+ instrument using a CM5 chip anti-His tag capture method to capture human or cynomolgus TfR ectodomain (ECD) with an N-terminal Hisx6 tag. Binding was performed in Hepes buffered saline, pH 7.4 (containing 3mM EDTA, 0.05% BSA, 0.005% P20). To test competition with transferrin, the surface-captured TfR ECD was pre-saturated with 1 micromolar holo-human transferrin prior to injection of the test article (protein index 2 or 4). Monovalent antibodies were titrated in a 4-fold dilution series from 0.2 to 200 nM. Multi-cycle kinetics were fitted to a 1:1 binding model to generate binding parameters. Binding of anti-TfR humanized ANTIBODY-A monovalent Fc fusions to TfR was not affected by transferrin (Table 23). [Table 23]

[0280] Binding of human holo-transferrin to cyno TfR ECD in the presence or absence of humanized ANTIBODY-A monovalent versions (protein index 2 and 4) was examined by surface plasmon resonance at 25 degrees Celsius on a Biacore 8K+ instrument using a CM5 chip anti-His tag capture method to capture human or cyno TfR ectodomains (ECDs) with an N-terminal Hisx6 tag. Binding was performed in Hepes buffered saline, pH 7.4 (containing 3mM EDTA, 0.05% BSA, 0.005% P20). To test competition at TfR, the surface-captured TfR ECD was pre-saturated with 200nM humanized ANTIBODY-A (protein index 2 or 4) before injection of a 4-fold dilution series of human holo-transferrin from 0.2 to 200nM. Multi-cycle kinetics were fitted to a 1:1 binding model to generate binding parameters. In an upside-down format (USD, Fc-Fab), monovalent anti-TfR humanized ANTIBODY-A attenuated binding of transferrin to TfR (Table 24). In a right-side-up format (RSU, Fab-Fc), monovalent anti-TfR humanized ANTIBODY-A did not affect binding of transferrin to TfR (Table 24). [Table 24]

[0281] CHO cells lacking hamster TfR and overexpressing human TfR were incubated with 3 nM AlexaFluor647-conjugated transferrin and titrated with the required concentrations of test article (antibody--protein index 3 or 14) for 1 hour on ice, washed 3 times, fixed with 1% paraformaldehyde, and analyzed by flow cytometry for clearance of fluorescent transferrin. Humanized ANTIBODY-A in USD format, in which the Fab is linked to the C-terminus of the Fc through the Fab VH N-terminus, induces approximately 40% clearance of transferrin (Tf-AF647) from cell surface huTfR (Figure 10, protein index 3 identified with an "x"). This was minimized by linking the Fab to the Fc through the N-terminus of the VL domain and shortening the linker between the Fc and Fab (Figure 10, protein index 14 identified with a triangle).

[0282] In flow cytometry experiments, anti-TfR antibodies were tested for binding using CHO cells lacking hamster TfR and overexpressing full-length human TfR. Flow cytometry was performed by incubating CHO cells expressing human TfR with titrated test articles (protein index 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, or 20) at approximately 50,000 cells per well (100 microliters) for 1 hour on ice, followed by two washes, incubation with secondary anti-human IgG PE conjugate (Jackson Immunoresearch), two washes, and fixation with 1% paraformaldehyde before analysis on the flow cytometer.

[0283] Similar binding results were obtained with humanized ANTIBODY-A Fab fused to the C-terminus of Fc via either the VH N-terminus (protein index 5, 6, 7, 8, 9, 10, 11, and 12) or the VL N-terminus (protein index 13, 14, 16, 17, 18, 19, and 20) (Table 25). Furthermore, the linker between Fc and Fab can vary from 15 amino acids (3xSG4) to "minus 2" (the last two residues of the CH3 domain (Pro-Gly) are removed). Note that the maximum mean fluorescence intensity changes when Fc is fused to the VL domain of Fab and the linker is shorter. This is believed to be due to steric hindrance of the secondary antibody on the test article. [Table 25]

[0284] In flow cytometry experiments, anti-TfR antibodies were tested for binding using CHO cells lacking hamster TfR and overexpressing full-length human TfR. Flow cytometry was performed by incubating CHO cells expressing human TfR with titrated test articles (protein index 3, 21, or 22) at approximately 50,000 cells per well (100 microliters) for 1 hour on ice, followed by two washes, incubation with secondary anti-human IgG PE conjugate (Jackson Immunoresearch), two washes, and fixation with 1% paraformaldehyde before analysis on a flow cytometer.

[0285] When the VH and VL domains of humanized ANTIBODY-A were converted to scFv format and fused to the C-terminus of human IgG Fc, binding to the human TfR was retained (Table 26). The apparent difference in MFI is likely due to the larger epitope available to the secondary detection antibody in the non-scFv format. [Table 26]

[0286] Other embodiments While the invention has been described in conjunction with a detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the claims.

Claims

1. 1. An antibody that binds to the human transferrin receptor, comprising a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR) 1, a VH CDR2, and a VH CDR3, the VH CDR1 comprises the amino acid sequence GIDFSSSSGYMX (SEQ ID NO: 149), where X is any amino acid; the VH CDR2 comprises the amino acid sequence XIYTYSSNTYYAXXXKG (SEQ ID NO: 151), where X is any amino acid; the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106); the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107); the VL CDR2 comprises the amino acid sequence RASXLXS (SEQ ID NO: 153), where X is any amino acid; The antibody, wherein the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110).

2. The VH CDR1 has the amino acid sequence GIDFSSSSGYMX １ (SEQ ID NO: 150), wherein X １ is C, A, or H, The VH CDR2 has the amino acid sequence X ２ IYTYSSSNTYYAX ３ X ４ X ５ KG (SEQ ID NO: 152), where X ２ is C or A, and X ３ is S or A, and X ４ is W or S, and X ５ is A or V, the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106); the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107); The VL CDR2 has the amino acid sequence RASX ６ LX ７ S (SEQ ID NO: 154), wherein X ６ is T or S, and X ７ is A or E, The antibody of claim 1, wherein the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110).

3. The antibody of claim 1, wherein the VH-CDR1, the VH-CDR2, and the VH-CDR3 each correspond to the VH CDRs of a single VH clone shown in any one of SEQ ID NOs: 4 to 18, and the VL-CDR1, the VL-CDR2, and the VL-CDR3 each correspond to the VL CDRs of a single VL clone shown in any one of SEQ ID NOs: 34 to 37.

4. The antibody of claim 1, wherein the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 correspond to the VH CDRs and VL CDRs of a single clone set forth in any one of SEQ ID NOs: 101 to 110, respectively.

5. (a) the VH CDR1 comprises the amino acid sequence GIDFSSSSGYMC (SEQ ID NO: 101), the VH CDR2 comprises the amino acid sequence CIYTYSSNTYYASWAKG (SEQ ID NO: 104), the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASTLAS (SEQ ID NO: 109), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110); (b) the VH CDR1 comprises the amino acid sequence GIDFSSSSGYMH (SEQ ID NO: 102), the VH CDR2 comprises the amino acid sequence AIYTYSSNTYYASWAKG (SEQ ID NO: 105), the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASSLES (SEQ ID NO: 108), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110); (c) the VH CDR1 comprises the amino acid sequence GIDFSSSSGYMC (SEQ ID NO: 101), the VH CDR2 comprises the amino acid sequence CIYTYSSNTYYAASVKG (SEQ ID NO: 103), the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASSLES (SEQ ID NO: 108), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110), or (d) The antibody of claim 1, wherein the VH CDR1 comprises the amino acid sequence GIDFSSSSGYMC (SEQ ID NO: 101), the VH CDR2 comprises the amino acid sequence CIYTYSSNTYYASWAKG (SEQ ID NO: 104), the VH CDR3 comprises the amino acid sequence GTYGYTGYTYTMGYFSL (SEQ ID NO: 106), the VL CDR1 comprises the amino acid sequence QASQNINSYLA (SEQ ID NO: 107), the VL CDR2 comprises the amino acid sequence RASSLES (SEQ ID NO: 108), and the VL CDR3 comprises the amino acid sequence QSYYYSGSSNYNA (SEQ ID NO: 110).

6. (i) the VH is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 12, or 15-18; and (ii) The antibody of claim 1, wherein the VL is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 35-37.

7. The antibody of claim 1, wherein the VH comprises the amino acid sequence of any one of SEQ ID NOs: 4, 12, or 15-18, and the VL comprises the amino acid sequence of SEQ ID NOs: 34-37.

8. (a) the VH comprises the amino acid sequence of SEQ ID NO: 4 and the VL comprises the amino acid sequence of SEQ ID NO: 34; (b) the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO: 35; (c) the VH comprises the amino acid sequence of SEQ ID NO: 15 and the VL comprises the amino acid sequence of SEQ ID NO: 35; (d) the VH comprises the amino acid sequence of SEQ ID NO: 16 and the VL comprises the amino acid sequence of SEQ ID NO: 35; (e) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 35; or (f) The antibody of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 18 and the VL comprises the amino acid sequence of SEQ ID NO:

35.

9. Binds to the human transferrin receptor and exhibits the following binding to the human transferrin receptor: (a) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 34; (b) a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 35; (c) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 35; (d) a VH comprising the amino acid sequence of SEQ ID NO: 16 and a VL comprising the amino acid sequence of SEQ ID NO: 35; (e) a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 35; or (f) An antibody that competes with an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO:

35.

10. 1. An antibody that binds to the human transferrin receptor, comprising a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR) 1, a VH CDR2, and a VH CDR3, the VH CDR1 comprises the amino acid sequence GFSFSNSYWIX (SEQ ID NO: 155), where X is any amino acid; the VH CDR2 comprises the amino acid sequence XINTDADSTNYAXXXXG (SEQ ID NO: 157), where X is any amino acid; the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119); the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; the VL CDR1 comprises the amino acid sequence XASQNIGSNLA (SEQ ID NO: 159); the VL CDR2 comprises the amino acid sequence DASKLXS (SEQ ID NO: 161), where X is any amino acid; The antibody, wherein the VL CDR3 comprises the amino acid sequence QXTVRGGAYGXA (SEQ ID NO: 163), where X is any amino acid.

11. The VH CDR1 has the amino acid sequence GFSFSNSYWIX １ (SEQ ID NO: 156), wherein X １ is C, A, or H, The VH CDR2 has the amino acid sequence X ２ INTDADSTNYAX ３ X ４ X ５ X ６ G (SEQ ID NO: 158), wherein X ２ is C or A, and X ３ is S or D, and X ４ is W or S, and X ５ is A or V, and X ６ is R or K, the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119); The VL CDR1 has the amino acid sequence X ７ ASQNIGSNLA (SEQ ID NO: 160), wherein X ７ is Q or R, The VL CDR2 has the amino acid sequence DASKLX ８ S (SEQ ID NO: 162), wherein X ８ is A or E, The VL CDR3 has the amino acid sequence QX ９ TVRGGAYGX １０ A (SEQ ID NO: 164), wherein X ９ is C, Q, A, S, T, or V, and X １０ The antibody of claim 10, wherein is N or L.

12. The antibody of claim 10, wherein the VH-CDR1, the VH-CDR2, and the VH-CDR3 each correspond to the VH CDRs of a single VH clone shown in any one of SEQ ID NOs: 19 to 33, and the VL-CDR1, the VL-CDR2, and the VL-CDR3 each correspond to the VL CDRs of a single VL clone shown in any one of SEQ ID NOs: 38 to 44.

13. The antibody of claim 10, wherein the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 correspond to the VH and VL CDRs of a single clone set forth in any one of SEQ ID NOs: 116 to 127, respectively.

14. (a) the VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), the VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), the VL CDR1 comprises the amino acid sequence QASQNIGSNLA (SEQ ID NO: 121), the VL CDR2 comprises the amino acid sequence DASKLAS (SEQ ID NO: 123), and the VL CDR3 comprises the amino acid sequence QCTVRGGAYGNA (SEQ ID NO: 124); (b) the VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), the VH CDR2 comprises the amino acid sequence CINTDADSTNYADSVKG (SEQ ID NO: 118), the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), the VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), the VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and the VL CDR3 comprises the amino acid sequence QCTVRGGAYGNA (SEQ ID NO: 124); (c) the VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), the VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), the VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), the VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and the VL CDR3 comprises the amino acid sequence QCTVRGGAYGNA (SEQ ID NO: 124); (d) the VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), the VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), the VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), the VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and the VL CDR3 comprises the amino acid sequence QSTVRGGAYGNA (SEQ ID NO: 125); (e) the VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), the VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), the VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), the VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and the VL CDR3 comprises the amino acid sequence QTTVRGGAYGNA (SEQ ID NO: 126); or (f) The antibody of claim 10, wherein the VH CDR1 comprises the amino acid sequence GFSFSNSYWIC (SEQ ID NO: 116), the VH CDR2 comprises the amino acid sequence CINTDADSTNYASWARG (SEQ ID NO: 117), the VH CDR3 comprises the amino acid sequence QNNVFDPGYNL (SEQ ID NO: 119), the VL CDR1 comprises the amino acid sequence RASQNIGSNLA (SEQ ID NO: 120), the VL CDR2 comprises the amino acid sequence DASKLES (SEQ ID NO: 122), and the VL CDR3 comprises the amino acid sequence QVTVRGGAYGNA (SEQ ID NO: 127).

15. (i) the VH is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 30-33; (ii) The antibody of any one of claims 10 to 14, wherein the VL is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 41 to 44.

16. The antibody of claim 10, wherein the VH comprises the amino acid sequence of any one of SEQ ID NOs: 30 to 33, and the VL comprises the amino acid sequence of any one of SEQ ID NOs: 41 to 44.

17. (a) the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 38; (b) the VH comprises the amino acid sequence of SEQ ID NO: 30 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (c) the VH comprises the amino acid sequence of SEQ ID NO: 31 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (d) the VH comprises the amino acid sequence of SEQ ID NO: 32 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (e) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (f) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 42; (g) the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO: 43; or (h) The antibody of claim 10, wherein the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO:

44.

18. Binds to the human transferrin receptor and exhibits the following binding to the human transferrin receptor: (a) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 38; (b) a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (c) a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (d) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (e) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 41; (f) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 42; (g) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (h) An antibody that competes with an antibody comprising a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO:

44.

19. The antibody according to any one of claims 1 to 14 and 18, which is a Fab fragment or a Fab' fragment.

20. Bispecific antibodies, single chain antibodies, Fab fragments, F(ab') ２ fragment, Fab' fragment, Fsc fragment, Fv fragment, scFv, sc(Fv) ２ 19. The antibody of any one of claims 1 to 14 and 18, which is a diabody.

21. 19. The antibody of any one of claims 1 to 14 and 18, comprising a constant heavy (CH) domain and a constant light (CL) domain.

22. The antibody of claim 21, wherein the CH domain comprises a CH1 domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 52.

23. The antibody of claim 22, wherein the CH1 domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 45, 47, and 49.

24. The antibody of claim 23, wherein the CH1 domain is fused to a hinge comprising the amino acid sequence set forth in any one of SEQ ID NOs: 53 to 55 and 57.

25. The antibody of claim 22, wherein the CH1 domain comprises the amino acid sequence set forth in SEQ ID NO:

51.

26. The antibody of claim 25, wherein the CH1 domain is fused to a hinge comprising the amino acid sequence ES or the amino acid sequence set forth in SEQ ID NO:

56.

27. The antibody of claim 22, wherein the CH1 domain is fused to a hinge comprising the amino acid sequence ES, ESK, or an amino acid sequence set forth in any one of SEQ ID NOs: 53 to 90.

28. The antibody of claim 21 , wherein the CL domain comprises the amino acid sequence set forth in SEQ ID NO: 91 or 92.

29. 22. The antibody of claim 21 , (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 93, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 94; (ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 95, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96; (iii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 97, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96; (iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 96; or (v) The antibody, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

100.

30. Nucleic acid(s) encoding the antibody of any one of claims 1 to 14 and 18.

31. 31. An expression vector(s) comprising the nucleic acid(s) of claim 30 operably linked to a promoter.

32. 31. An isolated cell comprising the nucleic acid(s) of claim 30.

33. An isolated cell comprising the expression vector(s) described in claim 31.

34. 19. An isolated cell comprising: a first expression vector comprising a first nucleic acid operably linked to a promoter, the first nucleic acid encoding a first polypeptide comprising the VH of the antibody of any one of claims 1 to 14 and 18; and a second expression vector comprising a second nucleic acid operably linked to a promoter, the second nucleic acid encoding a second polypeptide comprising the VL of the antibody of any one of claims 1 to 14 and 18.

35. A method for producing an antibody according to any one of claims 1, 9, 10, and 18, comprising: An isolated cell comprising nucleic acid(s) encoding the antibody of any one of claims 1 to 14 and 18. An isolated cell comprising an expression vector(s) comprising nucleic acid(s) encoding the antibody of any one of claims 1 to 14 and 18 operably linked to a promoter; or 19. An isolated cell comprising: a first expression vector comprising a first nucleic acid operably linked to a promoter, the first nucleic acid encoding a first polypeptide comprising the VH of the antibody of any one of claims 1 to 14 and 18; and a second expression vector comprising a second nucleic acid operably linked to a promoter, the second nucleic acid encoding a second polypeptide comprising the VL of the antibody of any one of claims 1 to 14 and 18. and isolating the antibody.

36. A pharmaceutical composition comprising the antibody of any one of claims 1 to 14 and 18 and a pharmaceutically acceptable carrier.

37. A conjugate comprising the antibody of any one of claims 1 to 14 and 18 and a drug.

38. 38. The conjugate of claim 37, wherein the agent is a nucleic acid.

39. 39. The conjugate of claim 38, wherein the nucleic acid is an mRNA, an siRNA, an antisense oligonucleotide, a microRNA (miRNA), a guide RNA (gRNA), or a phosphoramidate morpholino oligomer (PMO).

40. 39. The conjugate of claim 38, wherein the nucleic acid is an antisense oligonucleotide.

41. 38. The conjugate of claim 37, wherein the drug is linked to the antibody via a linker.

42. 38. A composition for use in an in vivo drug delivery method, comprising the conjugate of claim 37, said method comprising administering said composition to a human subject.

43. (a) the human subject has a muscle disease, muscle wasting disease, arteriosclerotic disease, heart-related disease, or lysosomal storage disease, and the method delivers the agent to muscle tissue; (b) the human subject has a brain disease, and the method delivers the agent to brain tissue. (c) the human subject has liver disease and the method delivers the agent to a liver cell. (d) the human subject has cancer and the method delivers the agent to tumor cells; or (e) the human subject has an immune disorder and the method delivers the agent to an immune cell.

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