Compositions and methods for transferrin receptor (TFR)-mediated delivery to brain and muscle

Abstract

The present invention provides, in part, protein-drug conjugates comprising an antigen transferrin receptor (e.g., human transferrin receptor) antigen binding protein (e.g., scFv, Fab) conjugated to a molecular cargo (e.g., a polynucleotide, liposome, or lipid nanoparticle) for delivery of the molecular cargo to a target tissue (e.g., brain or muscle). Methods of using the conjugates to treat various diseases or disorders, such as neurological or muscular diseases, are provided.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 393,749, filed July 29, 2022, which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy, created on July 26, 2023, is named 250298000507SEQLIST.XML and is 547,729 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to protein-drug conjugates comprising an antigen-binding protein conjugated to a molecular cargo, as well as methods of treating disease using such protein-drug conjugates. [Background technology]

[0004] Iron delivery to the brain is achieved through the binding and intracellular transport of the iron-binding protein transferrin (Tf). The Tf receptor (TfR) is the target of several studies for drug delivery to the brain. For example, approaches include the use of Tf-decorated liposomes, which are used to deliver imaging agents and DNA (Sharma et al., (2013) Cell penetrating peptide tethered biligand liposomes for delivery to the brain in vivo: biodistribution and transfection. J. Control. Release 167, 1-10.), or the use of an iron-mimetic peptide as a ligand (Staquicini et al., (2011)). A correlation between increased antibody affinity and lysosomal degradation has also been suggested (Bien-Ly et al., (2014) Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J. Exp. Med. 211, 233-244), supporting the idea that lower antibody affinity helps transported complexes avoid intracellular degradation. Bien-Ly et al. found that a bispecific antibody against TfR and β-secretase (BACE1) crossed the blood-brain barrier (BBB) ​​and effectively reduced brain amyloid-β levels, but high-affinity binding to TfR also caused a dose-dependent decrease in brain TfR levels. Similarly, Moos & Morgan (2001) compared the ability of an anti-TfR antibody, OX26, and transferrin to cross the rat BBB. They found that OX26 did not recirculate from the brain like transferrin because the antibody exhibited a high-affinity antibody-antigen interaction with TfR, but that TfR was not readily reversed, whereas Tf was readily reversed depending on the pH and iron content of Tf (Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat, J Neurochem 2001 Oct;79(1):119-29). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sharma et al.,(2013)Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo:biodistribution and transfection.J.Control.Release 167,1-10. [Non-patent document 2] Restricted transport of anti-transferrin receptor antibody(OX26)through the blood-brain barrier in the rat,J Neurochem 2001 Oct;79(1):119-29 Summary of the Invention

[0006] In one aspect, provided herein is a protein-drug conjugate comprising an antigen binding protein that specifically binds to human transferrin receptor (TfR), or a variant or antigenic fragment thereof, conjugated to a molecular cargo.

[0007] The antigen-binding protein may have a K of about 41 nM or greater affinity, e.g., about 30 nM or greater affinity, about 20 nM or greater affinity, about 10 nM or greater affinity, about 5 nM or greater affinity, about 3 nM or greater affinity, or about 1 nM or greater affinity. D In some embodiments, the antigen binding protein can bind to the human transferrin receptor with a K of about 3 nM. D In some embodiments, the antigen binding protein binds to the human transferrin receptor with an affinity of about 0.45 nM to 3 nM or greater. D Such binding affinity can be measured, for example, in a surface plasmon resonance assay at 25°C.

[0008] In some embodiments, the antigen binding protein comprises a heavy chain variable region (HCVR or V H ) and light chain variable region (LCVR or V L ) and the Fab having the HCVR and LCVR has a K D or binds to the human transferrin receptor with greater affinity.

[0009] In some embodiments, the antigen-binding protein comprises an antibody or antigen-binding fragment thereof. The antigen-binding fragment may be chosen from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain fragment variable (scFv), a bis-scFv, an (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, or a chemically modified derivative thereof.

[0010] In some embodiments, the antigen binding protein of the protein-drug conjugate comprises an antigen binding fragment that is a fragment antigen binding region (Fab).

[0011] In some embodiments, the antigen-binding protein of the protein-drug conjugate comprises a single-chain fragment variable (scFv). In some embodiments, the protein-drug conjugate is a single-chain fragment variable (scFv) comprising domains arranged in the following orientation from N-terminus to C-terminus: heavy chain variable region (HCVR)-light chain variable region (LCVR). In some embodiments, the protein-drug conjugate comprises a single-chain fragment variable (scFv) comprising domains arranged in the following orientation from N-terminus to C-terminus: light chain variable region (LCVR)-heavy chain variable region (HCVR). In some embodiments, the scFv variable regions are linked by a peptide linker. In some embodiments, the scFv variable region is -(GGGGS) n -(SEQ ID NO: 426), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the antigen binding protein of the protein-drug conjugate is about 1 x 10 -7 K of M D or binds to the human transferrin receptor with greater affinity.

[0012] In some embodiments, the antigen binding protein of the protein-drug conjugate comprises (i) a HC of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 or 312 (or a variant thereof). and / or (ii) an HCVR comprising LCDR1, HCDR2 and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 527; 317 or 484 (or a variant thereof).

[0013] In some embodiments, the antigen binding protein of the protein-drug conjugate is selected from the group consisting of: (1) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (2) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (3) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (4) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof). (5) an HCVR comprising HCDR1, HCDR2 and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2 and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (6) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof). (7) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (8) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof);(8) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence shown in SEQ ID NO: 72 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence shown in SEQ ID NO: 77 (or a variant thereof); (9) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence shown in SEQ ID NO: 82 (or a variant thereof); and LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence shown in SEQ ID NO: 87 (or a variant thereof). (10) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (11) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof). (12) LCVR comprising HCDR1, HCDR2 and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and LCVR comprising LCDR1, LCDR2 and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (13) HCVR comprising HCDR1, HCDR2 and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and (14) an LCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (15) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof);and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (16) an HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (17) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (18) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (19) an amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof). and an LCVR comprising an LCDR1, an LCDR2, and an LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (20) an HCVR comprising an HCDR1, an HCDR2, and an HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and an LCVR comprising an LCDR1, an LCDR2, and an LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (21) an LCVR comprising an HCDR1, an HCDR2, and an HCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 (21) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 207; (22) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 212; and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or a variant thereof) set forth in SEQ ID NO: 217;(23) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (24) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof). (25) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (26) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and an amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof). (27) an LCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (28) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising an LCDR1, HCDR2, and LCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (29) an LCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (30) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof);and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (31) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 or 527 (or a variant thereof); and / or (32) an HCVR comprising an HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and an LCVR comprising an LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof).

[0014] In some embodiments, the antigen binding protein of the protein-drug conjugate is selected from the group consisting of: (a) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (b) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (or a variant thereof); and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 (or a variant thereof). (c) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (d) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 (or a variant thereof);and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); (e) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 43 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 45 (or a variant thereof); and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (f) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 53 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 55 (or a variant thereof); and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 60 (or a variant thereof); (g) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 63 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 65 (or a variant thereof); and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (h) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 73 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 75 (or a variant thereof);and LCVRs comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 (or a variant thereof); (i) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85 (or a variant thereof); and LCVRs comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (j) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 (or a variant thereof); and sequences (k) an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100 (or a variant thereof); (k) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105 (or a variant thereof); and (I) an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 109 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (I) an HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 113 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 115 (or a variant thereof);and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 118 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 120 (or a variant thereof); (m) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 123 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 125 (or a variant thereof); and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (n) HCDR1 having the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), (i) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof); (o) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof);(p) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 155 (or a variant thereof); and LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 158 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 160 (or a variant thereof); (q) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof); and LCDR1 having the amino acid sequence set forth in SEQ ID NO: 168 (or a variant thereof), an amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof). (r) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof); (s) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof);and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 188 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 190 (or a variant thereof); (t) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 195 (or a variant thereof); and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof); (u) HCDR1 having the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof), and the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); (v) HCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof); and LCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof); (w) LCVRs comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof). (x) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (x) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof);(y) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (z) HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); and LCDR1 having the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof). (aa) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof); (ab) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof);and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof); (ac) HCVRs comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof); and LCVRs comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof); (ad) HCDR1 having the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof), an amino acid sequence set forth in SEQ ID NO: 294 (or (ae) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof);and / or (af) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof);

[0015] In some embodiments, the antigen binding protein of the protein-drug conjugate is selected from the group consisting of: (i) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (ii) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (v) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (vi) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof). (vii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (viii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (ix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (x) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (xi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (xii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof);(xiii) HCVR comprising the amino acid sequence shown in SEQ ID NO: 122 (or a variant thereof); and LCVR comprising the amino acid sequence shown in SEQ ID NO: 127 (or a variant thereof); (xiv) HCVR comprising the amino acid sequence shown in SEQ ID NO: 132 (or a variant thereof); and LCVR comprising the amino acid sequence shown in SEQ ID NO: 137 (or a variant thereof); (xv) HCVR comprising the amino acid sequence shown in SEQ ID NO: 142 (or a variant thereof); and LCVR comprising the amino acid sequence shown in SEQ ID NO: 147 (or a variant thereof); ( (xvi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (xvii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (xviii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (xix) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xx) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (xxi) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (xxii) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (xxiii) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (xxiv) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof);(xxv) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (xxvi) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xxvii) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (xxviii) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xxix) a sequence (xxx) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (xxxi) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 or 527 (or a variant thereof); and / or (xxxii) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof).

[0016] In some embodiments, the antigen binding protein of the protein-drug conjugate comprises: i. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 329 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 328 (or a variant thereof); ii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 331 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 330 (or a variant thereof); iii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 333 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 332 a light chain comprising the amino acid sequence set forth in SEQ ID NO: 334 (or a variant thereof); iv. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 335 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 334 (or a variant thereof); v. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 337 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 336 (or a variant thereof); vi. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 339 (or a variant thereof); and the amino acid sequence set forth in SEQ ID NO: 338 (or a variant thereof). a light chain comprising the amino acid sequence set forth in SEQ ID NO: 340 (or a variant thereof); vii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 341 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 340 (or a variant thereof); viii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 343 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 342 (or a variant thereof); ix. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 345 (or a variant thereof); and the amino acid sequence set forth in SEQ ID NO: 344 (or a variant thereof). a light chain comprising the amino acid sequence set forth in SEQ ID NO: 348 (or a variant thereof); x. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 347 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 346 (or a variant thereof); xi. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 349 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 348 (or a variant thereof); xii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 351 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 350 (or a variant thereof); xiii.a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 353 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 352 (or a variant thereof); xiv. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 355 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 354 (or a variant thereof); xv. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 357 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 356 (or a variant thereof); xvi. a sequence a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 359 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 358 (or a variant thereof); xvii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 361 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 360 (or a variant thereof); xviii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 363 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 362 (or a variant thereof); xix. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 365 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 364 (or a variant thereof); xx. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 367 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 366 (or a variant thereof); xxi. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 369 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 368 (or a variant thereof); xxii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 369 (or a variant thereof); A heavy chain region comprising the amino acid sequence set forth in sequence number 371 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in sequence number 370 (or a variant thereof); xxiii. A heavy chain region comprising the amino acid sequence set forth in sequence number 373 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in sequence number 372 (or a variant thereof); xxiv. A heavy chain region comprising the amino acid sequence set forth in sequence number 375 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in sequence number 374 (or a variant thereof); xxv.a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 377 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 376 (or a variant thereof); xxvi. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 379 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 378 (or a variant thereof); xxvii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 381 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 380 (or a variant thereof); xxviii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 383 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof); xxi x. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 385 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 384 (or a variant thereof); xxx. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 387 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 386 (or a variant thereof); xxxi. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 389 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 388 (or a variant thereof); or xxxii. a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 391 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 390 (or a variant thereof).

[0017] In some embodiments, the antigen binding protein of the protein-drug conjugate comprises: i. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 543 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 328 (or a variant thereof); ii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 544 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 330 (or a variant thereof); iii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 545 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 332 (or a variant thereof); iv. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 546 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 334 (or a variant thereof); v. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 547 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 336 (or a variant thereof); vi. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 548 (or a variant thereof); and the amino acid sequence set forth in SEQ ID NO: 338 (or a variant thereof). a light chain comprising the amino acid sequence set forth in SEQ ID NO: 340 (or a variant thereof); vii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 549 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 340 (or a variant thereof); viii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 550 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 342 (or a variant thereof); ix. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 551 (or a variant thereof); and the amino acid sequence set forth in SEQ ID NO: 344 (or a variant thereof). a light chain comprising the amino acid sequence set forth in SEQ ID NO: 346 (or a variant thereof); x. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 553 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 348 (or a variant thereof); xii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 554 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 350 (or a variant thereof); xiii.a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 352 (or a variant thereof); xiv. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 354 (or a variant thereof); xv. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 356 (or a variant thereof); xvi. an amino acid sequence set forth in SEQ ID NO: 558 (or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 358 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 358 (or a variant thereof); xvii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 559 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 360 (or a variant thereof); xviii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 362 (or a variant thereof); xix. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof); and a sequence a light chain comprising the amino acid sequence set forth in SEQ ID NO: 364 (or a variant thereof); xx. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 366 (or a variant thereof); xxi. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 368 (or a variant thereof); xxii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 564 (or a variant thereof); and an amino acid sequence set forth in SEQ ID NO: 370 (or a variant thereof). a light chain comprising the amino acid sequence set forth in SEQ ID NO: 372 (or a variant thereof); xxiv. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 566 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 374 (or a variant thereof); XXV. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 567 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 376 (or a variant thereof); xxvi.a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 568 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 378 (or a variant thereof); xxvii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 569 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 380 (or a variant thereof); xxviii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 570 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof); xxix. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 571 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 384 (or a variant thereof); xxx. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 572 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 386 (or a variant thereof); xxxi. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 573 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 388 (or a variant thereof); or xxxii. a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 574 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 390 (or a variant thereof).

[0018] In some embodiments, the antigen binding protein of the protein-drug conjugate is selected from the group consisting of: (1) a HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (2) a HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (3) a HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). (4) an HCVR comprising HCDR1, HCDR2 and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2 and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (5) an HCVR comprising HCDR1, HCDR2 and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2 and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); or (6) an HCVR comprising HCDR1, HCDR2 and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2 and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).

[0019] In some embodiments, the antigen binding protein of the protein-drug conjugate is selected from the group consisting of: (a) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof); (b) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), (c) an LCVR comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof); (c) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (d) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof);and an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (e) an HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof). or (f) an LCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof);

[0020] In some embodiments, the protein-drug conjugate comprises: (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). (iv) an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (v) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); or (vi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).

[0021] In some embodiments, the antigen binding protein of the protein-drug conjugate comprises: (A) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 355 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 354 (or a variant thereof); (B) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 363 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 362 (or a variant thereof); (C) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 373 (or a variant thereof); and the amino acid sequence set forth in SEQ ID NO: 372 (D) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 377 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 376 (or a variant thereof); (E) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 381 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 380 (or a variant thereof); or (F) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 383 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof).

[0022] In some embodiments, the antigen binding protein of the protein-drug conjugate comprises: (I) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 354 (or a variant thereof); (II) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 362 (or a variant thereof); (III) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 565 (or a variant thereof); and the amino acid sequence set forth in SEQ ID NO: 372 (IV) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 367 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 376 (or a variant thereof); (V) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 569 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 380 (or a variant thereof); or (VI) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 570 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 382 (or a variant thereof).

[0023] In some embodiments, the antigen binding protein binds to the same epitope on the human transferrin receptor as an antibody comprising the HCVR / LCVR amino acid sequence pair shown in Table 1-1.

[0024] In some embodiments, the antigen binding protein competes for binding to the human transferrin receptor with an antibody comprising an HCVR / LCVR amino acid sequence pair shown in Table 1-1.

[0025] In another aspect, provided herein is a protein-drug conjugate comprising an antigen binding protein that specifically binds to the human transferrin receptor (hTfR), wherein the antigen binding protein is conjugated to a molecular cargo and comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a. an epitope comprising the sequence LLNE (SEQ ID NO: 529) and / or an epitope comprising the sequence TYKEL (SEQ ID NO: 509); b. an epitope comprising the sequence DSTDFTGT (SEQ ID NO: 530) and / or an epitope comprising the sequence VKHPVTGQF (SEQ ID NO: 531) and / or an epitope comprising the sequence IERIPEL (SEQ ID NO: 532); c. an epitope comprising the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 533); d. an epitope comprising the sequence FEDL (SEQ ID NO: 521); e. an epitope comprising the sequence IVDKNGRL (SEQ ID NO: 534); f. an epitope comprising the sequence IVDKNGRLVY (SEQ ID NO: 535); g. an epitope comprising the sequence DQTKF (SEQ ID NO: 536); h. an epitope comprising the sequence LVENPGGY (SEQ ID NO: 537) and / or an epitope comprising the sequence PIVNAELSF (SEQ ID NO: 538) and / or an epitope comprising the sequence PYLGTTMDT (SEQ ID NO: 539); i. an epitope comprising the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope comprising the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope comprising the sequence TYKEL (SEQ ID NO: 509); j. an epitope comprising the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 510) and / or an epitope comprising the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 511) and / or an epitope comprising the sequence LIERIPELNKVARAAAE (SEQ ID NO: 512); k. an epitope comprising the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 513); l. an epitope comprising the sequence GTKKDFEDL (SEQ ID NO: 514); m. an epitope comprising the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 515); n. an epitope comprising the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope comprising the sequence DQTKFPIVNAEL (SEQ ID NO: 517) and / or an epitope comprising the sequence TYKELIERIPELNK (SEQ ID NO: 518); o. an epitope comprising the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope comprising the sequence TYKELIERIPELNK (SEQ ID NO: 518); p. an epitope comprising the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 519); q. an epitope comprising the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope comprising the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 520); r. an epitope comprising the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope comprising the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 522) and / or an epitope comprising the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 523) and / or an epitope comprising the sequence IISRAAAEKL (SEQ ID NO: 524) and / or an epitope comprising the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 525) and / or an epitope comprising the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 526); s. an epitope contained within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope contained within or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope contained within or overlapping with the sequence TYKEL (SEQ ID NO: 509); t. an epitope contained within or overlapping with the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 510) and / or an epitope contained within or overlapping with the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 511) and / or an epitope contained within or overlapping with the sequence LIERIPELNKVARAAAE (SEQ ID NO: 512); u. an epitope contained within or overlapping with the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 513); v. an epitope contained within or overlapping with the sequence GTKKDFEDL (SEQ ID NO: 514); w. an epitope contained within or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 515); x. an epitope contained within or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope contained within or overlapping with the sequence DQTKFPIVNAEL (SEQ ID NO: 517) and / or an epitope contained within or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 518); y. an epitope contained within or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope contained within or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 518); z. an epitope contained within or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 519); an epitope contained within or overlapping with the aa sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope contained within or overlapping with the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 520); and bb. an epitope contained within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope contained within or overlapping with the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 522) and / or an epitope contained within or overlapping with the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 523) and / or an epitope contained within or overlapping with the sequence SRAAAEKL (SEQ ID NO: 524) and / or an epitope contained within or overlapping with the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 525) and / or an epitope contained within or overlapping with the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 526). The antibody binds to one or more epitopes of hTfR selected from the group consisting of:

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a. an epitope consisting of the sequence LLNE (SEQ ID NO: 529) and / or an epitope consisting of the sequence TYKEL (SEQ ID NO: 509); b. an epitope consisting of the sequence DSTDFTGT (SEQ ID NO: 530) and / or an epitope consisting of the sequence VKHPVTGQF (SEQ ID NO: 531) and / or an epitope consisting of the sequence IERIPEL (SEQ ID NO: 532); c. an epitope consisting of the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 533); d. an epitope consisting of the sequence FEDL (SEQ ID NO: 521); e. an epitope consisting of the sequence IVDKNGRL (SEQ ID NO: 534); f. an epitope consisting of the sequence IVDKNGRLVY (SEQ ID NO: 535); g. an epitope consisting of the sequence DQTKF (SEQ ID NO: 536); h. an epitope consisting of the sequence LVENPGGY (SEQ ID NO: 537) and / or an epitope consisting of the sequence PIVNAELSF (SEQ ID NO: 538) and / or an epitope consisting of the sequence PYLGTTMDT (SEQ ID NO: 539); i. an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope consisting of the sequence TYKEL (SEQ ID NO: 509); j. an epitope consisting of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 510) and / or an epitope consisting of the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 511) and / or an epitope consisting of the sequence LIERIPELNKVARAAAE (SEQ ID NO: 512); k. an epitope consisting of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 513); l. an epitope consisting of the sequence GTKKDFEDL (SEQ ID NO: 514); m. an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 515); n. an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope consisting of the sequence DQTKFPIVNAEL (SEQ ID NO: 517) and / or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 518); o. an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 518); p. an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 519); q. an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope consisting of the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 520); and r. an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope consisting of the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 522) and / or an epitope consisting of the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 523) and / or an epitope consisting of the sequence ISRAAAEKL (SEQ ID NO: 524) and / or an epitope consisting of the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 525) and / or an epitope consisting of the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 526) The antibody binds to one or more epitopes of hTfR selected from the group consisting of:

[0027] In some embodiments, the antigen binding protein is selected from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain fragment variable (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, or a chemically modified derivative thereof.

[0028] In some embodiments, the protein-drug conjugate comprises an scFv comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and a molecular cargo, wherein the molecular cargo is conjugated to the HCVR. In some embodiments, the protein-drug conjugate comprises an scFv comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and a molecular cargo, wherein the molecular cargo is conjugated to the LCVR. In some embodiments, the scFv and the molecular cargo are conjugated via a linker.

[0029] In some embodiments, the molecular cargo is conjugated to (i) the HCVR of the antigen binding protein, (ii) the LCVR of the antigen binding protein, (iii) the heavy chain of the antigen binding protein, and / or (iv) the light chain of the antigen binding protein.

[0030] In some embodiments, the molecular cargo is conjugated to (i) one or both HCVRs of the antigen binding protein, (ii) one or both LCVRs of the antigen binding protein, (iii) one or both heavy chains of the antigen binding protein, and / or (iv) one or both light chains of the antigen binding protein.

[0031] In some embodiments, the molecular cargo is conjugated to the antigen binding protein via glutamine and / or lysine residues.

[0032] In some embodiments, the glutamine residue is (i) introduced at the N-terminus and / or C-terminus of the heavy chain of the antigen binding protein, (ii) introduced at the N-terminus and / or C-terminus of the light chain of the antigen binding protein, (iii) naturally occurring in the CH2 or CH3 domain of the antigen binding protein, (iv) introduced into the antigen binding protein by modifying one or more amino acids, and / or (v) Q295, or mutated from N297 to Q297 (N297Q). In one embodiment, the glutamine residue is Q295.

[0033] In some embodiments, the antigen binding protein comprises a glutamine-containing tag, and the molecular cargo is conjugated to the antigen binding protein via a glutamine residue of the glutamine-containing tag. In some embodiments, the glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO:439), LLQG (SEQ ID NO:440), LSLSQG (SEQ ID NO:441), gGGLLQGG (SEQ ID NO:442), gLLQG (SEQ ID NO:443), LLQ (SEQ ID NO:444), gSPLAQSHGG (SEQ ID NO:445), gLLQGGG (SEQ ID NO:446), gLLQGG (SEQ ID NO:447), gLLQ (SEQ ID NO:448), LLQLLQGA (SEQ ID NO:449), LLQGA (SEQ ID NO:450), LLQYQGA (SEQ ID NO:451), LLQGSG (SEQ ID NO:452), LLQYQG (SEQ ID NO:453), LLQLLQG (SEQ ID NO:454), SLLQG (SEQ ID NO:455), LLQLQ (SEQ ID NO:456), LLQLLQ (SEQ ID NO:457), and LLQGR (SEQ ID NO:458).

[0034] In some embodiments, the antigen binding protein and the molecular cargo are conjugated via a linker, which can be a cleavable or non-cleavable linker.

[0035] In some embodiments, the protein-drug conjugate comprises a molecular cargo comprising a polynucleotide molecule, a carrier, or a small molecule.

[0036] In some embodiments, the protein-drug conjugate comprises a polynucleotide molecule. In some embodiments, the polynucleotide molecule is an interfering nucleic acid molecule, a guide RNA, a ribozyme, an aptamer, a mixer, a multimer, or an mRNA. In some embodiments, the interfering nucleic acid is an siRNA, an shRNA, an miRNA, a gapmer, or an antisense oligonucleotide. In some embodiments, the interfering nucleic acid is an siRNA. In some embodiments, the interfering nucleic acid is an antisense oligonucleotide. In some embodiments, the polynucleotide molecule is a guide RNA. In various embodiments, the polynucleotide molecule comprises one or more modified nucleotides.

[0037] In some embodiments, the molecular cargo is an siRNA that inhibits the DMPK, CNBP, dystrophin, DUX4, ApoE, MAPT, APP, HTT, SOD1, C9orf72, SNCA, LRRK2, PRNP, SLC5A1, SLC16A3, HDAC6, MMP27, MFAP5, FAM64A, BAIAP3, MYH7, TPM1, RBM20, KLHL24, MYL2 or TNNT2 gene or a mutant thereof.

[0038] In some embodiments, the siRNA comprises a sense strand 21 nucleotides in length. In some embodiments, the siRNA comprises an antisense strand 23 nucleotides in length. In some embodiments, the siRNA comprises two phosphorothioate linkages in the first and second internucleoside linkages at the 5'-end of the sense strand. In some embodiments, the siRNA comprises two phosphorothioate linkages in the first and second internucleoside linkages at the 3'-end and / or 5'-end of the antisense strand.

[0039] In some embodiments, the molecular cargo comprises a carrier, such as a lipid-based carrier. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP), a liposome, a lipidoid, or a lipoplex. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle (LNP) further comprises a polynucleotide molecule and / or a polypeptide molecule.

[0040] In some embodiments, a lipid nanoparticle (LNP) comprises one or more components of a gene editing system. In some embodiments, the lipid nanoparticle (LNP) comprises (a) a Cas nuclease or a nucleic acid encoding a Cas nuclease, and / or (b) a guide RNA or one or more DNAs encoding the guide RNA. In some embodiments, the Cas nuclease is a Cas9 protein. In some embodiments, the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein. In some embodiments, the nucleic acid encoding the Cas protein is codon-optimized for expression in mammalian cells. In some embodiments, the nucleic acid encoding the Cas protein is codon-optimized for expression in human cells. In some embodiments, the nucleic acid encoding the Cas nuclease comprises an mRNA encoding the Cas protein. In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the lipid nanoparticle (LNP) comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).

[0041] In some embodiments, the lipid nanoparticles comprise a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some embodiments, the neutral lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the helper lipid is cholesterol. In some embodiments, the stealth lipid is PEG2k-DMG.

[0042] In some embodiments, the antigen binding protein, when not conjugated to a molecular cargo, does not inhibit greater than 50% of the binding of a human transferrin receptor C-terminal fragment to human holo-transferrin that occurs in the absence of such single-chain fragment variable (scFv), antibody, or antigen-binding fragment. In some embodiments, such inhibition is measured in an enzyme-linked immunosorbent assay (ELISA) plate assay in which a human transferrin receptor extracellular domain fused to a His6-myc-myc tag is pre-bound to the scFv, antibody, or antigen-binding fragment and then contacted with holo-transferrin immobilized on the surface of a plate by binding of a plate-bound anti-holo-transferrin antibody.

[0043] In some embodiments, the binding of holo-transferrin and human transferrin receptor extracellular domain in the absence of an antigen binding protein is measured at a concentration of human transferrin receptor extracellular domain of about 300 pM.

[0044] In various embodiments, the protein-drug conjugates described herein exhibit the following characteristics: a) affinity for binding to human TfR at 25° C. in a surface plasmon resonance format with an affinity of about 41 nM or higher (K D b) affinity (K) for binding to monkey TfR at 25°C in a surface plasmon resonance format of about 0 nM (no detectable binding) or higher affinity D ), c) K binding to monkey TfR at 25°C in a surface plasmon resonance format of 0–278 D / K binding to human TfR D] ratio; d) Fab format (IgG1) blocks approximately 3-13% of hTfR binding to human Holo-Tf; e) scFv (V K -V H ) format, blocks approximately 6-13% of hTfR binding to human Holo-Tf; and / or f) scFv (V H -V L ) format, blocks approximately 11-26% of hTfR binding to human Holo-Tf;

[0045] In another aspect, provided herein is a pharmaceutical composition comprising a protein-drug conjugate described herein and a pharmaceutically acceptable carrier.

[0046] In another aspect, provided herein are compositions or kits comprising a protein-drug conjugate described herein or a pharmaceutical composition thereof in combination with an additional therapeutic agent. In one embodiment, the additional therapeutic agent is selected from alglucosidase alfa, rituximab, methotrexate, intravenous immunoglobulin (IVIG), avalglucosidase alfa, levalbuterol, antibiotics, cortisone, prednisone, bisphosphonates, and palivizumab. In one embodiment, the additional therapeutic agent is selected from beta2-adrenergic agonists, steroids, bisphosphonates, infectious disease treatments, vaccines, and pneumococcal vaccines.

[0047] In another aspect, provided herein is a complex comprising a protein-drug conjugate described herein linked to a human transferrin receptor polypeptide, or an antigenic fragment thereof.

[0048] In another aspect, provided herein is a method of making a protein-drug conjugate described herein, comprising: (a) contacting an antigen-binding protein with a molecular cargo under conditions favoring conjugation of the antigen-binding protein to the molecular cargo; and (b) optionally isolating the protein-drug conjugate produced in step (a). In another aspect, provided herein is a protein-drug conjugate that is the product of such a method.

[0049] In another aspect, provided herein is a container or injection device comprising a protein-drug conjugate described herein.

[0050] In another aspect, provided herein are methods of administering a protein-drug conjugate described herein to a subject, the method comprising introducing the protein-drug conjugate into the subject's body (e.g., brain or muscle). In some embodiments, the protein-drug conjugate is introduced into the subject's body parenterally (e.g., intravenously). In some embodiments, the protein-drug conjugate is introduced into the subject's body via intrathecal, intraventricular, or intraparenchymal injection into the central nervous system.

[0051] In another aspect, provided herein are methods for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a protein-drug conjugate described herein. In some embodiments, the disease or disorder is a lysosomal storage disease or disorder, a cardiac disease or disorder, a central nervous system (CNS) disease or disorder, an eye disease or disorder, a brain disease or disorder, a spinal cord disease or disorder, a peripheral nervous system (PNS) disease or disorder, a muscle disease or disorder, a cartilage disease or disorder, a bone growth plate disease or disorder, a kidney disease or disorder, or a blood disease or disorder.

[0052] In some embodiments, the disease or disorder is a neurological disease or disorder, hi some embodiments, the disease or disorder is a lysosomal storage disease, amyloidosis, a neurological disorder, a neurodegenerative disease, stroke, a behavioral disorder, a leukodystrophy, a neuropsychiatric disorder, a traumatic brain injury, a neurodevelopmental disorder, a neuromuscular disorder, an ocular disease or disorder, a viral or microbial infection, inflammation, ischemia, and cancer.

[0053] In some embodiments, the disease or disorder is a lysosomal storage disease.

[0054] In some embodiments, the disease or disorder is a neurodegenerative disease such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, or a prion disease. In some embodiments, the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit the ApoE, MAPT, APP, HTT, SOD1, C9orf72, SNCA, LRRK2, or PRNP gene or mutants thereof.

[0055] In some embodiments, the disease or disorder is a cardiac disease or disorder. In some embodiments, the cardiac disease or disorder is heart failure. In some embodiments, the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit the SLC5A1, SLC16A3, HDAC6, MMP27, MFAP5, FAM64A, BAIAP3, MYH7, TPM1, RBM20, KLHL24, MYL2, or TNNT2 gene or a mutant thereof.

[0056] In some embodiments, the disease or disorder is a muscular disease or disorder. In some embodiments, the muscular disease or disorder is myotonic dystrophy, Duchenne muscular dystrophy, fascioscapulohumeral muscular dystrophy, facioscapulohumeral muscular dystrophy type 1, or muscle atrophy. In some embodiments, the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit the DMPK, CNBP, dystrophin, or DUX4 gene or a mutant thereof.

[0057] In various embodiments, the subject is administered the protein-drug conjugate in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from alglucosidase alfa, rituximab, methotrexate, intravenous immunoglobulin (IVIG), avalglucosidase alfa, levalbuterol, antibiotics, cortisone, prednisone, bisphosphonates, and palivizumab. In some embodiments, the additional therapeutic agent is selected from beta2-adrenergic agonists, steroids, bisphosphonates, infectious disease treatments, vaccines, and pneumococcal vaccines.

[0058] In another aspect, provided herein are methods for treating or preventing myotonic dystrophy, Duchenne muscular dystrophy, fascioscapulohumeral muscular dystrophy, or facioscapulohumeral muscular dystrophy type 1 in a subject in need thereof, comprising administering to the subject an effective amount of a protein-drug conjugate described herein. In some embodiments, the molecular cargo of the protein-drug conjugate is an interfering RNA (e.g., siRNA) selected from the group consisting of interfering RNAs (e.g., siRNAs) that inhibit the DMPK, CNBP, dystrophin, or DUX4 gene or a mutant thereof.

[0059] In another aspect, the present disclosure provides methods for treating or preventing a neurodegenerative disease, such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, or a prion disease, in a subject in need thereof, the method comprising administering to the subject an effective amount of a protein-drug conjugate described herein. In some embodiments, the molecular cargo of the protein-drug conjugate is an interfering RNA (e.g., siRNA) selected from the group consisting of interfering RNAs (e.g., siRNAs) that inhibit the ApoE, MAPT, APP, HTT, SOD1, C9orf72, SNCA, LRRK2, or PRNP gene, or a mutant thereof.

[0060] In another aspect, the disclosure provides methods for treating or preventing a cardiac disease or disorder, such as heart failure, in a subject in need thereof, the method comprising administering to the subject an effective amount of a protein-drug conjugate described herein. In some embodiments, the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit the SLC5A1, SLC16A3, HDAC6, MMP27, MFAP5, FAM64A, BAIAP3, MYH7, TPM1, RBM20, KLHL24, MYL2, or TNNT2 gene or a mutant thereof.

[0061] In another aspect, provided herein are methods of delivering a molecular cargo to a tissue or cell type within a subject's body, comprising administering to the subject an antigen binding protein that specifically binds to the human transferrin receptor, or an antigenic fragment or variant thereof, conjugated to the molecular cargo. In some embodiments, the molecular cargo comprises a polynucleotide molecule, a carrier, or a small molecule. In some embodiments, the tissue is brain / spinal cord / CNS; eye; skeletal muscle; adipose tissue; blood / bone marrow; breast; lung / bronchi; colon; uterus; esophagus; heart; kidney; liver; lymph node; ovary; pancreas; placenta; prostate; rectum; skin; peripheral blood mononuclear cells (PBMCs); small intestine; spleen; stomach; testis; peripheral nervous system; and / or bone / cartilage / joint. In some embodiments, the cell type and tissue associated with the cell type are selected from Tables 1-4 herein. In some embodiments, the method comprises puncturing the body of a subject with a needle of a syringe and injecting into the body of the subject an antigen binding protein that specifically binds to transferrin receptor, or an antigenic fragment or variant thereof, conjugated to a molecular cargo, hi some embodiments, the subject is suffering from a muscle wasting condition, metabolic disease, sarcopenia or cachexia. [Brief explanation of the drawings]

[0062] [Figure 1A]Western blot showing that anti-human TFRC antibody clones deliver GAA to the cerebrum of Tfrchum mice. Each lane = 1 mouse. Anti-mouse mTfR:GAA from Wt mice was used as a positive control. Anti-mouse mTfR:GAA from Tfrchum mice was used as a negative control. [Figure 1B] Western blot showing that anti-human TFRC antibody clones deliver GAA to the cerebrum of Tfrchum mice. Each lane = 1 mouse. Anti-mouse mTfR:GAA from Wt mice was used as a positive control. Anti-mouse mTfR:GAA from Tfrchum mice was used as a negative control. [Figure 1C] Western blot showing that anti-human TFRC antibody clones deliver GAA to the cerebrum of Tfrchum mice. Each lane = 1 mouse. Anti-mouse mTfR:GAA from Wt mice was used as a positive control. Anti-mouse mTfR:GAA from Tfrchum mice was used as a negative control.

[0063] [Figure 2] Western blots show that a subset of anti-hTFRC antibody clones deliver mature GAA to the brain parenchyma in the scfv:GAA format (delivered by HDD). Anti-mouse mTfR:GAA in wild-type mice was used as a positive control. Anti-mouse mTfR:GAA in Tfrchum mice was used as a negative control.

[0064] [Figure 3] Western blots show that four selected anti-hTFRC antibody clones deliver mature GAA to the brain parenchyma in the scfv:GAA format (AAV8 episomal liver depot gene therapy). GAA in anti-mouse mTfR:Wt mice was used as a positive control. GAA in anti-mouse mTfR:Tfrchum mice was used as a negative control.

[0065] [Figure 4]Western blot showing that three selected episomal AAV8 liver depot anti-hTFRC antibody clones deliver mature GAA to the CNS, heart, and muscle of Gaa- / -Tfrchum mice.

[0066] [Figure 5] We show that three selected episomal AAV8 liver depot anti-hTFRC antibody clones rescue glycogen stores in the CNS, heart, and muscle of Gaa- / - Tfrchum mice. Wt naive mice served as a positive control, and Gaa- / - naive mice served as a negative control.

[0067] [Figure 6A] We show that three selected episomal AAV8 liver depot anti-hTFRC antibody clones rescue glycogen stores in the thalamus of Gaa- / - Tfrchum mice (Figure 6A). Wt naive mice served as a positive control, and Gaa- / - naive mice served as a negative control. [Figure 6B] We show that three selected episomal AAV8 liver depot anti-hTFRC antibody clones rescue glycogen stores in the cerebral cortex of Gaa- / - Tfrchum mice (Figure 6B). Wt naive mice served as a positive control, and Gaa- / - naive mice served as a negative control. [Figure 6C] We show that three selected episomal AAV8 liver depot anti-hTFRC antibody clones rescue glycogen stores in the hippocampal CA1 region of Gaa- / -Tfrchum mice (Figure 6C). Wt naive mice served as a positive control, and Gaa- / - naive mice served as a negative control. [Figure 6D] We show that three selected episomal AAV8 liver depot anti-hTFRC antibody clones rescue glycogen stores in the quadriceps muscle of Gaa- / - Tfrchum mice (Figure 6D). Wt naive mice served as a positive control, and Gaa- / - naive mice served as a negative control.

[0068] [Figure 7A]Figure 1 shows that insertion of anti-hTFRC 12847scfv:GAA delivers mature GAA protein to the CNS and muscle of Pompe model mice. [Figure 7B] Anti-hTFRC 12847scfv insertion:GAA rescues glycogen stores in the CNS and muscle of Pompe model mice. One-way ANOVA *p<0.01; **p<0.001; ***p<0.0001. Untreated Pompe disease model mice and wild-type mice were used as controls. Mice injected with recombinant AAV8 anti-TfR:GAA episomal template was used as a positive control. Mice injected with recombinant AAV8 anti-TfR:GAA insertion template without LNP-g666 was used as a negative control.

[0069] [Figure 8] The figure shows the interactions of the Mammarenavirus machupoense GP1 protein (PDB 3KAS), human ferritin (PDB 6GSR), Plasmodium vivax Sal-1 PvRBP2b protein (PDB 6D04), human HFE protein (PDB 1DE4), and human transferrin (PDB 1SUV) molecules superimposed in the symmetry unit on two TfR molecules. For the Mammarenavirus machupoense GP1 protein and human ferritin, only a single copy in the symmetry unit has been shown to reduce the complexity of the diagram for clarity.

[0070] [Figure 9] We show that hydrogen-deuterium exchange mass spectrometry (HDX) protection of the antibodies tested in HDX-MS experiments can be assigned to five regions of TfR (PDB 1SUV).

[0071] [Figure 10]Figure 1 shows the TfR region protected by REGN17513. Representation of an antibody that elicits HDX protection in the TfR apical domain overlapping with the Mammarenavirus machupoense GP1 protein, human ferritin, and Plasmodium vivax PvRBP2b protein binding sites.

[0072] [Figure 11] Shown are the TfR regions protected by REGN17510, a representation of an antibody with HDX protection in the TfR apical domain that is not shared by other TfR binding partners shown in Figure 11 .

[0073] [Figure 12] Figure 1 shows the TfR region protected by REGN17515. Representation of an antibody with HDX protection in the TfR apical domain, which shares binding sites with human ferritin and Plasmodium vivax Sal-1 PvRBP2b protein.

[0074] [Figure 13] 1 shows the TfR region protected by REGN17514, a representation of an antibody that has HDX protection in the TfR protease-like domain and shares a binding site with the Plasmodium vivax Sal-1 PvRBP2b protein.

[0075] [Figure 14] 14 shows the region of TfR protected by REGN17508, a representation of an antibody with HDX protection in the TfR protease-like domain. This region is not utilized by other TfR-interacting molecules shown in FIG. 14. DETAILED DESCRIPTION OF THE INVENTION

[0076] Provided herein are anti-transferrin receptor (TfR) antigen-binding proteins conjugated to molecular cargoes. Such conjugates are useful for delivering molecular cargoes to various tissues in the body, including, for example, the brain and muscle. For example, anti-TfR protein-drug conjugates are provided that exhibit high affinity for the transferrin receptor and excellent blood-brain barrier crossing. Surprisingly, anti-TfR scFvs that exhibit high binding affinity for TfR crossed the BBB more efficiently than low-affinity binders. This contrasts with previous findings with monovalent and bivalent anti-TfR antibodies, in which low-affinity antibodies crossed the BBB more effectively. The conjugates described herein are capable of efficiently delivering molecular cargoes to the brain and muscle and can therefore be used for the treatment of diseases and disorders, such as neurological or muscular diseases and disorders.

[0077] In accordance with the present disclosure, one may employ conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being explained fully in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (herein referred to as "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985); Oligonucleotide Synthesis (MJ Gait ed.1984);Nucleic Acid Hybridization(BDHames & SJHiggins eds.(1985));Transcription And Translation(BDHames & SJHiggins,eds.1984;Animal Cell Culture(RIFreshney,ed.(1986));Immobilized Cells And Enzymes(IRL Press,(1986));B.Perbal,A Practical Guide To Molecular Cloning(1984);FMAusubel,et al.(eds.),Current Protocols See in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0078] Polynucleotides include DNA and RNA. The present disclosure includes any polynucleotide described herein operably linked to a promoter or other expression control sequence.

[0079] Transferrin receptor 1 (TfR) is a membrane receptor involved in regulating iron supply to cells through binding of transferrin, a major iron carrier protein. Transferrin receptor 1 is expressed from the TFRC gene. Transferrin receptor 1 may also be referred to herein as TFRC. Because iron is essential for maintaining ribonucleotide reductase activity, the only enzyme catalyzing the conversion of ribonucleotides to deoxyribonucleotides, this receptor plays an important role in regulating cell proliferation. Preferably, the TfR is human TfR (hTfR). See, e.g., accession numbers NP_001121620.1; BAD92491.1; and NP_001300894.1; and e!Ensembl entry: ENSG00000072274. Human transferrin receptor 1 is expressed in several tissues, including, but not limited to, the cerebral cortex, cerebellum, hippocampus, caudate nucleus, parathyroid glands, adrenal glands, bronchi, pulmonary oral mucosa, esophagus, stomach, duodenum, small intestine, colon, rectum, liver, gallbladder, pancreas, kidney, urinary bladder, testis, epididymis, prostate, vagina, ovaries, fallopian tubes, endometrium, cervix, placenta, breast, cardiac muscle, smooth muscle, soft tissue, skin, appendix, lymph nodes, tonsils, and bone marrow. See also the tissues and cell types in Tables 1-4 herein. A related transferrin receptor is transferrin receptor 2 (TfR2). Human transferrin receptor 2 shares approximately 45% sequence identity with human transferrin receptor 1. Trinder & Baker, Transferrin receptor 2: a new molecule in iron metabolism. Int J Biochem Cell Biol. 2003 Mar;35(3):292-6. Unless otherwise specified, transferrin receptor as used herein generally refers to transferrin receptor 1 (e.g., human transferrin receptor 1).

[0080] Human transferrin (Tf) is a single-chain, 80 kDa member of the anion-binding superfamily of proteins. Transferrin is a 698-amino acid precursor divided into a 19-amino acid signal sequence and a 679-amino acid mature segment, typically containing 19 intrachain disulfide bonds. The N- and C-terminal flanking regions (or domains) bind ferric iron through interactions with obligate anions (e.g., bicarbonate) and four amino acids (His, Asp, and two Tyr). Apotransferrin (or iron-free) first binds a single atom of iron at the C-terminus, followed by iron binding by the N-terminus to form holotransferrin (diferric Tf, holo-Tf). Via its C-terminal iron-binding domain, holotransferrin interacts with TfR on the cell surface, where it is internalized into acidifying endosomes. Iron dissociates from the Tf molecule within these endosomes and is transported to the cytosol as ferrous iron. In addition to TfR, transferrin has been reported to bind to cubrin, IGFBP3, microbial iron-binding proteins, and liver-specific TfR2.

[0081] The blood-brain barrier (BBB) ​​is located within the brain's microvasculature and regulates the passage of molecules from the blood to the brain. Burkhart et al., "Accessing targeted nanoparticles to the brain: the vascular route." Curr Med Chem. 2014;21(36):4092-9. Cellular passage through brain capillary endothelial cells can occur via: 1) cell invasion by leukocytes; 2) carrier-mediated influx of glucose, e.g., via glucose transporter 1 (GLUT-1), amino acids, e.g., via L-type amino acid transporter 1 (LAT-1), and small peptides, e.g., via organic anion transport peptide-B (OATP-B); 3) paracellular passage of small hydrophobic molecules; 4) adsorptive-mediated transcytosis of albumin and cationized molecules; 5) passive diffusion of lipid-soluble nonpolar solutes, including CO2 and O2; and 5) receptor-mediated transcytosis of insulin, e.g., via the insulin receptor, and Tf, e.g., via the TfR. Johnsen et al.,Targeting the transferrin receptor for brain drug delivery,Prog Neurobiol.2019 Oct;181:101665. Anti-human transferrin receptor antigen-binding protein conjugate

[0082] Anti-hTfR protein-drug conjugates are provided herein. The anti-hTfR protein-drug conjugates comprise an optional signal peptide linked to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment of an antibody, such as a Fab or scFv) that specifically binds to the transferrin receptor, preferably human transferrin receptor 1 (hTfR), conjugated (optionally via a linker) to a molecular cargo. The anti-hTfR antigen-binding proteins described herein can efficiently cross the blood-brain barrier (BBB), thereby delivering the conjugated molecular cargo to the brain.

[0083] Antigen binding proteins that specifically bind to transferrin receptors and their protein-drug conjugates, e.g., tags such as His6 and / or myc (e.g., human transferrin receptor (e.g., REGN2431) or monkey transferrin receptor (e.g., REGN2054)), have a K of about 20 nM or higher affinity at about 25° C., e.g., in a surface plasmon resonance assay. D Such antigen-binding proteins may be referred to as "anti-TfR."

[0084] The term "conjugate" refers to an entity in which two substances are covalently or non-covalently linked. The term "covalent bond" refers to the characteristic of at least two molecules being linked to each other by one or more covalent bonds. In various embodiments, the two molecules can be covalently linked to each other by a single bond, such as a disulfide bridge or disulfide bond, that acts as a linker between the molecules. In some embodiments, two or more molecules can be covalently linked together by a molecule that acts as a linker, binding at least two molecules together through multiple covalent bonds. In certain embodiments, the linker can be a cleavable linker or a non-cleavable linker. A conjugate can be a direct link between two substances or a linker between two substances. In certain embodiments, one of the two substances is an antigen-binding protein, such as an antibody or antigen-binding fragment thereof, and the other is a drug (e.g., a polynucleotide disclosed herein, or a liposome or LNP). In certain embodiments, the linker can be a cleavable linker or a non-cleavable linker.

[0085] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a conjugate of an antibody or antigen-binding fragment thereof and a drug (e.g., a polynucleotide disclosed herein, or a liposome or LNP). Affinity for an antigen is imparted to the drug by conjugating the antibody or antigen-binding fragment thereof to the drug (e.g., a polynucleotide disclosed herein, or a liposome or LNP), thereby increasing the efficiency of delivery of the drug to a target site in vivo.

[0086] In one embodiment, the assignment of amino acids to each framework or CDR domain in an immunoglobulin follows the definitions in Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. Thus, V H and V L comprises the amino acid sequence set forth herein (see, e.g., the sequences in Table 1-1 or variants thereof), and the CDRs are as defined according to Kabat and / or Chothia; H CDR and V L Also included herein are antibodies and antigen-binding fragments comprising the CDRs of

[0087] The protein-drug conjugates described herein include antibodies that specifically bind to human transferrin receptor 1. As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds. In one embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region ("HCVR" or "V"). H " ) (e.g., comprising SEQ ID NOs: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 and / or 312 or variants thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4), and each antibody light chain (LC) comprises a light chain variable region ("LCVR" or "VVR"). L " ) (e.g., SEQ ID NOs: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 527; 317 and / or 484 or variants thereof) and a light chain constant region (e.g., human kappa or human lambda). H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). H and V L comprises three CDRs and four FRs. The anti-TfR antibodies described herein can also be conjugated to a molecular cargo.

[0088] The anti-TfR antigen-binding proteins described herein may be antigen-binding fragments of antibodies that can be conjugated to molecular cargo. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, refers to an immunoglobulin molecule that binds to an antigen but does not contain all of the sequence of a complete antibody (preferably, the complete antibody is an IgG). Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; and (vi) dAb fragments; consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules such as domain-specific antibodies, single domain antibodies, one-arm antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies and small modular immunopharmaceuticals (SMIPs) are also encompassed by the term "antigen-binding fragment" as used herein.

[0089] In some embodiments, the anti-TfR protein-drug conjugates described herein may comprise an scFv conjugated to a molecular cargo. The scFv (single chain fragment variable) comprises a heavy (V H ) domain and light (V L) domain variable regions (in any order), which are preferably connected to each other by a flexible linker (e.g., a peptide linker). The length of the flexible linker used to connect both V regions can be important for ensuring correct folding of the polypeptide chain. Previously, it has been estimated that a peptide linker must span 3.5 nm (35 Å) between the carboxy terminus of one variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods in Enzymology. 1991;203:46-88). In one embodiment, the linker comprises an amino acid sequence of such length that it separates the variable domains by approximately 3.5 nm. In one embodiment of the present invention, the anti-TfR scFv-drug conjugate comprises an scFv comprising a variable region arrangement such as the following: LCVR-HCVR or HCVR-LCVR, where the HCVR and LCVR are optionally linked by a linker, and the scFv is optionally linked by a linker to a molecular cargo (e.g., LCVR-(Gly4Ser)3-HCVR-molecular cargo); or LCVR-((Gly4Ser)3-HCVR-molecular cargo).

[0090] In some embodiments, the anti-TfR protein-drug conjugates described herein may comprise a Fab conjugated to a molecular cargo.

[0091] In some embodiments, the anti-TfR protein-drug conjugates described herein comprise a bivalent antibody conjugated to a molecular cargo.

[0092] In some embodiments, the anti-TfR protein-drug conjugates described herein comprise a monovalent or "one-arm" antibody conjugated to a molecular cargo. As used herein, a monovalent or "one-arm" antibody refers to an immunoglobulin protein comprising a single variable domain. For example, a one-arm antibody may comprise a single variable domain within a Fab, where the Fab is linked to at least one Fc fragment. In certain embodiments, a one-arm antibody comprises a polypeptide comprising: (i) a heavy chain comprising a heavy chain constant region and a heavy chain variable region; (ii) a light chain comprising a light chain constant region and a light chain variable region; and (iii) an Fc fragment or truncated heavy chain. In certain embodiments, the Fc fragment or truncated heavy chain comprised in a separate polypeptide is a "dummy Fc," which refers to an Fc fragment not linked to an antigen-binding domain. The one-arm antibodies described herein may comprise any of the HCVR / LCVR pairs or CDR amino acid sequences set forth in Table 1-1 herein. One-arm antibodies comprising a full-length heavy chain, a full-length light chain, and an additional Fc domain polypeptide can be constructed using standard methodology (see, e.g., WO2010151792, incorporated herein by reference in its entirety), where the heavy chain constant region differs from the Fc domain polypeptide by at least two amino acids (e.g., H95R and Y96F according to the IMGT exon numbering system; or H435R and Y436F according to the EU numbering system). Such modifications are useful for purifying monovalent antibodies (see WO2010151792).

[0093] An antigen-binding fragment of an antibody, in one embodiment, comprises at least one variable domain. A variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H For antigen-binding fragments containing domains, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer, with the V H -V H , VH -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomer V H or V L It may contain domains.

[0094] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragments of antibodies described herein include: (i) a V H -CH1;(ii)V H -CH2;(ill)VH-CH3;(iv)V H -CH1-CH2;(v)V H -CH1-CH2-CH3;(vi)VH-CH2-CH3;(vii)V H -CL;(viii)VL-CH1;(ix)VL-CH2;(x)VL-CH3;(xi)VL-CH1-CH2;(xii)VL-CH1-CH2-CH3;(xiii)V L -CH2-CH3; and (xiv) V L -CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provides a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies described herein may be linked to each other and / or to one or more monomeric V H or V L The domains may be non-covalently associated (e.g., by disulfide bonds) to comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above. The present disclosure includes antigen-binding fragments of antigen-binding proteins, such as antibodies, described herein.

[0095] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. The present disclosure includes monospecific and multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains from the antigen-binding proteins specifically described herein.

[0096] The terms "specifically binds" or "binds specifically" refer to a specificity of at least about 10 M (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 nM) as measured by a real-time label-free biolayer interferometry assay, e.g., at 25°C or 37°C, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution affinity ELISA. D "Anti-TfR" refers to an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that has binding affinity for an antigen, such as the human TfR protein, mouse TfR protein, or monkey TfR protein, represented as:

[0097] "Isolated" antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides, and vectors are at least partially free from other biological molecules from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other materials such as cell debris and growth medium. Isolated antigen binding proteins may also be at least partially free from expression system components such as biological molecules from the host cell or its growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules (e.g., trace or insignificant amounts of impurities may remain), or the absence of water, buffers, or salts, or components of a pharmaceutical formulation that comprises the antigen binding protein (e.g., antibody or antigen-binding fragment).

[0098] The present disclosure includes antigen binding proteins, such as antibodies or antigen-binding fragments, that bind to the same epitope as the antigen binding proteins described herein.

[0099] An antigen is a molecule, such as a peptide (e.g., TfR or a fragment thereof (antigenic fragment)) to which an antibody or antigen-binding fragment thereof binds. The specific region on an antigen that an antibody recognizes and binds to is called an epitope. Antigen-binding proteins (e.g., antibodies) described herein that specifically bind to such antigens are part of the present disclosure.

[0100] The term "epitope" refers to a specific antigen-binding site of an antigen-binding protein, e.g., an antigenic determinant that interacts with the variable region of an antibody known as the paratope (e.g., TfR). A single antigen can have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" can also refer to the site on an antigen to which B cells and / or T cells respond and / or the region of an antigen bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. The epitopes to which the antigen binding proteins described herein bind may be contained in a fragment of the TfR, such as its extracellular domain. Antigen binding proteins (e.g., antibodies) described herein that bind to such epitopes are also contemplated.

[0101] Methods for determining the epitope of an antigen-binding protein, such as an antibody or fragment or polypeptide, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antigen-binding protein (e.g., an antibody or fragment or polypeptide) interacts is hydrogen / deuterium exchange as detected by mass spectrometry. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0102] The present disclosure includes antigen binding proteins that compete with the antigen binding proteins described herein for binding to the TfR epitopes described herein. As used herein, the term "compete" refers to an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., TfR) and inhibits or blocks the binding of another antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) to the antigen. Unless otherwise specified, the term also includes competition between two antigen binding proteins, e.g., antibodies, in both orientations, i.e., a first antibody binding to an antigen and blocking binding by a second antibody, and vice versa. Thus, in one embodiment, competition occurs in one such orientation. In certain embodiments, the first antigen binding protein (e.g., an antibody) and the second antigen binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen binding proteins (e.g., antibodies) may bind to different, but overlapping or non-overlapping, epitopes, such that binding of one inhibits or blocks binding of the second antibody, e.g., through steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, such as real-time label-free biolayer interferometry assays, and binding competition between TfR-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using real-time label-free biolayer interferometry assays on an Octet RED384 biosensor (Pall ForteBio Corp.).

[0103] Typically, antibodies or antigen-binding fragments described herein that have been modified in some way retain the ability to specifically bind to TfR, e.g., retain at least 10% of their TfR binding activity (compared to the parent antibody) when that activity is expressed on a molar basis. Preferably, antibodies or antigen-binding fragments described herein retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the TfR binding affinity of the parent antibody. It is also intended that the antibodies or antigen-binding fragments described herein may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of antibodies) that do not substantially alter their biological activity.

[0104] The anti-TfR antigen-binding proteins described herein can be monoclonal antibodies or antigen-binding fragments of monoclonal antibodies that can be conjugated to a molecular cargo. The present disclosure includes monoclonal anti-TfR antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" or "mAb" refers to a member of a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible minor naturally occurring mutations. A "plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., in amino acid sequence except for possible minor naturally occurring mutations, as discussed above) antibodies and fragments that exceeds the concentration that normally occurs in nature in the blood of a host organism, e.g., a mouse or human.

[0105] In one embodiment, the anti-TfR antigen binding protein, e.g., antibody or antigen-binding fragment (which may be conjugated to a molecular cargo), comprises a heavy chain constant domain, e.g., of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4) or IgM type. In one embodiment, the antigen binding protein, e.g., antibody or antigen-binding fragment, comprises a light chain constant domain, e.g., of the kappa or lambda type. In one embodiment, the V H is linked to a human heavy chain constant domain (e.g., IgG), and L is linked to a human light chain constant domain (e.g., kappa). The present disclosure includes antigen binding proteins comprising, e.g., a variable domain as described herein linked to a heavy and / or light chain constant domain as described herein.

[0106] The present disclosure includes human anti-TfR antigen-binding proteins that can be conjugated to molecular cargoes. The term "human" antigen-binding protein (e.g., antibody or antigen-binding fragment), as used herein, includes antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences, whether in a human cell or grafted into a non-human cell (e.g., a mouse cell). See, e.g., U.S. Patent Nos. 8,502,018; 6,596,541; or 5,789,215. The anti-TfR human mAbs described herein may contain amino acid residues, e.g., in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) are grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. The term is not intended to include natural antibodies isolated directly from a human subject. The present disclosure includes human antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof described herein).

[0107] The present disclosure includes anti-TfR chimeric antigen-binding proteins, such as antibodies and antigen-binding fragments thereof (which may be conjugated to molecular cargo), and methods of use thereof. As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). The present disclosure includes chimeric antibodies comprising the variable domains described herein and non-human constant domains.

[0108] A "recombinant" anti-TfR antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof (which may be conjugated to a molecular cargo), refers to such a molecule made, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. This term includes antibodies expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or cell (e.g., a CHO cell), e.g., in a cellular expression system, or antibodies isolated from a recombinant combinatorial human antibody library. The present disclosure includes recombinant antigen-binding proteins, such as the antibodies and antigen-binding fragments described herein.

[0109] Antigen-binding fragments of antibodies, in one embodiment, do not include the entire antibody, but specifically bind to an antigen, e.g., TfR, comprising at least one variable domain. The variable domain may be of any size or amino acid composition and generally comprises at least one (e.g., three) CDRs adjacent to or in-frame with one or more framework sequences. L V associated with the domain H For antigen-binding fragments containing domains, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer, with the V H -VH , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of non-covalently bound monomeric V H and / or V L It may include a domain.

[0110] "Variant" polypeptides, e.g., immunoglobulin chains, may be any of the amino acid sequences of a reference amino acid sequence described herein (e.g., SEQ ID NOs: 2; 3; 4; 5; 7; 8; 9; 10; 12; 13; 14; 15; 17; 18; 19; 20; 22; 23; 24; 25; 27; 28; 29; 30; 32; 33; 34; 35; 37; 38; 39; 40; 42; 43; 44; 45; 47; 48; 49; 50; 52; 53; 54; 55; 57; 58; 59; 60; 62; 63; 64; 65; 67; 68; 69; 70; 72; 73; 74; 75; 77; 78; 79; 80; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127; 12 4;85;87;88;89;90;92;93;94;95;97;98;99;100;102;103;104;105;107;108;109;110;112;113;114;115;117;118;119;120;122;123;124;125;127 ;128;129;130;132;133;134;135;137;138;139;140;142;143;144;145;147;148;149;150;152;153;154;155;157;158;159;160;162;163;164;165; 167;168;169;170;172;173;174;175;177;178;179;180;182;183;184;185;187;188;189;190;192;193;194;195;197;198;199;200;202;203;204;2 05;207;208;209;210;212;213;214;215;217;218;219;220;222;223;224;225;227;228;229;230;232;233;234;235;237;238;239;240;242;243;24 4;245;247;248;249;250;252;253;254;255;257;258;259;260;262;263;264;265;267;268;269;270;272;273;274;275;277;278;279;280;282;283 ;284;285;287;288;289;290;292;293;294;295;297;298;299;300;302;303;304;305;307;527;308;309;310;312;313;314;315;317;318;319;320;and 328 to 423) and 70 to 99.9% (e.g., at least about 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar amino acid sequences, and when the comparison is performed using the BLAST algorithm, the algorithm parameters are selected to maximize the match between the respective sequences over the entire length of each reference sequence (e.g., expectation threshold: 10; word size: 3; maximum match within query range: 0; BLOSUM 62 matrix; gap costs: presence 11, extension 1; conditional composition score matrix adjustment) and / or refers to a polypeptide comprising an amino acid sequence but having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations (e.g., point mutations, insertions, truncations, and / or deletions);

[0111] Furthermore, a variant of a polypeptide can include a polypeptide such as an immunoglobulin chain that can comprise the amino acid sequence of a reference polypeptide, which amino acid sequence is specifically described herein, but with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as one or more missense mutations (e.g., conservative substitutions), non-sense mutations, deletions, or insertions. For example, the present disclosure provides immunoglobulin light chains (or V) that include the amino acid sequences set forth in SEQ ID NOs: 7, 17, 27, 37, 465, 47, 466, 57, 468, 67, 469, 77, 471, 87, 97, 107, 117, 474, 127, 137, 147, 476, 157, 167, 177, 187, 479, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 527, 317, 484, but with one or more of such mutations. L) variants, and / or immunoglobulin heavy chains (or V) comprising the amino acid sequence set forth in SEQ ID NOs: 2, 462, 12, 463, 22, 464, 32, 42, 52, 467, 62, 492, 72, 470, 82, 92, 472, 102, 112, 473, 122, 132, 142, 475, 152, 162, 477, 172, 182, 478, 192, 480, 202, 481, 212, 222, 232, 242, 252, 482, 262, 272, 282, 292, 302, 483, 312, but having one or more of such mutations. H In one embodiment, the TfR binding protein comprises an immunoglobulin light chain variant comprising CDR-L1, CDR-L2, and CDR-L3, wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more of such mutations (e.g., conservative substitutions), and / or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2, and CDR-H3, wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more of such mutations (e.g., conservative substitutions).

[0112] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20):5101-5109; Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al. al.,(1993)Comput.Chem.17:149-163;Hancock,JMet al.,(1994)Comput.Appl.Biosci.10:67-70;ALIGNMENT SCORING SYSTEMS:Dayhoff,MO,et al.,"A model of evolutionary change in proteins."in Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.MODayhoff(ed.),pp.345-352,Natl.Biomed.Res.Found.,Washington,DC;Schwartz,RM,et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.”MODayhoff(ed.),pp.353-358,Natl.Biomed.Res.Found.,Washington,DC;Altschul,SF,(1991)J.Mol.Biol.219:555-565;States,DJ,et al.,(1991)Methods 3:66-70;Henikoff,S.,et al.,(1992)Proc.Natl.Acad.Sci.USA 89:10915-10919;Altschul,SF,et al.,(1993)J.Mol.Evol.36:290-300;ALIGNMENT STATISTICS:Karlin,S.,et al.,(1990)Proc.Natl.Acad.Sci.USA 87:2264-2268;Karlin,S.,et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, SF “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, NY. .

[0113] For example, "conservatively modified variants" or "conservative substitutions" of immunoglobulin chains described herein refer to variants in which there is a substitution of one or more amino acids in a polypeptide with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main-chain conformation, and rigidity, etc.). Such changes can frequently be made without significantly destroying the biological activity of the antibody or fragment. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to significantly destroy biological activity. The present disclosure includes TfR-binding proteins comprising such conservatively modified variant immunoglobulin chains.

[0114] Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45.

[0115] The antibodies and antigen-binding fragments described herein include immunoglobulin chains comprising the amino acid sequences specifically set forth herein (and variants thereof), as well as cellular and in vitro post-translational modifications to the antibodies or fragments. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to TfR comprising the heavy chain amino acid sequences and / or light chain amino acid sequences described herein, as well as antibodies and fragments in which one or more asparagine, serine, and / or threonine residues are glycosylated, one or more asparagine residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, the N-terminal glutamine is pyroglutamate (pyroE), and / or the C-terminal lysine or other amino acid is missing.

[0116] In one embodiment, the anti-hTfR protein-drug conjugate (eg, in scFv, Fab, or other antibody or antigen-binding fragment thereof format) may exhibit one or more of the following characteristics: An affinity (K) for binding to human TfR at 25°C in a surface plasmon resonance format of about 41 nM or higher (e.g., about 1 or 0.1 nM or about 0.18 to about 1.2 nM or higher). D ); Affinity (K) for binding to monkey TfR at 25°C in a surface plasmon resonance format of about 0 nM (no detectable binding) or higher affinity (e.g., about 20 nM or higher) D ); K for binding to monkey TfR / human TfR at 25°C in a surface plasmon resonance format of 0–278 D a ratio of (e.g., about 17 or 18); in the case of a Fab format (IgG1), inhibits about 3, 5, 10, or 13% of hTfR (e.g., Hmm-hTFRC, e.g., REGN2431) binding to human Holo-Tf, e.g., about 45% or less inhibition; scFv(V K -V H ) format, inhibits about 6, 8, 10, or 13% of hTfR (e.g., Hmm-hTFRC, e.g., REGN2431) binding to human Holo-Tf, e.g., about 45% or less inhibition; scFv(V H -V L ) format, inhibits about 11, 17, 23, or 26% of hTfR (e.g., Hmm-hTFRC, e.g., REGN2431) binding to human Holo-Tf, e.g., about 45% or less inhibition; * Tfrchum or Tfrchum / hum is a homozygous knock-in mouse.

[0117] The amino acid sequences of domains in the anti-human transferrin receptor antigen-binding proteins of the conjugates described herein are summarized in Table 1-1 below. Included herein, for example, are anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFv and Fab) that comprise the HCVR and LCVR of the molecules in Table 1-1; or those that comprise the CDRs thereof conjugated to a molecular cargo. [Table 1-1] [Table 8] [Table 9]

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

[0118] As discussed, anti-hTfR scFv protein-drug conjugates (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263) comprise scFvs (e.g., Vs) optionally linked by a linker. L and V Hand optionally a signal peptide (e.g., an mROR signal sequence) linked to a molecular cargo, linked to a selected linker, linked to a selected linker, including, for example, (I) The optional signal peptide is, for example, the signal peptide from Mus musculus R or 1 (e.g., consisting of the amino acids MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 528)); (II) the scFv comprising (i) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 or 312; and / or (ii) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 527; 317 or 484; or scFv (1) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 or 462 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (2) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 12 or 463 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (3) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 or 464 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (4) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 37 or 465 (or a variant thereof); (5) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 47 or 466 (or a variant thereof); (6) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 52 or 467 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 57 or 468 (or a variant thereof); (7) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 or 492 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 67 or 469 (or a variant thereof); (8) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 72 or 470 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 77 or 471 (or a variant thereof); (9) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (10) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92 or 472 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (11) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (12) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112 or 473 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 117 or 474 (or a variant thereof); (13) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (14) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (15) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 142 or 475 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 147 or 476 (or a variant thereof); (16) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (17) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 or 477 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (18) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (19) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 182 or 478 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 or 479 (or a variant thereof); (20) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 192 or 480 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (21) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 or 481 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (22) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (23) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (24) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (25) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (26) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 or 482 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (27) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (28) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (29) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (30) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (31) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 or 483 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 or 527 (or a variant thereof); (32) A HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 or 484 (or a variant thereof), or scFv (a) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof); and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); LCVR including (b) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20 (or a variant thereof); LCVR including (c) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); LCVR including (d) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); LCVR including (e) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); LCVR including (f) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 (or a variant thereof); LCVR including (g) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); LCVR including (h) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 (or a variant thereof); LCVR including (i) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); LCVR including (j) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 (or a variant thereof); Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100 (or a variant thereof); LCVR including (k) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); LCVR including (l) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120 (or a variant thereof); LCVR including (m) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 123 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 125 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof) LCVR including (n) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof) LCVR including (o) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof) LCVR including (p) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160 (or a variant thereof) LCVR including (q) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170 (or a variant thereof) LCVR including (r) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof) LCVR including (s) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 (or a variant thereof) LCVR including (t) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 194 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 195 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof) LCVR including (u) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 208 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 210 (or a variant thereof) LCVR including (v) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof) LCVR including (w) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof) LCVR including (x) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof) LCVR including (y) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof) LCVR including (z) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof) LCVR including (aa) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof) LCVR including (ab) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof) LCVR including (ac) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof) LCVR including (ad) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof) LCVR including (ae) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof) LCVR including and / or (af) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof); and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof) Including HCVR and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof); and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof) LCVR including Including, or scFv (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 or 462 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 12 or 463 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 or 464 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 37 or 465 (or a variant thereof); (v) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 47 or 466 (or a variant thereof); (vi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 52 or 467 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 57 or 468 (or a variant thereof); (vii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 or 492 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 67 or 469 (or a variant thereof); (viii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 72 or 470 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 77 or 471 (or a variant thereof); (ix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (x) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92 or 472 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (xi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (xii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112 or 473 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 117 or 474 (or a variant thereof); (xiii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (xiv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (xv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 142 or 475 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 147 or 476 (or a variant thereof); (xvi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (xvii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 or 477 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (xviii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (xix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 182 or 478 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 or 479 (or a variant thereof); (xx) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 192 or 480 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (xxi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 or 481 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (xxii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (xxiii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (xxiv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (xxv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (xxvi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 or 482 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xxvii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (xxviii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xxix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (xxx) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (xxxi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 or 483 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 or 527 (or a variant thereof); and / or (xxxii) HCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and LCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 or 484 (or a variant thereof). Including, For example, the HCVR and LCVR may be in either orientation (HCVR-LCVR or LCVR-HCVR), Optionally, the HCVR and LCVR may be linked together by a linker comprising, for example, an amino acid sequence, for example, about 10 amino acids in length, for example, (Gly4Ser) n (SEQ ID NO: 426), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0119] In some embodiments, V L -(Gly4Ser)3 (SEQ ID NO: 541)-V H The anti-hTfR scFvs described herein in the format comprise the amino acid sequences shown in Tables 1-2. In other embodiments, the scFvs described herein comprise the V H -(Gly4Ser)3 (SEQ ID NO: 541)-V L Optionally, the anti-hTfR scFv described herein further comprises an N-terminal LLQGSG (SEQ ID NO: 452) and / or a C-terminal HHHHHH (SEQ ID NO: 501). [Table 1-2-1] [Table 1-2-2] [Table 1-2-3]

[0120] Provided herein are Fab fragments that specifically bind to the human transferrin receptor (anti-TfR Fab-drug conjugates), optionally conjugated to a molecular cargo. The Fab fragment typically contains one complete light chain VL and constant light chain domain, e.g., kappa (e.g., [Table 38] Fab fragment antibodies can be generated by papain digestion of a whole IgG antibody to remove the entire Fc fragment, including the hinge region. For example, the Fab proteins described herein contain (1) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 2 or 462, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 7, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (2) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 12 or 463, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 17, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (3) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 22 or 464, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 27, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (4) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to the CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 37 or 465, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (5) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 42, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 47 or 466, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (6) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 52 or 467, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 57 or 468, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (7) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 62 or 492, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 67 or 469, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (8) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 72 or 470, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 77 or 471, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (9) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 82, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and (10) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 92 or 472, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 97, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (11) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 102, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 107, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (12) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 112 or 473, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 117 or 474, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (13) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 122, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 127, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (14) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 132, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 137, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (15) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 142 or 475, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 147 or 476, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (16) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 152, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 157, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (17) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 162 or 477, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 167, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (18) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 172, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 177, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (19) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 182 or 478, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 187 or 479, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (20) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 192 or 480, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 197, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (21) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 202 or 481, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 207, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (22) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 212, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 217, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (23) A heavy chain variable region comprising a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 222, or HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain, and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 227, or LCDR1, LCDR2, and LCDR3 of such an LCVR linked to a CL domain; (24) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 232, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 237, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (25) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 242, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 247, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (26) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 252 or 482, or a heavy chain variable region comprising HCDR1. HCDR2 and HCDR3 of such HCVR- linked to the CH1 domain, and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 257, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (27) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 262, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 267, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (28) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 272, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 277, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (29) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 282, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 287, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (30) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 292, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 297, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; (31) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 302 or 483, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 307 or 527, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain; and / or (32) A heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 312, or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of such an HCVR linked to a CH1 domain; and A light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 317 or 484, or LCDR1, LCDR2 and LCDR3 of such an LCVR linked to a CL domain. may include For example, CH1 is derived from IgG1 or IgG4, For example, CH1 is SEQ ID NO: 425, 459 or 493. The heavy and light chains of the anti-hTfR Fab in the anti-hTfR protein-drug conjugates described herein are shown below. [Table 39] [Table 40] [Table 41] [Table 42] [Table 43] [Table 44] [Table 45] [Table 46] [Table 47] [Table 48] [Table 49] [Table 50] [Table 51]

[0121] In some embodiments, the anti-hTfR proteins described herein can include any of the exemplary hIgG1 heavy chain sequences provided in Tables 1-3. [Table 1-3-1] [Table 1-3-2] [Table 1-3-3] [Table 1-3-4] [Table 1-3-5] [Table 1-3-6]

[0122] In some embodiments, an anti-TfR protein described herein may comprise an IgG1 heavy chain constant domain comprising the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 575), or a variant thereof.

[0123] "31874B"; "31863B"; "69348"; "69340"; "69331"; "69332"; "69326"; "69329"; "69323"; "69305"; "69307" ;"12795B";"12798B";"12799B";"12801B";"12802B";"12808B";"12812B";"12816B";"12833B";"12834B" ;"12835B";"12847B";"12848B";"12843B";"12844B";"12845B";"12839B";"12841B";"12850B";"69261"; and"69263" are SEQ ID NOs: 7;17;27;37;465;47;466;57;468;67;469;77;471;87;97;107;117;474;127;137;147; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 76;157;167;177;187;479;197;207;217;227;237;247;257;267;277;287;297;307;527;317 or 484 (or a variant thereof), and SEQ ID NOs: 2;462;12;463;22;464;32;42;52;467;62;492;72;470;82;92;472;1 483 or 312 (or a variant thereof), which in the case of an scFv can be fused together (in either order), for example, by a peptide linker (e.g., (G4S)3 (SEQ ID NO: 541)), or a VF comprising the CDRs thereof. H (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof) and CDR-H3 (or a variant thereof)) and / or V comprising the CDRs thereof L (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof), and CDR-L3 (or a variant thereof)), and in the case of an scFv, V LV fused with H or V H V fused with L can be conjugated to the molecular cargo, for example by a linker.

[0124] The terms "fused" or "tethered" with respect to a fusion polypeptide refer to polypeptides that are joined directly or indirectly (eg, via a linker or other polypeptide).

[0125] In some embodiments, the anti-TfR antigen binding proteins described herein comprise a humanized antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof (e.g., a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or antigen-binding fragment thereof, a bispecific antibody or binding fragment thereof (e.g., a bisscFv, or a bispecific T-cell engager (BiTE)), a triabody (e.g., a F(ab)'3 fragment or a triabody), or a chemically modified derivative thereof.

[0126] As used herein, the term "humanized antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences or otherwise modified to increase similarity to antibody variants that naturally occur in humans.

[0127] In some cases, the anti-TfR antigen binding protein is an antibody comprising one or more mutations in a framework region, e.g., the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the one or more mutations stabilize the antibody and / or increase half-life. In some embodiments, the one or more mutations modulate Fc receptor interactions, reducing or eliminating Fc effector function, e.g., FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further embodiments, the one or more mutations modulate glycosylation.

[0128] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1) and / or hinge region, numbering according to the Kabat numbering system (e.g., EU index of Kabat)) to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0129] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. See, e.g., PCT Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, which provide examples of mutations that alter (e.g., decrease or increase) the half-life of an antibody in vivo. In some embodiments, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutation comprises L234 and L235. In some embodiments, the mutations include L234A and L235A.

[0130] The anti-TfR antibodies and antigen-binding fragments described herein may be post-translationally modified (eg, glycosylated).

[0131] For example, the antibodies and antigen-binding fragments described herein can be glycosylated (e.g., N-glycosylated and / or O-glycosylated) or non-glycosylated. Typically, the antibodies and antigen-binding fragments are glycosylated at the conserved residue N297 of the IgG Fc domain. Some antibodies and fragments contain one or more additional glycosylation sites in the variable region. In one embodiment, the glycosylation site is in the following context: FN 297 S or YN 297 S.

[0132] In one embodiment, the glycosylation is any one or more of the three different N-glycan types found on IgG with each linkage: high mannose, complex, and / or hybrid. The complex and hybrid types exist with core fucosylation, the addition of a fucose residue to the innermost N-acetylglucosamine, and without core fucosylation.

[0133] In some cases, the anti-TfR antigen binding protein is an aglycosylated antibody, i.e., an antibody that does not contain a glycosylation sequence that could interfere with the transglutamination reaction, for example, an antibody that does not have a sugar group at N297 on one or more heavy chains according to the EU numbering system (or at position N180 relative to the amino acid sequence of SEQ ID NO: 575). In certain embodiments, the antibody heavy chain has an N297 mutation (or at position N180 relative to the amino acid sequence of SEQ ID NO: 575). In certain embodiments, the antibody heavy chain has an N297Q or N297D mutation (or an N180Q or N180D mutation relative to the amino acid sequence of SEQ ID NO: 575). The N-linked glycan found at position 297 can be found as a core structure common to all IgGs found in humans and rodents. Antibodies containing such above-mentioned mutations can be prepared by site-directed mutagenesis to remove or disable glycosylation sequences or by site-directed mutagenesis to insert glutamine residues at sites away from any interfering glycosylation sites or any other interfering structures. Such antibodies can also be isolated from natural or artificial sources. Non-glycosylated antibodies also include antibodies containing T299 or S298P or other mutations, or combinations of mutations that result in the absence of glycosylation.

[0134] In some cases, the antigen-binding protein is a deglycosylated antibody, i.e., an antibody in which saccharide groups have been removed to facilitate transglutaminase-mediated conjugation. The sugars include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N180 (with reference to the amino acid sequence of SEQ ID NO: 575). In some embodiments, deglycosylation is performed at residue N297 according to the EU numbering system. In some embodiments, removal of saccharide groups is achieved enzymatically via, but not limited to, PNGase.

[0135] In one embodiment, the antibody or fragment described herein is fucosylated.

[0136] The antibodies and antigen-binding fragments described herein may also be post-translationally modified in other ways, including, for example, Glu or Gln cyclization at the N-terminus; loss of a positive N-terminal charge; Lys variants at the C-terminus; deamidation (Asn to Asp); isomerization (Asp to isoAsp); deamidation (Gln to Glu); oxidation (Cys, His, Met, Tyr, Trp); and / or disulfide bond heterogeneity (shuffling, thioether and trisulfide formation)).

[0137] In some embodiments, the antibodies disclosed herein comprise Q295, which may be naturally occurring in the antibody heavy chain sequence. In some embodiments, the antibody heavy chains disclosed herein may comprise Q295. In some embodiments, the antibody heavy chains disclosed herein may comprise Q295 and the amino acid substitution N297D.

[0138] Certain embodiments of the present disclosure provide anti-TfR antibodies and antigen-binding fragments comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH relative to neutral pH. For example, the present disclosure includes anti-TfR antibodies containing mutations in the CH2 or CH3 region of the Fc domain that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations may result in increased serum half-life of the antibody when administered to an animal.

[0139] Non-limiting examples of such Fc modifications include, for example, modifications at the following positions: 250 (for example, E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and / or 256 (e.g., S / R / Q / E / D or T); and / or modifications at the following positions: 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or 434 (e.g., A, W, H, F or Y); and / or modifications at the following positions: · 250 and / or 428; and / or modifications at the following positions: 307 or 308 (e.g., 308F, V308F), and / or 434.

[0140] In one embodiment, the modification is 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and / or 307 and / or 308 modifications (e.g., 308F or 308P) Includes.

[0141] For example, the disclosure includes an anti-TfR antibody comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g. M252Y, S254T and T256E); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g. T307A, E380A and N434A); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F).

[0142] In yet another embodiment, the modifications include a 265A (eg, D265A) and / or a 297A (eg, N297A) modification.

[0143] In one embodiment, the heavy chain constant domain is gamma 4 containing an S228P and / or S108P mutation. See Angal et al., A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody, Mol Immunol. 1993 Jan;30(1):105-108.

[0144] All possible combinations of the aforementioned Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated within the scope of this disclosure.

[0145] The anti-TfR antibodies described herein may comprise an altered Fc domain with reduced effector function. As used herein, an "altered Fc domain with reduced effector function" refers to any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc., relative to a wild-type, naturally occurring Fc domain, such that the molecule comprising the modified Fc exhibits a reduced severity or degree of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, relative to a comparison molecule comprising a wild-type, naturally occurring Fc portion. In certain embodiments, an "altered Fc domain with reduced effector function" is an Fc domain that has reduced or weakened binding to an Fc receptor (e.g., FcγR).

[0146] In certain embodiments, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc comprising a substitution in the hinge region. For example, a modified Fc for use in the context of the present disclosure may comprise a variant IgG1 Fc in which at least one amino acid in the IgG1 Fc hinge region has been replaced with the corresponding amino acid from an IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present disclosure may comprise a variant IgG4 Fc in which at least one amino acid in the IgG4 Fc hinge region has been replaced with the corresponding amino acid from an IgG2 Fc hinge region. Non-limiting exemplary modified Fc regions that can be used in the context of the present disclosure are described in U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions described therein.

[0147] The present disclosure also includes antigen-binding proteins, antibodies, or antigen-binding fragments comprising the HCVRs and chimeric heavy chain constant (CH) regions described herein, where the chimeric CH region comprises segments derived from CH regions of more than one immunoglobulin isotype. For example, an antibody of the present disclosure may comprise a chimeric CH region comprising part or all of the CH2 domain from a human IgG1, human IgG2, or human IgG4 molecule combined with part or all of the CH3 domain from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, an antibody of the present disclosure comprises a chimeric CH region with a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216 to 227, EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228 to 236, EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Antibodies comprising the chimeric CH regions described herein can, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., WO 2014 / 022540.)

[0148] Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications described in U.S. Patent Application Publication Nos. 2014 / 0171623; 8697396; 2014 / 0134162; and WO 2014 / 043361, the disclosures of which are incorporated herein by reference in their entireties. Methods for constructing antibodies or other antigen-binding fusion proteins comprising the modified Fc domains described herein are known in the art.

[0149] In some embodiments, the anti-TfR antibodies and antigen-binding fragments described herein comprise an Fc domain comprising one or more mutations in the CH2 and / or CH3 regions that create distinct TfR binding sites.

[0150] In one embodiment, the CH2 region has any of the following: a) position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; position 49 is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; position 56 is Asp, Pro, Met, Leu, Ala, Asn, or Phe; position 58 is Arg, Ser, Ala, or Gly; position 59 is Tyr, Trp, Arg, or Val; position 60 is Glu; a) position 62 is Gln, Tyr, His, Ile, Phe, Val, or Asp; b) position 63 is Leu, Trp, Arg, Asn, Tyr, Val; b) position 39 is Pro, Phe, Ala, Met, or Asp; position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; and position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp, or Le. position 42 is Pro, Val, Ala, Thr, or Asp, position 43 is Pro, Val, or Phe, position 44 is Trp, Gln, Thr, or Glu, position 68 is Glu, Val, Thr, Leu, or Trp, position 70 is Tyr, His, Val, or Asp, position 71 is Thr, His, Gln, Arg, Asn, or Val; position 72 is Tyr, Asn, Asp, Ser, or Pro; c) position 41 is Val or Asp, position 42 is Pro, Met, or Asp, position 43 is Pro or Trp, position 44 is Arg, Trp, Glu, or Thr, position 45 is Met, Tyr, or Trp, position 65 is Leu or Trp, position 66 is Thr, Val, Ile, or Lys, position 67 is Ser, Lys, Ala, or Leu, position 69 is His, Leu, or Pro; position 73 is Val or Trp;or d) comprising one or more amino acid mutations selected from: at position 45 is Trp, Val, Ile, or Ala, at position 47 is Trp or Gly, at position 49 is Tyr, Arg, or Glu, at position 95 is Ser, Arg, or Gln, at position 97 is Val, Ser, or Phe, at position 99 is Ile, Ser, or Trp, at position 102 is Trp, Thr, Ser, Arg, or Asp, at position 103 is Trp, and at position 104 is Ser, Lys, Arg, or Val, or a combination thereof; The substitutions and positions are determined with reference to amino acids 4-113 of SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 540).

[0151] In one embodiment, the CH3 region has one of the following: position 153 is Trp, Leu, or Glu, position 157 is Tyr or Phe, position 159 is Thr, position 160 is Glu, position 161 is Trp, position 162 is Ser, Ala, Val, or Asn; position 163 is Ser or Asn, position 186 is Thr or Ser, position 188 is Glu or Ser, position 189 is Glu, position 194 is Phe, b) position 118 is Phe or Ile, position 122 is Thr or Ala; position 210 is Gly; position 211 is Phe; position 212 is His, Tyr, Ser, or Phe; position 213 is Asp; or a combination thereof, wherein the substitutions and positions are determined with reference to amino acids 114 to 220 of SEQ ID NO: 540.

[0152] In some embodiments, the CH3 region comprises one or more amino acid mutations selected from the following: position 384 is Leu, Tyr, Met, or Val; position 386 is Leu, Thr, His, or Pro; position 387 is Val, Pro, or an acidic amino acid; position 388 is Trp; position 389 is Val, Ser, or Ala; position 413 is Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His, or Phe (EU numbering), or a combination thereof. In one embodiment, the CH3 region comprises one or more amino acid mutations selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe (EU numbering), or a combination thereof.

[0153] In some embodiments, the CH3 region comprises: a) Phe at position 382, ​​Tyr at position 383, Asp at position 384, Asp at position 385, Ser at position 386, Lys at position 387, Leu at position 388, Thr at position 389, Pro at position 419, Arg at position 420, Gly at position 421, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440, Gly at position 442, and Gly at position 443. u; b) Phe at position 382, ​​Tyr at position 383, Gly at position 384, N at position 385, Ala at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, and Leu at position 440; c) Phe at position 382, ​​Tyr at position 383, Glu at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Leu at position 422, and A at position 424 a) at position 382, ​​Glu at position 384, Tyr at position 438, Leu at position 440; d) at position 382, ​​Phe at position 384, Glu at position 386, Ser at position 387, Lys at position 389, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; e) at position 382, ​​Gly at position 384, Ala at position 385, Lys at position 387, Ser at position 389, Leu at position 422, f) one or more mutations selected from Phe at position 382, ​​Gly at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440, or a combination thereof, wherein the positions are determined according to EU numbering.

[0154] Additional mutations in the CH2 and / or CH3 regions that can introduce a non-native TfR binding site into the antigen binding proteins described herein include those described in U.S. Patent Application Publication Nos. 2020 / 0223935, 2020 / 0369746, 2021 / 0130485, 2022 / 0017634, WO 2023 / 279099, WO 2023 / 114499, and WO 2023 / 114510, which are incorporated by reference in their entireties.

[0155] A container (e.g., a plastic or glass vial, e.g., equipped with a cap or a chromatography column, hollow bore needle, or syringe cylinder) containing an anti-TfR protein-drug conjugate described herein, e.g., an anti-TfR scFv-drug conjugate or an anti-TfR Fab-drug conjugate, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 1 2795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261 and 69263 are also provided herein.

[0156] anti-TfR protein-drug conjugates, such as the anti-TfR scFv drug conjugates described herein or anti-TfR Also provided herein is an injection device comprising a Fab drug conjugate, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, or a pharmaceutical composition thereof. The injection device may be packaged in a kit. An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g., intramuscularly, subcutaneously, or intravenously. For example, an injection device can be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector) that includes, for example, a cylinder or barrel for holding the fluid to be injected (e.g., comprising a protein-drug conjugate or a pharmaceutical composition thereof), a needle for puncturing the skin and / or blood vessel or other tissue for injection of the fluid, and a plunger for forcing the fluid from the cylinder through the needle bore and into the body of the subject.

[0157] Anti-TfR protein-drug conjugates, such as anti-TfR scFv-drug conjugates or anti-TfR Fab-drug conjugates described herein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 128 Further provided herein are methods for administering 35B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263 to a subject, and include introducing the protein-drug conjugate into the body of a subject (e.g., a human), e.g., parenterally (e.g., intravenously). For example, the method includes puncturing the subject's body with a syringe needle and injecting the antigen-binding protein into the subject's body, e.g., into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0158] The molecular cargo may be, for example, an antigen binding protein described herein, e.g., an anti-TfR scFv anti-TfR Fab described herein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 1 The molecular cargo conjugated to 2834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263 is administered to a target tissue of interest (e.g., a corresponding tissue or corresponding set of tissues as set forth in Tables 1-4 below). Further provided herein are methods for delivering a protein-drug conjugate to tissues (e.g., tissues or cell types associated with tissues or any cell type); or brain, cerebral cortex; cerebellum hippocampus; caudate nucleus; parathyroid glands; adrenal glands; bronchi; lung oral mucosa; esophagus; stomach; duodenum; small intestine; colon; rectum; liver; gallbladder; pancreas; kidney; bladder; testes; epididymis; prostate; vagina; ovaries; fallopian tubes; endometrium; cervix; placenta; breast; muscle (e.g., cardiac muscle; skeletal muscle, smooth muscle, and / or their endothelial vasculature); soft tissue; skin; appendix; lymph nodes; tonsils, and / or bone marrow), comprising, for example, parenterally (e.g., intravenously), introducing the protein-drug conjugate into the body of a subject (e.g., a human). For example, this method comprises puncturing the subject's body with a syringe needle and injecting the protein-drug conjugate into the subject's body, e.g., a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject. For example, the protein-drug conjugate may be introduced into a subject via intrathecal, intracerebroventricular, or intraparenchymal injection into the central nervous system. [Table 1-4-1] [Table 1-4-2] [Table 1-4-3] [Table 1-4-4] Molecular Cargo

[0159] In some aspects, the present disclosure includes methods and compositions for delivering conjugated molecular cargo to cells or tissues. In certain aspects, antigen binding proteins, e.g., scFvs, antibodies, or antigen-binding fragments thereof, that specifically bind to the transferrin receptor (TfR) disclosed herein can be conjugated (e.g., covalently conjugated) to the molecular cargo.

[0160] As used herein, the term "molecular cargo" refers to a molecule that acts to produce a biological result. By way of non-limiting example, the molecular cargo may act to regulate the transcription of a DNA sequence, regulate the expression of a protein, or regulate the activity of a protein, delete or disrupt an endogenous gene (or fragment thereof), insert an exogenous gene (or fragment thereof), or replace an endogenous gene (or fragment thereof) with an exogenous gene (or fragment thereof). In various embodiments, the molecular cargo may comprise a polynucleotide. In various embodiments, the molecular cargo comprises a lipid nanoparticle, liposome, or non-lipid nanoparticle described herein, optionally comprising one or more polynucleotide and / or protein molecules. In various embodiments, the molecular cargo may comprise a small molecule.

[0161] In some embodiments, the anti-TfR antibodies or antigen-binding fragments thereof disclosed herein can be used to deliver conjugated molecular cargo to cells or tissues expressing TfR1 (e.g., brain or muscle), e.g., to diagnose and / or treat a disease (e.g., a neurological or muscular disease). In some embodiments, the molecular cargo conjugated to the anti-TfR antibody or antigen-binding fragment thereof can be taken up, e.g., by endothelial cells, via binding to the transferrin receptor, which can be endocytosed, e.g., via clathrin-mediated endocytosis. In some embodiments, the anti-TfR antibodies or antigen-binding fragments thereof described herein can exhibit superior activity, e.g., in delivering molecular cargo to a target tissue (e.g., brain, spinal cord, muscle, spleen, heart, or lung) or target cell (e.g., brain cell or muscle cell). In some embodiments, the anti-TfR antibody or antigen-binding fragment thereof may be effective in delivering a molecular cargo to one or more brain cells, such as neurons (e.g., motor neurons, sensory neurons), astrocytes, glial cells (e.g., oligodendrocytes, microglia), and / or other cells of the brain and / or spinal cord.

[0162] In some embodiments, the molecular cargo comprises a polynucleotide molecule. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs linked together along the backbone, including polymers of traditional RNA, DNA, mixed RNA-DNA, and their analogs. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with any substitution, such as methoxy or 2' halide substitutions. In some embodiments, polynucleotides up to about 30 nucleotides in length can be referred to herein as "oligonucleotides." Oligonucleotides can be a variety of different lengths, depending, for example, on the form. In some embodiments, the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length, hi some embodiments, the oligonucleotide is 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0163] In some embodiments, the molecular cargo described herein can comprise a carrier such as a liposome or lipid nanoparticle (LNP). Lipid particles, such as liposomes or lipid nanoparticles disclosed herein, can comprise lipid formulations that can be used to deliver therapeutic nucleic acids (e.g., gRNA) to a target site of interest (e.g., a cell, tissue, organ, etc.). Without wishing to be bound by theory, carriers can be used, for example, as a means for delivery of the polynucleotides disclosed herein and / or the proteins disclosed herein. In some embodiments, carriers (e.g., liposomes or LNPs) can be useful for delivery of nucleic acids (e.g., DNA or RNA), proteins (e.g., RNA-guided DNA binders), or combinations thereof. As a non-limiting example, carriers (e.g., liposomes or LNPs) can be used to deliver various components of a gene editing system, such as a CRISPR / Cas system or additional gene editing systems described herein.

[0164] In some embodiments, the molecular cargo comprises a small molecule. Small molecules (SMs) have a low molecular weight (typically up to about 1 kDa) and therefore easily enter cells. Once inside the cell, they can affect other molecules, such as proteins, potentially killing cancer cells. This differs from many high-molecular-weight molecules, such as antibodies. Examples of small molecules that can be conjugated to anti-TfR antigen-binding proteins to form anti-TfR: SM conjugates. Furthermore, anti-cancer SMs can be delivered via anti-TfR-mediated delivery. Such anti-cancer SMs can include, for example, cytotoxic agents, alkylating agents (e.g., platinum-containing drugs), antimetabolites (5-fluorouracil), topoisomerase inhibitors (e.g., topotecan), anthracyclines (e.g., doxorubicin), and plant alkaloids (e.g., vinblastine). Other small molecule cargoes can include miglustat.

[0165] Exemplary molecular cargoes are described in further detail herein, however, it should be understood that the exemplary molecular cargoes provided herein are not intended to be limiting. Polynucleotide molecules

[0166] Non-limiting examples of polynucleotide molecules useful as molecular cargos in the protein-drug conjugates described herein include, but are not limited to, interfering nucleic acids (e.g., shRNA, siRNA, microRNA, antisense oligonucleotides), gapmers, mixmers, ribozymes, phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, and guide nucleic acids (e.g., Cas9 guide RNA), mRNA, and the like. In various embodiments, the polynucleotide may comprise one or more modified nucleotides. In various embodiments, the polynucleotide may comprise one or more modified internucleotide linkages. The polynucleotide may be single-stranded or double-stranded.

[0167] In some embodiments, the molecular cargo comprises at least one polynucleotide molecule, hi some embodiments, the molecular cargo comprises at least two, at least three, at least four, at least five, or at least ten polynucleotide molecules.

[0168] In some embodiments, the polynucleotide molecule is DNA. In some embodiments, the polynucleotide molecule is RNA.

[0169] In various embodiments, a polynucleotide (e.g., an interfering nucleic acid or a guide RNA) described herein may comprise a region of complementarity to a target nucleic acid that may range from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In certain embodiments, the region of complementarity of a polynucleotide to a target nucleic acid may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity may be complementary to at least 10 consecutive nucleotides of the target nucleic acid. In some embodiments, a polynucleotide may contain 1, 2, 3, 4, or 5 base mismatches compared to a portion of contiguous nucleotides of a target nucleic acid. In some embodiments, a polynucleotide may have up to 3 mismatches of more than 15 bases, or up to 4 mismatches of more than 10 bases. In some embodiments, a polynucleotide is complementary (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100%) to a target sequence of any one of the polynucleotides described herein. In various embodiments, such a target sequence may be 100% complementary to a polynucleotide described herein. In some embodiments, any one or more thymine bases (T) in any one of the polynucleotides described herein may be uracil bases (U), and / or any one or more U may be T. A target sequence described herein may include a sequence of a nucleic acid within a target gene that has complementarity to a guide sequence of a gRNA. Interaction of the target sequence with the guide sequence directs the RNA-guided DNA binding agent (e.g., a Cas protein) to bind and potentially nick or cleave (depending on the activity of the agent) within the target sequence.

[0170] The polynucleotides described herein may be modified, for example, by incorporating modified nucleotides, modified internucleoside linkages, and / or modified sugar moieties, or a combination thereof. Furthermore, the polynucleotides may have one or more of the following properties: improved cellular uptake compared to unmodified polynucleotides; not toxic to cells or immunostimulatory to mammals; avoiding pattern recognition receptors from mediating alternative splicing; nuclease-resistant, improved endosomal exit within cells; or minimized TLR stimulation. Any of the various modified chemistries or formats of polynucleotides disclosed herein may be combined together. As a non-limiting example, one, two, three, four, five, six, seven, eight, or more different types of modifications may be included within the same polynucleotide.

[0171] In various embodiments, specific nucleotide modifications can be used that make the polynucleotide incorporating the modifications more resistant to nuclease digestion than natural oligoribonucleotide or oligodeoxynucleotide molecules. Such modified polynucleotides remain intact for longer periods than unmodified polynucleotides. Exemplary modified polynucleotides include those containing modified backbones, such as modified internucleoside linkages, e.g., methylphosphonate, phosphotriester, phosphorothioate, short alkyl or cycloalkyl intersugar linkages, heterocyclic intersugar linkages, or short heteroatoms. Thus, the polynucleotides described herein can be stabilized against nucleolytic degradation, for example, through the incorporation of modifications, such as nucleotide modifications.

[0172] In various embodiments, a polynucleotide can be up to 50 nucleotides in length, and 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, or 2 to 45 nucleotides of the polynucleotide can be modified nucleotides. A polynucleotide can be 8 to 30 nucleotides in length, and 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, or 2 to 30 nucleotides of the polynucleotide can be modified nucleotides. In some embodiments, a polynucleotide can be 8 to 15 nucleotides in length, and 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, or 2 to 14 nucleotides of the polynucleotide can be modified nucleotides. In some embodiments, a polynucleotide can have all nucleotides except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides that are modified.

[0173] In various embodiments, the polynucleotides disclosed herein can include at least one nucleoside modified, for example, at the 2'-position of the sugar. In some embodiments, all nucleosides in the polynucleotide are 2'-modified nucleosides. In some embodiments, the polynucleotide includes at least one 2'-modified nucleoside.

[0174] In various embodiments, the polynucleotides disclosed herein can comprise one or more non-bicyclic 2'-modified nucleosides, such as 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-methyl (2'-O-Me), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-methoxyethyl (2'-MOE), 2'-deoxy, 2'-ON-methylacetamide (2'-O-NMA) modified nucleosides, 2'-fluoro (2'-F), 2'-O-aminopropyl (2'-O-AP), or 2'-O-dimethylaminopropyl (2'-O-DMAP).

[0175] In some embodiments, the polynucleotides described herein may comprise one or more 2'-4' bicyclic nucleosides in which the ribose ring may comprise a bridging moiety, e.g., a moiety connecting two atoms in the ring (e.g., connecting the 2'-O atom to the 4'-C atom via an ethylene (ENA) bridge, a methylene (LNA) bridge, or an (S)-constrained ethyl (cEt) bridge). Non-limiting examples of ENAs are disclosed in PCT Publication No. WO 2005 / 042777; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Koizumi, Curr. Opin. Mol. Then, 8:144-149, 2006; Surono et al., Hum. Gene Then, 15:749-757, 2004; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the disclosures of which are incorporated herein by reference in their entirety. Non-limiting examples of LNAs are disclosed in PCT Patent Application Publication No. WO 2008 / 043753, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of cEt are disclosed in US Pat. Nos. 7,569,686, 7,101,993, and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0176] In various embodiments, the polynucleotides described herein may include modified nucleosides as disclosed, for example, in U.S. Patent Nos. 8,022,193; 7,569,686; 7,399,845; 7,741,457; 7,335,765; 7,816,333; 8,957,201; and 7,314,923, the entire contents of each of which are incorporated herein by reference for all purposes.

[0177] In various embodiments, a polynucleotide comprises at least one modified nucleoside that results in an increase in the Tm of the polynucleotide in the range of 1° C. to 10° C. compared to a polynucleotide that does not contain at least one modified nucleoside. A polynucleotide can have multiple modified nucleosides that result in a total increase in the Tm of the polynucleotide in the range of 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C. or more compared to a polynucleotide that does not have the modified nucleoside.

[0178] In some embodiments, a polynucleotide may comprise a mixture of different types of nucleosides. A polynucleotide may comprise a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. A polynucleotide may comprise a mixture of 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. A polynucleotide may comprise a mixture of non-bicyclic 2' modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). A polynucleotide may comprise a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. A polynucleotide can comprise a mixture of 2'-fluoro and 2'-O-Me modified nucleosides.

[0179] In various embodiments, an oligonucleotide may comprise alternating nucleosides of different types. In certain embodiments, an oligonucleotide may comprise alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. In certain embodiments, a polynucleotide may comprise alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. In certain embodiments, an oligonucleotide may comprise alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. In certain embodiments, an oligonucleotide may comprise alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. In certain embodiments, oligonucleotides may comprise alternating non-bicyclic 2' modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0180] In various embodiments, the polynucleotides described herein may contain one or more abasic residues, 5-vinylphosphonate modifications, and / or one or more reverse abasic residues.

[0181] In various embodiments, the oligonucleotide may comprise phosphorothioate or other modified internucleoside linkages. In various embodiments, the oligonucleotide may comprise phosphorothioate internucleoside linkages. In various embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleotides. In various embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides. As a non-limiting example, in certain embodiments, the oligonucleotide comprises a first, second, and / or (e.g., and) third internucleoside linkage modified internucleoside linkage at the 5'-end or 3'-end of the nucleotide sequence.

[0182] Non-limiting examples of phosphorus-containing linkages include aminoalkyl phosphotriester phosphorothioates, chiral phosphorothioates, phosphotriesters, phosphorodithioates, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionoalkyl phosphonates, thionophosphoramidates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reverse polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. U.S. Patent Nos. 5,625,050; 4,469,863; 4,476,301; 5,023,243; 5,550,111; 5,177,196; 5,587,361; 5,188,897; 5,264,423; 5,276,019; 5,519,126; 5,278,302; 5,286,71 See Nos. 7; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,536,821; 5,541,306; 5,563,253; 5,571,799 and 3,687,808.

[0183] In various embodiments, the polynucleotides described herein can have a heteroatom backbone, such as a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone and the nucleotides are bound directly or indirectly to aza nitrogen atoms of the polyamide backbone (see Nielsen et al., Science 1991, 254, 1497)), a morpholino backbone (see Summerton and Weller in U.S. Pat. No. 5,034,506); an amide backbone (De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); or an MMI or methylene (methylimino) backbone.

[0184] The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., modified uridine such as 5-methoxyuridine, pseudouridine, or N1-methylpseudounine, or others); inosine; a purine or pyrimidine derivative (e.g., N4-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having a substituent at the 5- or 6-position (e.g., 5-methylcytosine), purine bases having a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, 6-O-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and 4-O-alkyl-pyrimidine; U.S. Pat. No. 5,378,825 and PCT Publication No. WO 93 / 13121). For a general discussion, see Adams et al., The Biochemistry of the Nucleic Acids 5-36, 11th ed., 1992. Nucleic acids can contain one or more "abasic" residues, where the backbone does not contain a nitrogenous base relative to the position of the polymer (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases, and linkages, or can contain both conventional components and substitutions (e.g., conventional nucleosides with 2'-methoxy substituents, or polymers containing both conventional nucleotides and one or more nucleotide analogs). Nucleic acids include "locked nucleic acids" (LNAs), analogs containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked to an RNA sugar conformation that enhances hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or its analogues in RNA and thymine or its analogues in DNA. Interfering nucleic acids

[0185] In some embodiments, the conjugated molecular cargo may comprise a polynucleotide molecule capable of altering the expression of one or more genes in a target cell (e.g., inhibiting gene expression and / or translation, modulating RNA splicing, or inducing exon skipping). In some embodiments, the polynucleotide molecule may be an interfering nucleic acid molecule, such as an siRNA, shRNA, miRNA, or antisense oligonucleotide (ASO), that targets, for example, RNA (e.g., mRNA).

[0186] In certain embodiments, interfering nucleic acid molecules that selectively target and inhibit the activity or expression of a target gene product (e.g., mRNA product) are used in the compositions and methods described herein. Interfering nucleic acid molecules can inhibit the expression or activity of at least one target gene product (e.g., mRNA product) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The agents disclosed herein can include a nucleobase sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to at least the target gene product (e.g., mRNA product). Without wishing to be bound by theory, "complementarity" of a nucleic acid can mean that the nucleotide sequence of one strand of a nucleic acid forms hydrogen bonds with another sequence on an opposing nucleic acid strand due to the orientation of its nucleobase groups. Complementary bases in DNA are typically A and T and C and G. In RNA, they are typically C and G and U and A. Complementarity can be complete or substantial / sufficient. Complete complementarity between two nucleic acids means that the two nucleic acids can form a duplex, with all bases in the duplex binding to complementary bases through Watson-Crick pairing. "Substantial" or "sufficient" complementarity means that the sequence of one strand is not completely and / or perfectly complementary to the sequence of the opposite strand, but sufficient binding occurs between the bases of the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by predicting the Tm (melting temperature) of the hybridized strands using the sequences and standard mathematical calculations, or by empirically determining the Tm using routine methods. The Tm comprises the temperature at which the population of hybridization complexes formed between two nucleic acid strands becomes 50% denatured (ie, the population of double-stranded nucleic acid molecules becomes half-dissociated into single strands).Temperatures below the Tm favor the formation of hybridization complexes, while temperatures above the Tm favor the melting or separation of the strands in the hybridization complex. While other known Tm calculations take into account the structural characteristics of nucleic acids, the Tm can be estimated, for example, for a nucleic acid with a known G+C content in 1 M NaCl aqueous solution by using Tm = 81.5 + 0.41 (G+C%).

[0187] Interfering nucleic acids can comprise an array of circular subunits, each having a base-pairing portion linked by intersubunit linkages that allow the base-pairing portion to hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a hetero-oligomeric heteroduplex within the nucleic acid:target sequence.

[0188] Typically, at least 17, 18, 19, 20, 21, 22, or 23 nucleotides of the complement of the target mRNA sequence are sufficient to mediate inhibition of the target transcript. Perfect complementarity is not required. In some embodiments, the interfering nucleic acid molecule is single-stranded RNA. In some embodiments, the interfering nucleic acid molecule is double-stranded RNA. The double-stranded RNA molecule may have 1-3 nucleotide 3' and / or 5' overhangs on either the sense and / or antisense strands. In some embodiments, the double-stranded RNA molecule has a 2-nucleotide 3' overhang. In some embodiments, the two RNA strands are linked via a hairpin structure to form an shRNA molecule. The shRNA molecule may include a hairpin derived from a microRNA molecule.

[0189] The interfering nucleic acid molecule described herein can contain RNA base, non-RNA base, or a mixture of RNA base and non-RNA base.For example, the interfering nucleic acid molecule described herein can be mainly composed of RNA base or modified RNA base, but can also contain DNA base, modified DNA base, and / or non-naturally occurring nucleotide.The term "ribonucleotide" or "nucleotide" can also refer to the modified nucleotide or substituted moiety at one or more positions in the case of modified RNA or nucleotide surrogate.

[0190] In some embodiments, the interfering nucleic acid molecule is a small interfering RNA (siRNA), also known as small interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway within cells. Such siRNA molecules typically contain a region of sufficient homology to the target region and are of sufficient length, in terms of nucleotides, so that the siRNA molecule downregulates the target nucleic acid. While perfect complementarity between the siRNA molecule and the target need not exist, the correspondence must be sufficient to allow the siRNA molecule to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded character of the molecule.

[0191] The specificity of an siRNA molecule can be measured via binding between the antisense strand of the molecule and its target RNA. Effective siRNA molecules are often less than 30 to 35 base pairs in length to prevent stimulation of nonspecific RNA interference pathways in cells, for example, via the interferon response, although longer siRNAs can also be effective. In various embodiments, the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In various embodiments, the siRNA molecule is about 35 to about 70 base pairs in length. In some embodiments, the siRNA molecule is greater than 70 base pairs in length. In some embodiments, the siRNA molecule is 8 to 40 base pairs in length, 10 to 20 base pairs in length, 10 to 30 base pairs in length, 15 to 20 base pairs in length, 19 to 23 base pairs in length, or 21 to 24 base pairs in length. In some embodiments, the sense strand and antisense strand of the siRNA molecule are each independently about 19 to about 24 nucleotides in length. In some embodiments, the sense strand of the siRNA molecule is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, both the sense strand and the antisense strand of the siRNA molecule are 21 nucleotides in length.

[0192] After selecting an appropriate target RNA sequence, an siRNA molecule comprising a nucleotide sequence complementary to all or part of the target sequence, i.e., an antisense sequence, can be designed and prepared using a suitable method (see, for example, US Patent Application Publication No. 2004 / 0077574 and US Patent Application Publication No. 2008 / 0081791 and International Publication No. 2004 / 016735). In some embodiments, the siRNA molecule can be single-stranded (i.e., an ssRNA molecule comprising only an antisense strand) or double-stranded (i.e., a dsRNA molecule comprising an antisense strand and a complementary sense strand that hybridizes to form a dsRNA). In various embodiments, the siRNA molecule can comprise a duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure comprising a self-complementary sense strand and / or antisense strand.

[0193] In various embodiments, the antisense strand of the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiments, the antisense strand of the siRNA molecule is about 35 to about 70 nucleotides in length. In various embodiments, the antisense strand of the siRNA molecule is greater than 70 nucleotides in length. In some embodiments, the antisense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 24 nucleotides in length.

[0194] In some embodiments, the sense strand of the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiments, the sense strand of the siRNA molecule is about 30 to about 70 nucleotides in length. In various embodiments, the sense strand of the siRNA molecule is greater than 70 nucleotides in length. In some embodiments, the sense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 24 nucleotides in length.

[0195] In various embodiments, the siRNA molecule can comprise an antisense strand comprising a region of complementarity to the target region in the target mRNA. In some embodiments, the complementary region is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the target region in the target mRNA. In some embodiments, the target region can comprise a region of consecutive nucleotides in the target mRNA. In some embodiments, the complementary region being 100% complementary to the complementary region of its target may not be required to be specifically hybridizable or specific to the target RNA sequence.

[0196] In some embodiments, the siRNA molecules disclosed herein can comprise an antisense strand that includes a region of complementarity to a target RNA sequence, wherein the region of complementarity is between 8 and 20, 8 and 35, 8 and 45, 10 and 50, 5 and 55, or 5 and 40 nucleotides in length. In some embodiments, the region of complementarity is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35 or more consecutive nucleotides of the target RNA sequence. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer base mismatches compared to a portion of the consecutive nucleotides of the target RNA sequence. In some embodiments, the siRNA molecule comprises a nucleotide sequence having up to 3 mismatches of more than 15 bases or up to 4 mismatches of more than 10 bases with the target sequence. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with up to 0, 1, 2, or 3 mismatches with the target sequence over 15-22 bases. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with 0, 1, or 2 mismatches with the target sequence over more than 15-22 bases. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with 0 or 1 mismatch with the target sequence over 15-22 bases.In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with 0 mismatches of more than 15-22 bases with the target sequence.

[0197] In various embodiments, the siRNA molecule may comprise an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% complementary to the target RNA sequence of the antisense oligonucleotides disclosed herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% identical to any of the antisense oligonucleotides provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of any of the antisense oligonucleotides provided herein.

[0198] In some embodiments, double-stranded siRNAs can comprise sense and antisense RNA strands of different lengths or the same length. In some embodiments, double-stranded siRNA molecules can also be generated from a single oligonucleotide in a stem-loop structure. The self-complementary sense and antisense regions of siRNA molecules with a stem-loop structure can be linked by a nucleic acid-based or non-nucleic acid-based linker. In some embodiments, siRNAs with a stem-loop structure comprise a circular single-stranded RNA with two or more loop structures and a stem comprising self-complementary sense and antisense strands. In some embodiments, the circular RNA can be processed in vivo or in vitro to produce active siRNA molecules capable of mediating RNAi. Therefore, short hairpin RNA (shRNA) molecules are also contemplated herein. Such molecules can comprise a specific antisense sequence along with a reverse complement (sense) sequence, which in some instances can be separated by a spacer or loop sequence. The reverse complements described herein can comprise a sequence that is the complementary sequence of a reference sequence, with the complementary sequence written in reverse orientation. Due to codon usage redundancy, a reverse complement may diverge from a reference sequence that encodes the same polypeptide. As used herein, "reverse complement" also includes, for example, a sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reverse complement of a reference sequence. Cleavage of the spacer or loop can provide a single-stranded RNA molecule and its reverse complement, which can then anneal to form a dsRNA molecule. In various embodiments, further optional processing steps can result in the removal or addition of 1, 2, 3, 4, 5, or more nucleotides from the 3' and / or 5' ends of one or both strands. The spacer may be of a length suitable to allow the antisense and sense sequences to anneal and form a double-stranded structure or stem before cleavage of the spacer.In certain embodiments, any subsequent processing steps may result in the removal or addition of 1, 2, 3, 4, 5, or more nucleotides from the 3' and / or 5' ends of one or both strands. In some embodiments, the spacer sequence may be, for example, an unrelated nucleotide sequence that may be located between two complementary nucleotide sequence regions that may comprise an shRNA when annealed into a double-stranded nucleic acid.

[0199] The length of an siRNA molecule can vary from about 10 to about 120 nucleotides depending on the type of siRNA molecule designed. Generally, about 10 to about 55 of these nucleotides can be complementary to the RNA target sequence. For example, if the siRNA is a double-stranded siRNA or a single-stranded siRNA, the length can vary from about 10 to about 55 nucleotides, while if the siRNA is an shRNA or a circular molecule, the length can vary from about 30 to about 110 nucleotides.

[0200] In various embodiments, the siRNA molecule may comprise a 3' overhang at one end of the molecule. In some embodiments, the other end may be blunt or may comprise an overhang (e.g., 5' and / or 3'). When the siRNA molecule comprises an overhang at both ends of the molecule, the overhangs may be of different lengths or the same length. In some embodiments, the siRNA molecules described herein may comprise a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on both the sense strand and the antisense strand. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on the antisense strand. In some embodiments, the siRNA molecule may comprise a 3' overhang of about 1 to about 3 nucleotides on the sense strand.

[0201] In various embodiments, the siRNA molecule comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). In some embodiments, all nucleotides in the sense strand and / or antisense strand of the siRNA molecule are modified. In certain embodiments, the siRNA molecule may comprise one or more modified nucleotides and / or one or more modified internucleoside linkages. In some embodiments, the siRNA molecule may comprise a modified internucleoside linkage at the first and second internucleoside linkages at the 5'-end of the sense strand of the siRNA molecule. In some embodiments, the siRNA molecule may comprise a modified internucleoside linkage at the first and second internucleoside linkages at the 5'-end and 3'-end of the antisense strand of the siRNA molecule. In some embodiments, the siRNA molecule may comprise a modified internucleoside linkage at the first and second internucleoside linkages at the 5'-end of the sense strand of the siRNA molecule and at the first and second internucleoside linkages at the 5'-end and 3'-end of the antisense strand of the siRNA molecule.

[0202] In some embodiments, modified nucleotides may contain modified sugar moieties (e.g., 2'-modified nucleotides). In some embodiments, siRNA molecules may contain one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In various embodiments, each nucleotide of an siRNA molecule may be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, an siRNA molecule may contain one or more phosphorodiamidate morpholinos. In some embodiments, each nucleotide of an siRNA molecule consists of a phosphorodiamidate morpholino.

[0203] In various embodiments, siRNA molecules may contain phosphorothioate or other modified internucleoside linkages. In various embodiments, siRNA molecules may contain, for example, phosphorothioate internucleoside linkages. In some embodiments, siRNA molecules may contain phosphorothioate internucleoside linkages between two or more nucleotides. In some embodiments, siRNA molecules may contain phosphorothioate internucleoside linkages between all nucleotides. In some embodiments, siRNA molecules may contain modified internucleoside linkages at the first, second, and / or third internucleoside linkages at the 5'-end or 3'-end of the siRNA molecule. In some embodiments, siRNA molecules may contain modified internucleoside linkages at the first and second internucleoside linkages at the 5'-end and / or 3'-end of the siRNA molecule. In some embodiments, siRNA molecules may contain modified internucleoside linkages at the first and second internucleoside linkages at the 5'-end of the siRNA molecule sense strand. In some embodiments, the siRNA molecule may comprise a modified internucleotide linkage at the first and second internucleoside linkages at the 5'-end and 3'-end of the antisense strand of the siRNA molecule. In some embodiments, the siRNA molecule may comprise a modified internucleotide linkage at the first and second internucleoside linkages at the 5'-end of the sense strand of the siRNA molecule and at the first and second internucleoside linkages at the 5'-end and 3'-end of the antisense strand of the siRNA molecule. In some embodiments, the siRNA molecule may comprise a modified internucleotide linkage at the first internucleoside linkage at the 5'-end and 3'-end of the sense strand of the siRNA molecule, the first, second, and third internucleoside linkages at the 5'-end of the antisense strand of the siRNA molecule, and the first internucleoside linkage at the 3'-end of the antisense strand of the siRNA molecule.

[0204] In various embodiments, the modified internucleotide linkage may comprise a phosphorus-containing linkage. In some embodiments, the phosphorus-containing linkage that can be used in the methods or compositions described herein includes, but is not limited to, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, "normal 3'-5-aminophosphoramidates and aminoalkylphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters and boranophosphates," 3' alkylene phosphonates and phosphoramidates, including chiral phosphonates, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. U.S. Patent Nos. 5,625,050; 3,687,808; 4,469,863; 4,476,301; 5,177,196; 5,455,233; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,9 See Nos. 39; 5,519,126; 5,453,496; 5,466,677; 5,476,925; 5,536,821; 5,023,243; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 and 5,188,897.

[0205] Any of the various modified formats or chemistries of the siRNA molecules disclosed herein can be combined together, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications can be included in the same siRNA molecule.

[0206] In various embodiments, the antisense strand can comprise one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). In some embodiments, the antisense strand can comprise one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, a modified nucleotide can comprise a modified sugar moiety (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand comprises one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-methylacetamide (2'-O-NMA). In various embodiments, each nucleotide in the antisense strand can be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand can comprise one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand is comprised of phosphorodiamidate morpholino oligomers (PMOs).

[0207] In some embodiments, the antisense strand comprises a phosphorothioate or other modified internucleoside linkage. In some embodiments, the antisense strand may comprise a phosphorothioate internucleoside linkage. In some embodiments, the antisense strand may comprise a phosphorothioate internucleoside linkage between two or more nucleotides. In some embodiments, the antisense strand may comprise a phosphorothioate internucleoside linkage between all nucleotides. In some embodiments, the antisense strand may comprise a modified internucleoside linkage at the first, second, and / or third nucleotide at the 5'-end or 3'-end of the antisense strand. In some embodiments, the antisense strand may comprise a modified internucleoside linkage at the first and second nucleotide positions (e.g., between the first and second nucleotides and between the second and third nucleotides) at the 5'-end and 3'-end of the antisense strand.

[0208] In various embodiments, modified internucleotide linkages can include phosphorus-containing linkages in the antisense strand.In some embodiments, the phosphorus-containing linkages that can be used in the methods and compositions described herein include, but are not limited to, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, "normal 3'-5-aminophosphoramidates and aminoalkylphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters and boranophosphates," 3' alkylene phosphonates and phosphoramidates, including chiral phosphonates, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. U.S. Patent Nos. 5,625,050; 3,687,808; 4,469,863; 4,476,301; 5,177,196; 5,455,233; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,9 See Nos. 39; 5,519,126; 5,453,496; 5,466,677; 5,476,925; 5,536,821; 5,023,243; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 and 5,188,897.

[0209] Any of the antisense strand modified formats or chemistries disclosed herein can be combined together, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications can be included in the same antisense strand.

[0210] In some embodiments, the sense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15 or more). In some embodiments, the antisense strand can comprise one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, a modified nucleotide can comprise a modified sugar moiety (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand comprises one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-methylacetamide (2'-O-NMA). In various embodiments, each nucleotide in the antisense strand can be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand can comprise one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand is comprised of phosphorodiamidate morpholino oligomers (PMOs).

[0211] In some embodiments, the sense strand comprises a phosphorothioate or other modified internucleotide linkage. In some embodiments, the sense strand may comprise a phosphorothioate internucleoside linkage. In some embodiments, the sense strand may comprise a phosphorothioate internucleoside linkage between two or more nucleotides. In some embodiments, the sense strand may comprise a phosphorothioate internucleoside linkage between all nucleotides. For example, in some embodiments, the sense strand comprises a modified internucleotide linkage at the first, second, and / or third nucleotide at the 5'-end or 3'-end of the sense strand. In some embodiments, the sense strand may comprise a modified internucleotide linkage at the first and second nucleotide positions (e.g., between the first and second nucleotides and between the second and third nucleotides) at the 5'-end of the sense strand.

[0212] In various embodiments, the modified internucleotide linkage may comprise a phosphorus-containing linkage of the sense strand. In some embodiments, the phosphorus-containing linkage that can be used in the methods and compositions described herein includes, but is not limited to, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, "normal 3'-5-aminophosphoramidates and aminoalkylphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters and boranophosphates," 3' alkylene phosphonates and phosphoramidates, including chiral phosphonates, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. U.S. Patent Nos. 5,625,050; 3,687,808; 4,469,863; 4,476,301; 5,177,196; 5,455,233; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,93 See Nos. 9; 5,519,126; 5,453,496; 5,466,677; 5,476,925; 5,536,821; 5,023,243; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 and 5,188,897.

[0213] Any of the sense strand modified chemistries or formats described herein can be combined together, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications can be included within the same sense strand.

[0214] In various embodiments, the antisense and / or sense strands of the siRNA molecule may contain one or more modifications that can, for example, enhance or reduce RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule may contain one or more modifications that can enhance RISC loading. In various embodiments, the sense strand of the siRNA molecule may contain one or more modifications that can reduce RISC loading and / or reduce off-target effects. In various embodiments, the antisense strand of the siRNA molecule may contain a 2'-O-methoxyethyl (2'-MOE) modification. In some embodiments, the addition of a 2'-O-methoxyethyl (2'-MOE) group, for example, at the cleavage site, may improve the silencing activity and / or specificity of the siRNA by promoting directed RNA-induced silencing complex (RISC) loading of the modified strand, as disclosed, for example, in Song et al., (2017) Mol Ther Nucleic Acids 9:242-250, the entire contents of which are incorporated herein by reference. In various embodiments, the antisense strand of the siRNA molecule can comprise a 2'-O-Me-phosphorodithioate modification. In some embodiments, the 2'-O-Me-phosphorodithioate modification can increase RISC loading, as disclosed, for example, in Wu et al., (2014) Nat Commun 5:3459, the entire contents of which are incorporated herein by reference.

[0215] In various embodiments, the sense strand of the siRNA molecule may comprise a 5'-nitroindole modification. In some embodiments, the 5'-nitroindole modification may reduce the RNAi potency of the sense strand and / or reduce off-target effects, as disclosed, for example, in Zhang et al., (2012) Chembiochem 13(13):1940-1945, the entirety of which is incorporated herein by reference. In various embodiments, the sense strand may comprise a 2'-O-methyl (2'-O-Me) modification. In some embodiments, the 2'-O-Me modification may reduce RISC loading and / or off-target effects of the sense strand, as disclosed, for example, in Zheng et al., FASEB (2013) 27(10):4017-4026, the entirety of which is incorporated herein by reference. In various embodiments, the sense strand of the siRNA molecule may be fully substituted with morpholino, 2'-MOE and / or 2'-O-Me residues and may not be recognized by RISC, as described, for example, in Kole et al., (2012) Nature reviews. Drug Discovery 11(2):125-140, which is incorporated herein by reference in its entirety.

[0216] In various embodiments, the sense strand of the siRNA molecule may include a 5'-morpholino modification. In various embodiments, the 5'-morpholino modification can reduce RISC loading of the sense strand and / or improve RNAi activity and / or antisense strand selection, as disclosed, for example, in Kumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, the entire contents of which are incorporated herein by reference. In various embodiments, the sense strand of the siRNA molecule may be modified with a synthetic RNA-like high-affinity nucleotide analog called a locked nucleic acid (LNA), which can reduce RISC loading of the sense strand and / or promote RISC incorporation for RNAi activity and / or antisense strand selection, as disclosed, for example, in Elman et al., (2005) Nucleic Acids Res. 33(1):439-447. In various embodiments, the sense strand of the siRNA molecule may include a 5' non-locked nucleic acid (UNA) modification. In various embodiments, 5'-unlocked nucleic acid (UNA) modifications can reduce RISC loading of the sense strand and improve silencing function of the antisense strand, as disclosed, for example, in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103, which is incorporated herein by reference in its entirety.

[0217] In some embodiments, the antisense strand of the siRNA molecule may comprise a 2'-MOE modification and / or the sense strand may comprise a 2'-O-Me modification (see, e.g., Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 5, at least 8, at least 9, at least 10 or more) siRNA molecule may be covalently conjugated to an anti-TfR antigen binding protein, for example, as described herein. In some embodiments, the anti-TfR antigen binding protein may be conjugated to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen binding protein may be conjugated to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen binding protein may be internally conjugated to the sense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen binding protein may be conjugated to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen binding protein may be conjugated to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen binding protein is internally conjugated to the antisense strand of the siRNA molecule.

[0218] Furthermore, siRNA molecules may be modified or contain nucleoside surrogates. The single-stranded regions of an siRNA molecule may be modified or contain nucleoside surrogates, for example, one or more unpaired regions of a hairpin structure, such as the region connecting two complementary regions, may have modifications or nucleoside surrogates. Modifications to stabilize one or more 3' or 5' ends of an siRNA molecule, for example, against exonucleases or to aid in the entry of antisense siRNA agents into RISC, are also useful. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (e.g., C3-C12 (e.g., C3, C6, C9, C12), abasic, triethylene glycol, hexaethylene glycol), biotin or fluorescein reagents provided as phosphoramidites with additional DMT-protected hydroxyl groups to allow multiple couplings during RNA synthesis.

[0219] In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long, and the 3' and 5' terminal nucleotide positions of the sense strand are inverted abasic residues. The inverted abasic residues at the 3' and 5' ends of the sense strand can be overhangs. The inverted abasic residues can be linked via a 3'-3' phosphodiester bond. In some embodiments, the antisense strand of the siRNA molecule contains one to two phosphorothioate linkages at the 3' and / or 5' end. In some embodiments, the antisense strand contains two or three phosphorothioate internucleotide linkages at the 5' end and one phosphorothioate internucleotide linkage at the 3' end. The siRNA molecule can be linked to a targeting moiety at the 5' or 3' end of the sense strand.

[0220] In some embodiments, the sense strand is 21 nucleotides long, the antisense strand is 23 nucleotides long, and the antisense strand contains two nucleobase 3' overhangs. In some embodiments, the antisense strand of the siRNA molecule contains 1 to 3 phosphorothioate linkages at the 3' and 5' ends, and the sense strand of the siRNA molecule contains 1 to 2 phosphorothioate linkages at the 5' end. In some embodiments, the antisense strand of the siRNA molecule contains 2 to 3 phosphorothioate linkages at the 5' end and 2 phosphorothioate linkages at the 3' end, and the sense strand of the siRNA molecule contains 2 phosphorothioate linkages at the 5' end. The siRNA molecule can be linked to a targeting moiety at the 5' or 3' end of the sense strand.

[0221] In some embodiments, the interfering nucleic acid molecule is a short hairpin RNA (shRNA). The "small hairpin RNA" or "short hairpin RNA" or "shRNA" described herein may comprise a short RNA sequence that forms a tight hairpin turn, which can be used to silence gene expression through RNA interference. The shRNA provided herein can be chemically synthesized or transcribed from a transcription cassette in a DNA plasmid. The shRNA hairpin structure can be cleaved into siRNA by the cellular machinery, and the siRNA then binds to the RNA-induced silencing complex (RISC).

[0222] Non-limiting examples of shRNAs include double-stranded polynucleotide molecules assembled from single-stranded molecules, in which the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker. These include double-stranded polynucleotide molecules with a hairpin secondary structure having self-complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure containing about 1 to about 25 nucleotides, about 2 to about 20 nucleotides, about 4 to about 15 nucleotides, about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

[0223] Further embodiments related to shRNAs, as well as methods for designing and synthesizing such shRNAs, are described in U.S. Patent Application Publication No. 2011 / 0071208, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0224] In some embodiments, the interfering nucleic acid molecule is a microRNA (miRNA). miRNAs represent a large group of small RNAs naturally produced in organisms, some of which regulate the expression of target genes. miRNAs are short hairpin RNAs, approximately 18 to 25 nucleotides in length, that function in RNA silencing and post-translational regulation of gene expression. Typically, miRNAs are generated in the nucleus from larger RNA precursors (called pri-miRNAs), which are processed into approximately 70-nucleotide pre-miRNAs that fold into imperfect stem-loop structures. These pre-miRNAs typically undergo further processing steps in the cytoplasm, where the 18- to 25-nucleotide-long mature miRNAs are excised from one side of the pre-miRNA hairpin by the RNase III enzyme Dicer. miRNAs are not translated into proteins but instead bind to specific messenger RNAs, thereby blocking translation. In some embodiments, miRNAs imprecisely base-pair with their targets to inhibit translation.

[0225] The miRNAs described herein may include fragments of pri-miRNA, pre-miRNA, mature miRNA, or variants thereof that retain the biological activity of the mature miRNA. In some embodiments, the size range of the miRNA can be 21 nucleotides to 170 nucleotides. In one embodiment, the size range of the miRNA is 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of 21 nucleotides to 25 nucleotides in length can be used.

[0226] In certain embodiments, the interfering nucleic acid molecule is an antisense oligonucleotide (ASO). ASOs can downregulate targets by inducing RNase H endonuclease cleavage of target RNA, sterically hindering ribosomal activity, inhibiting 5' cap formation, or altering splicing. ASOs can be, but are not limited to, gapmers or morpholinos. Antisense oligonucleotides typically contain short nucleotide sequences that are substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, heterogeneous nuclear RNA (hnRNA), or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule containing the antisense sequence can form a stable double-stranded hybrid with the target nucleotide sequence in an RNA molecule under physiological conditions. Antisense oligonucleotides are often synthetic and chemically modified.

[0227] Antisense oligonucleotides can be 100% complementary to target sequences, or can contain mismatches to improve the selective targeting of alleles containing disease-related mutations, as long as the heteroduplex formed between the oligonucleotide and target sequence is stable enough to withstand the action of cellular nucleases and other degradation modes that may occur in vivo.Therefore, some oligonucleotides can have about or at least about 70% sequence complementarity between the oligonucleotide and target sequence, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity. The present invention is directed to an oligonucleotide backbone that is less susceptible to nuclease cleavage. When mismatches are present, they are typically less destabilizing toward the end regions of the hybrid double strand than toward the center. The number of mismatches that can be tolerated depends on the length of the oligonucleotide, the percentage of G. According to well-understood principles of double strand stability, the C base pairs in the double strand and the position of the mismatch in the double strand are determined.

[0228] In some embodiments, the interfering nucleic acid molecules described herein are gapmers. A "gapmer" is an oligonucleotide comprising an internal region having multiple nucleosides that support RNase H cleavage, separated by an external region having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from one or more nucleosides comprising the external region. The internal region is sometimes referred to as the "gap," and the external regions are sometimes referred to as the "wings." Gapmers can have 5' and 3' wings, each having 2-6 nucleotides, and a gap having 7-12 nucleotides. In some embodiments, gapmers can have a 3-10-3 or 5-10-5 configuration.

[0229] Gapmers generally have the formula 5'-XYZ-3', with X and Z as flanking regions around gap region Y. In some embodiments, flanking region X of formula 5'-XYZ-3' is also referred to as X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, flanking region Z of formula 5'-XYZ-3' is also referred to as Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, gap region Y of formula 5'-XYZ-3' is also referred to as Y region, Y segment, gap segment Y, gap segment, or gap region. In some embodiments, each nucleoside in gap region Y is a 2'-deoxyribonucleoside, and neither 5' wing region X nor 3' wing region Z contains any 2'-deoxyribonucleosides.

[0230] In some embodiments, the gap region of a gapmer polynucleotide may contain modified nucleotides known to be permissive for efficient RNase H action, in addition to DNA nucleotides such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-constituent nucleotides. In some embodiments, the gap region contains one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, or at least five or more nucleotides. In some embodiments, each internucleoside linkage in the gap segment comprises a phosphorothioate linkage. In some embodiments, the gap region and two flanking regions each independently contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, or at least five or more nucleotides. In some embodiments, each internucleotide linkage in the 5' wing region or the 3' wing region comprises a phosphorothioate linkage. In some embodiments, each internucleotide linkage in a gapmer comprises a phosphorothioate linkage.

[0231] In some embodiments, the Y region can comprise a stretch of contiguous nucleotides, e.g., a region of 5 or more DNA nucleotides that can recruit RNases, including, but not limited to, RNase H. In some embodiments, a gapmer can bind to a target nucleic acid such that RNases are recruited to cleave the target nucleic acid. In some embodiments, the Y region can be flanked on both the 5' and 3' ends by regions X and Z that comprise high-affinity modified nucleosides, e.g., 1 to 10 high-affinity modified nucleosides. Exemplary high-affinity modified nucleosides include, but are not limited to, 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA) and 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F). In some embodiments, the flanking sequences X and Z can be 1 to 30 nucleotides, 1 to 20 nucleotides, 1 to 10 nucleotides, or 1 to 5 nucleotides in length. The flanking sequences X and Z can be of similar or different lengths. In some embodiments, flanking sequences X and Z are each 5 nucleotides in length. In some embodiments, flanking sequences X and Z are each 3 nucleotides in length. In some embodiments, gap segment Y can be a nucleotide sequence between 5 and 30 nucleotides, between 5 and 20 nucleotides, or between 5 and 10 nucleotides in length. In some embodiments, the gap segment is 10 nucleotides in length.

[0232] Gapmers can be produced using any suitable method. The preparation of gapmers is described, for example, in U.S. Patent Nos. 10,260,069; 10,017,764; 9,695,418; 9,428,534; 9,428,534; 9,045,754; 8,580,756; 8,580,756; 7,750,131; 7,683,036; , 569,686; 7,432,250; 7,399,845; 7,101,993; 7,015,315; 5,898,031; 5,700,922; 5,652,356; 5,652,355; 5,623,065; 5,565,350; 5,491,133; Nos. 5,403,711; 5,366,878; 5,256,775; 5,220,007; 5,149,797 and 5,013,830; U.S. Patent Application Publication No. 2010 / 0197762, U.S. Patent Application Publication No. 2005 / 0074801, U.S. Patent Application Publication No. 2009 / 0221685, U.S. Patent Application Publication No. No. 2009 / 0286969, and U.S. Patent Application Publication No. 2011 / 0112170; PCT Publication Nos. WO 2005 / 023825, WO 2004 / 069991, WO 2008 / 049085, and WO 2009 / 090182, each of which is incorporated herein by reference in its entirety.

[0233] In some embodiments, gapmers are 10 to 50 nucleosides in length. For example, gapmers can be 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 40, 25 to 35, 25 to 30, 30 to 40, 30 to 35, or 35 to 40 nucleosides in length. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length. In some embodiments, the gapmer is about 16 to about 20 nucleosides in length. In some embodiments, the gapmer is 16 nucleotides in length. In some embodiments, the gapmer is 20 nucleotides in length.

[0234] In some embodiments, the 5' and 3' wing regions of a gapmer are independently 1 to 20 nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. For example, the 5' and 3' wing regions of a gapmer can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the 5' and 3' wing regions of a gapmer are the same length. In some embodiments, the 5' wing region and the 3' wing region of the gapmer are different lengths. In some embodiments, the 5' wing region is longer than the 3' wing region of the gapmer. In some embodiments, the 5' wing region is shorter than the 3' wing region of the gapmer.

[0235] In some embodiments, the gap region in a gapmer is 5 to 20 nucleosides in length. For example, gap region Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the gap region is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, one or more nucleosides in gap region Y are 2'-deoxyribonucleosides. In some embodiments, all nucleotides in the gap region are deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region are modified nucleosides (e.g., 2'-modified nucleosides such as those described herein). In some embodiments, one or more cytosines in gap region Y are 5-methyl-cytosine. In some embodiments, all cytosines in gap region Y are 5-methyl-cytosine. In some embodiments, all cytosines in the gapmer are 5-methyl-cytosine.

[0236] In some embodiments, one or more nucleosides in the 5' or 3' wing region of a gapmer are modified nucleotides. In some embodiments, the modified nucleotides can be 2'-modified nucleosides, such as 2'-4' bicyclic nucleosides or non-bicyclic 2'-modified nucleosides. In some embodiments, the nucleoside can be a 2'-4' bicyclic nucleoside (e.g., LNA, cET, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAE0E), or 2'-ON-methylacetamide (2'-O-NMA)). In some embodiments, all nucleotides in the wing region are modified nucleotides. In some embodiments, all nucleotides in the wing region are 2'-MOE, LNA, or cET nucleotides.

[0237] In some embodiments, gapmers described herein can include one or more modified internucleoside linkages in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage can include a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently includes a combination of phosphodiester and phosphorothioate linkages. In some embodiments, each internucleoside linkage in gap region Y can be a phosphorothioate linkage, 5' wing region X includes a combination of phosphorothioate and phosphodiester linkages, and 3' wing region Z includes a combination of phosphorothioate and phosphodiester linkages.

[0238] In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a 2'-MOE nucleotide. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a 2'-MOE nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a 2'-MOE nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines, and all internucleotide linkages are phosphorothioate linkages.

[0239] In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is an LNA nucleotide. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is an LNA nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is an LNA nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines, and all internucleotide linkages are phosphorothioate linkages. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a cET nucleotide. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a cET nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide, each nucleotide in the wing regions is a cET nucleotide, all cytosines in the gapmer are 5-methyl-cytosines, and all internucleotide linkages are phosphorothioate linkages.

[0240] Interfering nucleic acids can use various oligonucleotide chemistries. Examples of oligonucleotide chemistries include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate, 2'-O-Me modified oligonucleotide, and morpholino chemistry, including any combination thereof. Generally, PNA chemistry and LNA chemistry can utilize shorter target sequences due to relatively higher target binding strength compared to 2'-O-Me oligonucleotide. Phosphorothioate and 2'-O-Me modified chemistry are often combined to produce 2'-O-Me modified oligonucleotides with phosphorothioate backbones. For example, see PCT Publication Nos. WO / 2013 / 112053 and WO / 2009 / 008725, the entire contents of which are incorporated by reference.

[0241] Peptide nucleic acids (PNAs) are analogs of DNA whose backbone is structurally identical to a deoxyribose backbone consisting of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides according to Watson-Crick base-pairing rules, mimicking DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). PNA backbones are formed by peptide bonds rather than phosphodiester bonds, making them well suited for antisense applications. The backbone is uncharged, resulting in greater than normal thermal stability of PNA / DNA or PNA / RNA duplexes. PNAs are not recognized by nucleases or proteases.

[0242] Despite radical structural changes relative to their native structure, PNAs are capable of sequence-specific binding to DNA or RNA in a helical form. PNA features include high binding affinity to complementary DNA or RNA, no destabilizing effects caused by single-base mismatches, resistance to nucleases and proteases, salt-independent hybridization with DNA or RNA, and triplex formation with homopurine DNA. PANAGENE™ has a unique Bts PNA monomer (Bts; benzothiazole-2-sulfonyl group) and a unique oligomerization process. PNA oligomerization using the Bts PNA monomer consists of repeated cycles of deprotection, coupling, and capping. PNAs can be synthetically produced using any technique known in the art. See, for example, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. See also U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the above is incorporated by reference in its entirety.

[0243] The interfering nucleic acids described herein may also contain "locked nucleic acid" subunits (LNAs). "LNAs" are members of a class of modifications called bridged nucleic acids (BNAs). BNAs are characterized by a covalent bond that locks the conformation of the ribose ring in the C30-endo (north) sugar pucker. In the case of LNAs, the bridge consists of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking to enhance hybridization and thermal stability.

[0244] The structure of LNA can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301; Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230. The compounds provided herein can incorporate one or more LNAs. In some cases, the compounds may be composed entirely of LNA. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, for example, in U.S. Patent No. 5,290,654. These are described in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers may be used. In some embodiments, the antisense oligonucleotide comprises an LNA-containing compound in which each LNA subunit is separated by a DNA subunit. Certain compounds are composed of alternating LNA and DNA subunits, and the intersubunit linker is phosphorothioate.

[0245] "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens is replaced with sulfur. Sulfuration of the internucleotide bond reduces the action of endonucleases and exonucleases, including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases SI and PI, RNases, serum nucleases, and snake venom phosphodiesterases. Phosphorothioates are prepared by two major routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by sulfurization of a phosphite triester with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter method avoids the problems of elemental sulfur insolubility in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield phosphorothioates of higher purity.

[0246] A "2'O-Me oligonucleotide" molecule has a methyl group at the 2'-OH residue of the ribose molecule. 2'-O-Me-RNA behaves the same as (or similar to) DNA, but is protected from nuclease degradation. 2'-O-Me-RNA can also be combined with phosphorothioate oligonucleotides (PTO) for further stabilization. 2'-O-Me oligonucleotides (phosphodiester or phosphorothioate) can be synthesized according to routine techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004).

[0247] Interfering nucleic acid molecules can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA with RNase III or Dicer. They can be introduced into cells by transfection, electroporation, or other methods known in the art. Hannon,GJ,2002,Nature 418:244-251;Bernstein E et al.,2002,RNA 7:1509-1521;Hutvagner G et al.,Curr.Opin.Genetics & Development 12:225-232;Brummelkamp,2002,Science 296:550-553;Lee NS,et al.2002.Nature Biotechnol.20:500-505;Miyagishi M,and Taira K.2002.Nature Biotechnol.20:497-500;Paddison PJ,et al.,2002.Genes & Dev.16:948-958;Paul CP,et al.,2002.Nature Biotechnol.20:505-508;Sui G et al., 2002. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu JY et al., 2002. Proc. Natl. Acad. Sci. USA 99(9):6047-6052, each of which is incorporated by reference in its entirety. guide RNA

[0248] In some embodiments, the conjugated molecular cargo comprises a guide RNA or DNA encoding a guide RNA. A "guide RNA" or "gRNA" is an RNA molecule that binds to a Cas protein (e.g., a Cas9 protein) and targets the Cas protein to a specific location within a target DNA. A guide RNA may comprise two segments: a "DNA-targeting segment" (also called a "guide sequence") and a "protein-binding segment." A "segment" comprises a section or region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA. Some gRNAs, such as those for Cas9, may comprise two separate RNA molecules: an "activator RNA" (e.g., tracrRNA) and a "targeter RNA" (e.g., CRISPR RNA or crRNA). Other gRNAs are single RNA molecules (single RNA polynucleotides), which may also be referred to as "single-molecule gRNA," "single guide RNA," or "sgRNA." See, for example, International Publication Nos. WO 2013 / 176772, WO 2014 / 065596, WO 2014 / 089290, WO 2014 / 093622, WO 2014 / 099750, WO 2013 / 142578, and WO 2014 / 131833, each of which is incorporated by reference in its entirety for all purposes. Guide RNA can refer to either CRISPR RNA (crRNA) or a combination of crRNA and trans-activating CRISPR RNA (tracrRNA). The crRNA and tracrRNA can be associated as a single RNA molecule (single guide RNA or sgRNA) or in two separate RNA molecules (dual guide RNA or dgRNA). For example, in the case of Cas9, a single guide RNA can include a crRNA fused to a tracrRNA (e.g., via a linker). For example, in the case of Cpfl and CasΦ, only the crRNA is required to achieve binding to the target sequence. The terms "guide RNA" and "gRNA" include both double-molecule (i.e., modular) gRNAs and single-molecule gRNAs.In some of the methods and compositions disclosed herein, the gRNA is a Streptococcus pyogenes (S. pyogenes) Cas9 gRNA or its equivalent. In some of the methods and compositions disclosed herein, the gRNA is a Staphylococcus aureus (S. aureus) Cas9 gRNA or its equivalent.

[0249] Exemplary bimolecular gRNAs include a crRNA-like ("CRISPR RNA" or "targeter RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-activating CRISPR RNA" or "activator RNA" or "tracrRNA") molecule. The crRNA comprises both the DNA-targeting segment (single strand) of the gRNA and a stretch of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the gRNA. An example of a crRNA tail located downstream (3') of the DNA-targeting segment (e.g., for use with S. pyogenes Cas9) comprises, consists essentially of, or consists of GUUUUAGAGCUAUGCU (SEQ ID NO: 321) or GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 322). Any of the DNA-targeting segments disclosed herein can be attached to the 5' end of SEQ ID NO: 321 or 322 to form a crRNA.

[0250] The corresponding tracrRNA (activator-RNA) contains a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. The stretch of nucleotides in the crRNA is complementary to and hybridizes with the stretch of nucleotides in the tracrRNA, forming the dsRNA duplex of the protein-binding domain of the gRNA. Thus, each crRNA can be said to have a corresponding tracrRNA. Exemplary tracrRNA sequences (e.g., for use with S. pyogenes Cas9) comprise, consist essentially of, or consist of any one of AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUU (SEQ ID NO: 323), AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUUUU (SEQ ID NO: 324), or GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA CUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 325).

[0251] In systems where both a crRNA and a tracrRNA are required, the crRNA and the corresponding tracrRNA hybridize to form a gRNA. In systems where only a crRNA is required, the crRNA can be the gRNA. The crRNA additionally provides a single-stranded DNA targeting segment that hybridizes to the complementary strand of the target DNA. When used for intracellular modification...

Claims

1. A protein-drug conjugate comprising an anti-human transferrin receptor (hTfR) antigen-binding protein, wherein the anti-hTfR antigen-binding protein is conjugated to a molecular cargo, and the anti-hTfR antigen-binding protein has a K2 of approximately 41 nM Ｄ Alternatively, a protein-drug conjugate that binds to the human transferrin receptor with stronger affinity.

2. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

3. The protein-drug conjugate according to claim 2, wherein the anti-hTfR antigen-binding protein is selected from a humanized antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-stranded fragment variable (scFv), a bis-scFv, (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a single-strand antibody, a bispecific antibody or its binding fragment, a bispecific T cell engager (BiTE), a triplicate antibody, or a chemically modified derivative thereof.

4. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein comprises a fragment antigen-binding region (Fab).

5. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein comprises a single-strand fragment variable (scFv).

6. The scFv is oriented from the N-terminus to the C-terminus as follows: heavy chain variable region (HCVR or V) arranged in HCVR-LCVR. Ｈ ) and light chain variable region (LCVR or V Ｌ The protein-drug conjugate according to claim 5, comprising )

7. The protein-drug conjugate according to claim 5, wherein the scFv comprises HCVR and LCVR arranged in the following orientation from the N-terminus to the C-terminus: LCVR-HCVR.

8. The protein-drug conjugate according to claim 5, wherein the scFv variable region is linked by a linker.

9. The protein-drug conjugate according to claim 8, wherein the linker is a peptide linker.

10. The aforementioned peptide linker is -(GGGGS) ｎ The protein-drug conjugate according to claim 9, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

11. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein comprises a bivalent antibody.

12. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein comprises a one-arm antibody.

13. The aforementioned anti-hTfR antigen-binding protein has a K content of approximately 3 nM. Ｄ The protein-drug conjugate according to claim 1, or which binds to a human transferrin receptor with stronger affinity.

14. The aforementioned anti-hTfR antigen-binding protein has a K content of approximately 0.45 nM to 3 nM. Ｄ The protein-drug conjugate according to claim 13, which binds to a human transferrin receptor.

15. The aforementioned anti-hTfR antigen-binding protein (i) HCVRs including HCDR1, HCDR2, and HCDR3, which include the amino acid sequence (or variant thereof) shown in SEQ ID NOs: 222;262;2;12;22;32;42;52;62;72;82;92;102;112;122;132;142;152;162;172;182;192;202;212;232;242;252;272;282;292;302; or 312; And / or (ii) LCVRs including LCDR1, LCDR2 and LCDR3, which include the amino acid sequence (or variant thereof) shown in SEQ ID NOs: 227;267;7;17;27;37;47;57;67;77;87;97;107;117;127;137;147;157;167;177;187;197;207;217;237;247;257;277;287;297;307; or 317 A protein-drug conjugate according to claim 1, comprising:

16. The aforementioned anti-hTfR antigen-binding protein (1) HCVRs including HCDR1, HCDR2, and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 222; and LCVRs including LCDR1, LCDR2, and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 227; (2) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 262; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 267; (3) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 2; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 7; (4) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 12; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 17; (5) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 22; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 27; (6) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 32; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 37 or 465; (7) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 42; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 47; (8) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 52; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 57; (9) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 62; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 67; (10) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 72; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 77; (11) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 82; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 87; (12) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 92; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 97; (13) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 102; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 107; (14) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 112; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 117; (15) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 122; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 127; (16) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 132; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 137; (17) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 142; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 147; (18) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 152; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 157; (19) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 162; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 167; (20) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 172; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 177; (21) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 182; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 187; (22) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 192; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 197; (23) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 202; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 207; (24) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 212; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 217; (25) HCVRs comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or variant thereof) shown in SEQ ID NO: 232; and LCVRs comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or variant thereof) shown in SEQ ID NO: 237; (26) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 242; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 247; (27) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 252; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 257; (28) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 272; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 277; (29) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 282; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 287; (30) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 292; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 297; (31) HCVRs including HCDR1, HCDR2 and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 302; and LCVRs including LCDR1, LCDR2 and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 307; and / or (32) HCVRs comprising HCDR1, HCDR2 and HCDR3 of an HCVR comprising the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 312; and LCVRs comprising LCDR1, LCDR2 and LCDR3 of an LCVR comprising the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

317. A protein-drug conjugate according to claim 1, comprising:

17. The aforementioned anti-hTfR antigen-binding protein (a) HCDR1 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 223; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 224; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 225 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 228; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 230 LCVR including (b) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 263; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 264; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 265 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 268; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 270 LCVR including (c) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 3; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 4; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 5 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 8; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 10 LCVR including (d) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 13; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 14; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 15 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 18; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 20 LCVR including (e) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 23; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 24; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 25 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 28; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 30 LCVR including (f) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 33; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 34; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 35 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 38; LCDR2 containing amino acid sequence DVS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 40 LCVR including (g) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 43; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 44; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 45 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 48; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 50 LCVR including (h) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 53; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 54; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 55 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 58; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 60 LCVR including (i) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 63; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 64; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 65 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 68; LCDR2 containing the amino acid sequence SAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 70 LCVR including (j) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 73; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 74; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 75 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 78; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 80 LCVR including (k) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 83; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 84; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 85 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 88; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 90 LCVR including (l) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 93; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 94; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 95; and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 98; LCDR2 containing the amino acid sequence TTS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 100 LCVR including (m) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 103; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 104; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 105 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 108; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 110 LCVR including (n) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 113; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 114; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 115 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 118; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 120 LCVR including (o) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 123; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 124; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 125 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 128; LCDR2 containing the amino acid sequence GSS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 130 LCVR including (p) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 133; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 134; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 135 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 138; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 140 LCVR including (q) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 143; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 144; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 145 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 148; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 150 LCVR including (r) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 153; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 154; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 155 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 158; LCDR2 containing amino acid sequence VAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 160 LCVR including (s) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 163; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 164; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 165 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 168; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 170 LCVR including (t) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 173; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 174; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 175 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 178; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 180 LCVR including (u) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 183; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 184; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 185 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 188; LCDR2 containing the amino acid sequence LGS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 190 LCVR including (v) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 193; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 194; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 195 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 198; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 200 LCVR including (w) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 203; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 204; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 205 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 208; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 210 LCVR including (x) HCDR1 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 213; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 214; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 215 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 218; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 220 LCVR including (y) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 233; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 234; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 235 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 238; LCDR2 containing the amino acid sequence GAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 240 LCVR including (z) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 243; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 244; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 245 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 248; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 250 LCVR including (aa) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 253; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 254; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 255 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 258; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 260 LCVR including (ab) HCDR1 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 273; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 274; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 275 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 278; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 280 LCVR including (ac) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 283; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 284; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 285 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 288; LCDR2 containing the amino acid sequence AAS (or its variant); and LCVR containing LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 290 (ad) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 293; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 294; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 295 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 298; LCDR2 containing the amino acid sequence GAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 300 LCVR including (ae) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 303; HCDR2 comprising the amino acid sequence (or variant thereof) shown in SEQ ID NO: 304; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 305 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 308; LCDR2 containing the amino acid sequence LGS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 310 LCVR including And / or (af) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 313; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 314; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 315 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 318; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 320 LCVR including A protein-drug conjugate according to claim 1, comprising:

18. The aforementioned anti-hTfR antigen-binding protein (i) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 222; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 227; (ii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 262; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 267; (iii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 2; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 7; (iv) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 12; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 17; (v) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 22; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 27; (vi) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 32; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 37; (vii) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 42; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 47; (viiii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 52; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 57; (ix) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 62; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 67; (x) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 72; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 77; (xi) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 82; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 87; (xi) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 92; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 97; (xiii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 102; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 107; (xiv) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 112; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 117; (xv) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 122; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 127; (xvi) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 132; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 137; (xvii) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 142; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 147; (xviiii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 152; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 157; (xix) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 162; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 167; (xx) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 172; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 177; (xxi) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 182; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 187; (xxii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 192; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 197; (xxiii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 202; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 207; (xxiv) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 212; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 217; (xxv) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 232; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 237; (xxvi) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 242; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 247; (xxvii) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 252; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 257; (xxviiii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 272; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 277; (xxix) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 282; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 287; (xxx) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 292; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 297; (xxxi) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 302; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 307; and / or (xxxii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 312; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 317 A protein-drug conjugate according to claim 1, comprising:

19. The aforementioned anti-hTfR antigen-binding protein i. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 373; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 372; ii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 381; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 380; iii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 329; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 328; iv. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 331; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 330; v. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 333; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 332; vi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 335; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 334; vii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 337; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 336; viiii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 339; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 338; ix. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 341; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 340; x. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 343; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 342; xi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 345; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 344; xi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 347; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 346; xiiii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 349; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 348; xiv. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 351; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 350; xv. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 353; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 352; xvi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 355; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 354; xvii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 357; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 356; xviiii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 359; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 358; xix. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 361; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 360; xx. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 363; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 362; xxi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 365; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 364; xxii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 367; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 366; xxiii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 369; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 368; xxiv. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 371; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 370; xxv. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 375; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 374; xxvi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 377; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 376; xxvii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 379; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 378; xxviiii. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 383; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 382; xxix. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 385; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 384; xxx. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 387; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 386; xxxi. A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 389; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 388; or xxxii. Heavy chain region containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 391; and light chain containing the amino acid sequence (or variant thereof) shown in SEQ ID NO:

390. A protein-drug conjugate according to claim 1, comprising:

20. The aforementioned anti-hTfR antigen-binding protein i. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 565; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 372; ii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 569; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 380; iii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 543; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 328; iv. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 544; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 330; v. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 545; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 332; vi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 546; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 334; vii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 547; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 336; viiii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 548; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 338; ix. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 549; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 340; x. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 550; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 342; xi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 551; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 344; xi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 552; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 346; xiiii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 553; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 348; xiv. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 554; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 350; xv. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 555; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 352; xvi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 556; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 354; xvii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 557; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 356; xviiii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 558; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 358; xix. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 559; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 360; xx. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 560; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 362; xxi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 561; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 364; xxii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 562; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 366; xxiii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 563; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 368; xxiv. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 564; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 370; xxv. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 566; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 374; xxvi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 567; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 376; xxvii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 568; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 378; xxviiii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 570; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 382; xxix. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 571; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 384; xxx. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 572; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 386; xxxi. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 573; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 388; or xxxii. A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 574; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

390. A protein-drug conjugate according to claim 1, comprising:

21. The aforementioned anti-hTfR antigen-binding protein (1) HCVRs including HCDR1, HCDR2, and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 222; and LCVRs including LCDR1, LCDR2, and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 227; (2) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 262; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 267; (3) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 132; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 137; (4) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 172; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 177; (5) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 242; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 247; or (6) HCVRs including HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 272; and LCVRs including LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO:

277. A protein-drug conjugate according to claim 1, comprising:

22. The aforementioned anti-hTfR antigen-binding protein (a) HCDR1 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 223; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 224; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 225 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 228; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 230 LCVR including (b) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 263; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 264; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 265 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 268; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 270 LCVR including (c) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 133; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 134; and Beauty HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 135 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 138; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 140 LCVR including (d) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 173; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 174; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 175 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 178; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 180 LCVR including (e) HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 243; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 244; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 245 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 248; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 250 LCVR including, or (f) HCDR1 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 273; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 274; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 275 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 278; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 280 LCVR including A protein-drug conjugate according to claim 1, comprising:

23. (i) an HCVR comprising the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 222; and an LCVR comprising the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 227; (ii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 262; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 267; (iii) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 132; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 137; (iv) HCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 172; and LCVR containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 177; (v) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 242; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 247; or (vi) HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 272; and LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 277 A protein-drug conjugate according to claim 1, comprising:

24. The aforementioned anti-hTfR antigen-binding protein (A) A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 355; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 354; (B) A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 363; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 362; (C) A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 373; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 372; (D) A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 377; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 376; (E) A heavy chain region containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 381; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 380; or (F) Heavy chain region containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 383; and light chain containing the amino acid sequence (or variant thereof) shown in SEQ ID NO:

382. A protein-drug conjugate according to claim 1, comprising:

25. The aforementioned anti-hTfR antigen-binding protein (I) A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 556; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 354; (II) A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 560; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 362; (III) A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 565; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 372; (IV) A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 367; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 376; (V) A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 569; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 380; or (VI) A heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 570; and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

382. A protein-drug conjugate according to claim 1, comprising:

26. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein binds to the same epitope on the human transferrin receptor as the antibody containing the HCVR / LCVR amino acid sequence pair as shown in Table 1-1.

27. The protein-drug conjugate according to claim 1, wherein the anti-hTfR antigen-binding protein competes with an antibody containing an HCVR / LCVR amino acid sequence pair as shown in Table 1-1 for binding to a human transferrin receptor.

28. A protein-drug conjugate comprising an anti-hTfR antigen-binding protein, wherein the anti-hTfR antigen-binding protein is conjugated to a molecular cargo, and comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a. Epitopes containing sequence LLNE (sequence number 529) and / or epitopes containing sequence TYKEL (sequence number 509); b. Epitopes containing sequence DSTDFTGT (SEQ ID NO: 530) and / or epitopes containing sequence VKHPVTGQF (SEQ ID NO: 531) and / or epitopes containing sequence IERIPEL (SEQ ID NO: 532); c. Epitope containing the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 533); d. Epitope containing sequence FEDL (sequence number 521); e. Epitope containing sequence IVDKNGRL (sequence number 534); f. Epitope containing sequence IVDKNGRLVY (sequence number 535); g. Epitope containing sequence DQTKF (sequence number 536); h. Epitopes containing the sequence LVENPGGY (SEQ ID NO: 537) and / or epitopes containing the sequence PIVNAELSF (SEQ ID NO: 538) and / or epitopes containing the sequence PYLGTTMDT (SEQ ID NO: 539); i. Epitopes containing the sequence LLINENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or epitopes containing the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or epitopes containing the sequence TYKEL (SEQ ID NO: 509); j. Epitopes containing the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 510) and / or epitopes containing the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 511) and / or epitopes containing the sequence LIERIPELNKVARAAAAE (SEQ ID NO: 512); k. Epitope containing the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 513); l. Epitope containing sequence GTKKDFEDL (sequence number 514); m. Epitope containing sequence SVIIVDKNGRRLVYLVENPGGYVAYSK (SEQ ID NO: 515); n. Epitopes containing the sequence LLINENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or epitopes containing the sequence DQTKFPIVNAEL (SEQ ID NO: 517) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 518); o. Epitopes containing the sequence LLINENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 518); p. Epitope containing sequence SVIIVDKNGRRLVYLVENPGGYVAY (SEQ ID NO: 519); q. Epitopes containing the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or epitopes containing the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 520); r. Epitopes containing the sequence LLINENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or epitopes containing the sequence LVENPGGYVAYSKAAATVTGKL (SEQ ID NO: 522) and / or epitopes containing the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 523) and / or epitopes containing the sequence ISRAAAAEKL (SEQ ID NO: 524) and / or epitopes containing the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 525) and / or epitopes containing the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 526); s. Epitopes contained in or overlapping with sequence LLINENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or epitopes contained in or overlapping with sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or epitopes contained in or overlapping with sequence TYKEL (SEQ ID NO: 509); t. Epitopes contained in or overlapping with sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 510), and / or epitopes contained in or overlapping with sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 511), and / or epitopes contained in or overlapping with sequence LIERIPELNKVARAAAAE (SEQ ID NO: 512); u. Epitopes contained within or overlapping with the sequence LNENSYVPREAGSQKDENL (sequence number 513); v. Epitopes (sequence number 514) that are contained within or overlap with the sequence GTKKDFEDL; w. Epitopes contained within or overlapping with sequence SVIIVDKNGRRLVYLVENPGGYVAYSK (sequence number 515); x. Epitopes contained in or overlapping with sequence LLINENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or epitopes contained in or overlapping with sequence DQTKFPIVNAEL (SEQ ID NO: 517) and / or epitopes contained in or overlapping with sequence TYKELIERIPELNK (SEQ ID NO: 518); y. Epitopes contained in or overlapping with the sequence LLINENSYVPREAGSQKDEN (SEQ ID NO: 516), and / or epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 518); z. Epitopes contained within or overlapping with sequence SVIIVDKNGRRLVYLVENPGGYVAY (sequence number 519); aa. Epitopes contained in or overlapping with sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508), and / or epitopes contained in or overlapping with sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 520); and bb. Epitopes contained in or overlapping with sequence LLINENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or epitopes contained in or overlapping with sequence LVENPGGYVAYSKAAATVTGKL (SEQ ID NO: 522) and / or epitopes contained in or overlapping with sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 523) and / or epitopes contained in or overlapping with sequence ISRAAAAEKL (SEQ ID NO: 524) and / or epitopes contained in or overlapping with sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 525) and / or epitopes contained in or overlapping with sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 526) A protein-drug conjugate that binds to one or more epitopes of hTfR selected from the available options.

29. The antibody or its antigen-binding fragment a. An epitope consisting of sequence LLNE (sequence number 529) and / or an epitope consisting of sequence TYKEL (sequence number 509); b. An epitope consisting of sequence DSTDFTGT (SEQ ID NO: 530) and / or an epitope consisting of sequence VKHPVTGQF (SEQ ID NO: 531) and / or an epitope consisting of sequence IERIPEL (SEQ ID NO: 532); c. An epitope consisting of the sequence LNENSYVPREAGSQKDEN (sequence number 533); d. An epitope consisting of sequence FEDL (sequence number 521); e. An epitope consisting of the sequence IVDKNGRL (sequence number 534); f. An epitope consisting of the sequence IVDKNGRLVY (sequence number 535); g. An epitope consisting of sequence DQTKF (sequence number 536); h. An epitope consisting of the sequence LVENPGGY (sequence number 537) and / or an epitope consisting of the sequence PIVNAELSF (sequence number 538) and / or an epitope consisting of the sequence PYLGTTMDT (sequence number 539); i. An epitope consisting of the sequence LLINENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope consisting of the sequence TYKEL (SEQ ID NO: 509); j. An epitope consisting of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 510) and / or an epitope consisting of the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 511) and / or an epitope consisting of the sequence LIERIPELNKVARAAAAE (SEQ ID NO: 512); k. An epitope consisting of the sequence LNENSYVPREAGSQKDENL (sequence number 513); l. An epitope consisting of the sequence GTKKDFEDL (sequence number 514); m. An epitope consisting of the sequence SVIIVDKNGRRLVYLVENPGGYVAYSK (sequence number 515); n. An epitope consisting of the sequence LLINENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope consisting of the sequence DQTKFPIVNAEL (SEQ ID NO: 517) and / or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 518); o. An epitope consisting of the sequence LLINENSYVPREAGSQKDEN (SEQ ID NO: 516) and / or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 518); p. An epitope consisting of the sequence SVIIVDKNGRRLVYLVENPGGYVAY (sequence number 519); q. An epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 508) and / or an epitope consisting of the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 520); and r. An epitope consisting of the sequence LLINENSYVPREAGSQKDENLAL (SEQ ID NO: 507) and / or an epitope consisting of the sequence LVENPGGYVAYSKAAATVTGKL (SEQ ID NO: 522) and / or an epitope consisting of the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 523) and / or an epitope consisting of the sequence ISRAAAAEKL (SEQ ID NO: 524) and / or an epitope consisting of the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 525) and / or an epitope consisting of the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 526) The protein-drug conjugate according to claim 28, which binds to one or more epitopes of hTfR selected from.

30. The protein-drug conjugate according to claim 28, wherein the anti-hTfR antigen-binding protein is selected from a humanized antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-stranded fragment variable (scFv), a bis-scFv, (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a single-strand antibody, a bispecific antibody or its binding fragment, a bispecific T cell engager (BiTE), a triplicate antibody, or a chemically modified derivative thereof.

31. The aforementioned molecular cargo (i) HCVR of the antigen-binding protein, (ii) LCVR of the antigen-binding protein, (iii) the heavy chain of the antigen-binding protein, and / or (iv) Light chain of the antigen-binding protein A protein-drug conjugate according to any one of claims 1 to 30, which is conjugated to a protein-drug conjugate.

32. The protein-drug conjugate according to any one of claims 1 to 30, wherein the molecular cargo is conjugated to the anti-hTfR antigen-binding protein via glutamine residues and / or lysine residues.

33. The aforementioned glutamine residue, (i) The heavy chain of the antigen-binding protein is introduced at the N-terminus and / or C-terminus, (ii) The light chain of the antigen-binding protein is introduced at the N-terminus and / or C-terminus, (iii) The CH2 or CH3 domain of the antigen-binding protein contains naturally occurring (iv) The anti-hTfR antigen-binding protein is introduced by modifying one or more amino acids, and / or (v) Mutation occurs from Q295 or N297 to Q297 (N297Q), The protein-drug conjugate according to claim 32.

34. The protein-drug conjugate according to claim 32, wherein the anti-hTfR antigen-binding protein comprises a glutamine-containing tag, and the molecular cargo is conjugated to the anti-hTfR antigen-binding protein via glutamine residues of the glutamine-containing tag.

35. The glutamine-containing tags are LLQGG (SEQ ID NO: 439), LLQG (SEQ ID NO: 440), LSLSQG (SEQ ID NO: 441), gGGLLQGG (SEQ ID NO: 442), gLLQG (SEQ ID NO: 443), LLQ (SEQ ID NO: 444), gSPLAQSHGG (SEQ ID NO: 445), gLLQGGGG (SEQ ID NO: 446), gLLQGG (SEQ ID NO: 447), gLLQ (SEQ ID NO: 448), LLQLLQGA (SEQ ID NO: 449), The protein-drug conjugate according to claim 34, comprising an amino acid sequence selected from the group consisting of LLQGA (SEQ ID NO: 450), LLQYQGA (SEQ ID NO: 451), LLQGSG (SEQ ID NO: 452), LLQYQG (SEQ ID NO: 453), LLQLLQG (SEQ ID NO: 454), SLLQG (SEQ ID NO: 455), LLQLQ (SEQ ID NO: 456), LLQLLQ (SEQ ID NO: 457), and LLQGR (SEQ ID NO: 458).

36. The protein-drug conjugate according to any one of claims 1 to 30, wherein the anti-hTfR antigen-binding protein and the molecular cargo are conjugated via a linker.

37. The protein-drug conjugate according to any one of claims 1 to 30, wherein the molecular cargo comprises a polynucleotide molecule, a carrier, or a small molecule.

38. The protein-drug conjugate according to any one of claims 1 to 30, wherein the molecular cargo comprises a polynucleotide molecule.

39. The protein-drug conjugate according to claim 38, wherein the polynucleotide molecule is an interfering nucleic acid molecule, guide RNA, ribozyme, aptamer, mixmer, multimer, or mRNA.

40. The protein-drug conjugate according to claim 39, wherein the interfering nucleic acid molecule is siRNA, shRNA, miRNA, antisense oligonucleotide, or gapmer.

41. The protein-drug conjugate according to claim 40, wherein the interfering nucleic acid is siRNA.

42. The protein-drug conjugate according to claim 40, wherein the siRNA inhibits the DMPK, CNBP, dystrophin, DUX4, ApoE, MAPT, APP, HTT, SOD1, C9orf72, SNCA, LRRK2, PRNP, SLC5A1, SLC16A3, HDAC6, MMP27, MFAP5, FAM64A, BAIAP3, MYH7, TPM1, RBM20, KLHL24, MYL2, or TNNT2 gene or a variant thereof.

43. The protein-drug conjugate according to claim 41, wherein the siRNA comprises a sense strand 21 nucleotides long.

44. The protein-drug conjugate according to claim 41, wherein the siRNA comprises a 23-nucleotide antisense chain.

45. The protein-drug conjugate according to claim 41, wherein the siRNA includes two phosphorothioate bonds in the internucleoside bond between the first and second nucleosides at the 5' end of the sense strand.

46. The protein-drug conjugate according to claim 41, wherein the siRNA includes two phosphorothioate bonds in the first and second nucleoside-to-nucleoside bonds at the 3' and / or 5' ends of the antisense strand.

47. The protein-drug conjugate according to claim 40, wherein the interfering nucleic acid is an antisense oligonucleotide.

48. The protein-drug conjugate according to claim 39, wherein the polynucleotide molecule is a guide RNA.

49. The protein-drug conjugate according to claim 39, wherein the polynucleotide molecule comprises one or more modified nucleotides.

50. The protein-drug conjugate according to any one of claims 1 to 30, wherein the molecular cargo comprises a carrier.

51. The protein-drug conjugate according to claim 50, wherein the molecular cargo comprises a lipid-based carrier.

52. The protein-drug conjugate according to claim 51, wherein the lipid-based carrier is a lipid nanoparticle (LNP), a liposome, a lipidoid, or a lipoplex.

53. The protein-drug conjugate according to claim 52, wherein the lipid-based carrier is an LNP.

54. The protein-drug conjugate according to claim 53, wherein the LNP further comprises a polynucleotide molecule and / or a polypeptide molecule.

55. The protein-drug conjugate according to claim 53, wherein the LNP comprises one or more components of a gene editing system.

56. LNP is (a) Cas nuclease, or nucleic acid encoding the said Cas nuclease, and / or (b) Guide RNA, or one or more DNAs encoding the guide RNA The protein-drug conjugate according to claim 55, comprising:

57. The protein-drug conjugate according to claim 56, wherein the Cas nuclease is a Cas9 protein.

58. The protein-drug conjugate according to claim 57, wherein the Cas9 protein is derived from Streptococcus pyogenes Cas9 protein, Staphylococcus aureus Cas9 protein, Campylobacter jejuni Cas9 protein, Streptococcus thermophilus Cas9 protein, or Neisseria meningitidis Cas9 protein.

59. The protein-drug conjugate according to claim 56, wherein the nucleic acid encoding the Cas protein is codon-optimized for expression in mammalian cells.

60. The protein-drug conjugate according to claim 59, wherein the nucleic acid encoding the Cas protein is codon-optimized for expression in human cells.

61. The protein-drug conjugate according to claim 56, wherein the nucleic acid encoding the Cas nuclease comprises mRNA.

62. The protein-drug conjugate according to claim 39, wherein the guide RNA is a single guide RNA (sgRNA).

63. The protein-drug conjugate according to claim 55, wherein the LNP comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).

64. The protein-drug conjugate according to claim 53, wherein the lipid nanoparticles include a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

65. The protein-drug conjugate according to claim 64, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC).

66. The protein-drug conjugate according to claim 64, wherein the helper lipid is cholesterol.

67. The protein-drug conjugate according to claim 64, wherein the stealth lipid is PEG2k-DMG.

68. The protein-drug conjugate according to any one of claims 1 to 30, wherein, if the antigen-binding protein is not conjugated to the molecular cargo, it does not block more than 50% of the binding of the human transferrin receptor C-terminal fragment to human holotransferrin that occurs in the absence of such antigen-binding protein.

69. The protein-drug conjugate according to claim 68, wherein the inhibition is measured by an enzyme-linked immunosorbent assay (ELISA) plate assay, in which the extracellular domain of a human transferrin receptor fused to a His6-myc-myc tag is pre-bound to the anti-hTfR antigen-binding protein, and then comes into contact with holotransferrin immobilized on the surface of the plate by binding of an anti-holotransferrin antibody bound to the plate.

70. The protein-drug conjugate according to claim 68, wherein the binding of holotransferrin and the extracellular domain of the human transferrin receptor in the absence of the anti-hTfR antigen-binding protein is measured at a concentration of the extracellular domain of the human transferrin receptor of about 300 pM.

71. The aforementioned anti-hTfR antigen-binding protein has the following characteristics: a. Affinity (K) for binding to monkey TfR at 25°C in surface plasmon resonance formats of approximately 0 nM (no detectable binding) or higher affinity. Ｄ ), b. Ratio of [K that binds to monkey TfR Ｄ / K that binds to human TfR Ｄ at 25°C in a surface plasmon resonance format of 0 to 278 c. In the case of the Fab format (IgG1), approximately 3-13% of hTfR binding to human Holo-Tf is blocked. d. scFv(V Ｋ -V Ｈ ) In the case of this format, it blocks approximately 6-13% of hTfR binding to human Holo-Tf, and / or e. scFv(V Ｈ -V Ｌ In this format, approximately 11-26% of hTfR binding to human Holo-Tf is blocked. A protein-drug conjugate according to any one of claims 1 to 30, having one or more of the above.

72. A pharmaceutical composition comprising a protein-drug conjugate according to any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

73. A composition or kit comprising, in combination with any one of claims 1 to 30, a protein-drug conjugate or a pharmaceutical composition thereof.

74. The composition or kit according to claim 73, wherein the further therapeutic agent is selected from alglucosidase alfa, rituximab, methotrexate, intravenous immunoglobulin (IVIG), avalglucosidase alfa, revalbuterol, antibiotics, cortisone, prednisone, bisphosphonates, and palivizumab.

75. The composition or kit according to claim 73, wherein the further therapeutic agent is selected from beta-2-adrenergic agonists, steroids, bisphosphonates, infectious agents, vaccines, and pneumococcal vaccines.

76. A complex comprising a protein-drug conjugate according to any one of claims 1 to 30, conjugated to a human transferrin receptor polypeptide or a fragment thereof.

77. (a) Contacting the antigen-binding protein with the molecular cargo under conditions favorable for the conjugation of the anti-hTfR antigen-binding protein to the molecular cargo, (b) If applicable, isolate the protein-drug conjugate produced in step (a), A method for producing a protein-drug conjugate according to any one of claims 1 to 30, including the above.

78. A protein-drug conjugate which is a product of the method according to claim 77.

79. A protein-drug conjugate according to any one of claims 1 to 30, or a protein-drug conjugate that is a product of a method for producing a protein-drug conjugate according to any one of claims 1 to 30, wherein the method is (a) Contacting the antigen-binding protein with the molecular cargo under conditions favorable for the conjugation of the anti-hTfR antigen-binding protein to the molecular cargo, (b) optionally including isolating the protein-drug conjugate produced in step (a), A container or injection device containing a protein-drug conjugate.

80. A protein-drug conjugate according to any one of claims 1 to 30, or a protein-drug conjugate that is a product of a method for producing a protein-drug conjugate according to any one of claims 1 to 30, wherein the method is (a) Contacting the antigen-binding protein with the molecular cargo under conditions favorable for the conjugation of the anti-hTfR antigen-binding protein to the molecular cargo, (b) optionally, isolating the protein-drug conjugate produced in step (a), a composition comprising a protein-drug conjugate, The composition is characterized by being administered to a subject, and the administration of the composition comprises introducing the protein-drug conjugate into the body of the subject.

81. The composition according to claim 80, characterized in that the protein-drug conjugate is introduced parenterally into the body of the subject.

82. The composition according to claim 81, characterized in that the protein-drug conjugate is intravenously introduced into the body of the subject.

83. The composition according to claim 80, characterized in that the protein-drug conjugate is introduced into the body of the subject via intrathecal, intraventricular, or intraparenchymal injection into the central nervous system.

84. A composition for treating or preventing a disease or disorder of which treatment or prevention is required, wherein the protein-drug conjugate is a product of a method for producing a protein-drug conjugate according to any one of claims 1 to 30, and the method is (a) Contacting the antigen-binding protein with the molecular cargo under conditions favorable for the conjugation of the anti-hTfR antigen-binding protein to the molecular cargo, (b) optionally including isolating the protein-drug conjugate produced in step (a), Contains protein-drug conjugates, The composition is characterized by being administered to the subject.

85. The composition according to claim 84, wherein the disease or disorder is a lysosomal storage disorder or disorder, a heart disease or disorder, a central nervous system (CNS) disease or disorder, an eye disease or disorder, a brain disease or disorder, a spinal cord disease or disorder, a peripheral nervous system (PNS) disease or disorder, a muscle disease or disorder, a cartilage disease or disorder, a bone growth plate disease or disorder, a kidney disease or disorder, or a blood disorder or disorder.

86. The composition according to claim 84, wherein the disease or disorder is a neurological disease or disorder.

87. The composition according to claim 86, wherein the neurological disease or disorder is lysosomal storage disorder, amyloidosis, neuropathy, neurodegenerative disease, seizure, behavioral disorder, leukodystrophy, neuropsychiatric disorder, traumatic brain injury, neurodevelopmental disorder, neuromuscular disease, eye disease or disorder, viral or microbial infection, inflammation, ischemia, and cancer.

88. The composition according to claim 84, wherein the disease or disorder is a lysosomal storage disorder.

89. The composition according to claim 87, wherein the neurodegenerative disease is Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, or prion disease.

90. The composition according to claim 89, wherein the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit ApoE, MAPT, APP, HTT, SOD1, C9orf72, SNCA, LRRK2, or PRNP genes or their variants.

91. The composition according to claim 85, wherein the disease or disorder is a heart disease or disorder.

92. The composition according to claim 91, wherein the aforementioned heart disease or disorder is heart failure.

93. The composition according to claim 92, wherein the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit the SLC5A1, SLC16A3, HDAC6, MMP27, MFAP5, FAM64A, BAIAP3, MYH7, TPM1, RBM20, KLHL24, MYL2, or TNNT2 gene or its variant.

94. The composition according to claim 85, wherein the disease or disorder is a muscle disease or disorder.

95. The composition according to claim 94, wherein the muscle disease or disorder is myotonic dystrophy, Duchenne muscular dystrophy, myofascial scapulohumeral muscular dystrophy, facioscapulohumeral muscular dystrophy type 1, or muscle atrophy.

96. The composition according to claim 95, wherein the molecular cargo of the protein-drug conjugate is an siRNA selected from the group consisting of siRNAs that inhibit DMPK, CNBP, dystrophin, or the DUX4 gene or its variants.

97. The composition according to claim 84, characterized in that the subject is administered the protein-drug conjugate in combination with a further therapeutic agent.

98. The composition according to claim 97, wherein the further therapeutic agent is selected from alglucosidase alfa, rituximab, methotrexate, intravenous immunoglobulin (IVIG), avalglucosidase alfa, revalbuterol, antibiotics, cortisone, prednisone, bisphosphonates, and palivizumab.

99. The composition according to claim 97, wherein the further therapeutic agent is selected from beta-2-adrenergic agonists, steroids, bisphosphonates, infectious agents, vaccines, and pneumococcal vaccines.

100. A composition for delivering a molecular cargo to a target tissue or cell type in the body, comprising an anti-hTfR antigen-binding protein conjugated to the molecular cargo.

101. The composition according to claim 100, wherein the molecular cargo comprises a polynucleotide molecule, a carrier, or a small molecule.

102. The aforementioned tissues include the brain / spinal cord / CNS; eyes; skeletal muscle; adipose tissue; blood / bone marrow; breasts; lungs / bronchioles; colon; uterus; esophagus; heart; kidneys; liver; lymph nodes; ovaries; pancreas; placenta; prostate; rectum; skin; peripheral blood mononuclear cells (PBMCs); small intestine; spleen; stomach; testes; peripheral nervous system; and / or bone / cartilage / joints. The composition according to claim 100 or 101.

103. The aforementioned cell types and associated tissues are as follows: Table 7-1 Table 7-2 Table 7-3 Table 7-4 The composition according to claim 100 or 101.

104. The composition according to claim 84, characterized in that the composition is administered in combination with puncturing the body of the subject with a syringe needle and injecting the anti-hTfR antigen-binding protein conjugated to the molecular cargo into the body of the subject, or a composition for delivering a molecular cargo to a tissue or cell type in the body of the subject, comprising an anti-hTfR antigen-binding protein conjugated to the molecular cargo.

105. The composition according to claim 84, wherein the subject is suffering from a muscle atrophy, metabolic disease, sarcopenia, or cachexia, or a composition for delivering a molecular cargo to a tissue or cell type in the body of the subject, comprising an anti-hTfR antigen-binding protein conjugated to the molecular cargo.

106. A protein-drug conjugate comprising an anti-human transferrin receptor (hTfR) antigen-binding protein conjugated to a molecular cargo, The anti-hTfR antigen-binding protein comprises an HCVR containing HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 222, and an LCVR containing LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO:

227. Protein-drug conjugates.

107. The anti-hTfR antigen-binding protein is HCDR1 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 223; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 224; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 225 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 228; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 230 LCVR including A protein-drug conjugate according to claim 106, comprising:

108. The protein-drug conjugate according to claim 106, wherein the anti-hTfR antigen-binding protein comprises an HCVR having the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 222 and an LCVR having the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

227.

109. The protein-drug conjugate according to claim 106, wherein the anti-hTfR antigen-binding protein comprises a heavy chain region including the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 373, and a light chain including the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

372.

110. The protein-drug conjugate according to claim 106, wherein the anti-hTfR antigen-binding protein comprises a heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 565 and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

372.

111. A protein-drug conjugate comprising an anti-human transferrin receptor (hTfR) antigen-binding protein conjugated to a molecular cargo, The anti-hTfR antigen-binding protein comprises an HCVR containing HCDR1, HCDR2, and HCDR3 of an HCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 262, and an LCVR containing LCDR1, LCDR2, and LCDR3 of an LCVR containing the amino acid sequence (or variant thereof) shown in SEQ ID NO:

267. Protein-drug conjugates.

112. The anti-hTfR antigen-binding protein is HCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 263; HCDR2 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 264; and HCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 265 HCVR and LCDR1 containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 268; LCDR2 containing the amino acid sequence AAS (or its variant); and LCDR3 containing the amino acid sequence (or variant thereof) shown in SEQ ID NO: 270 LCVR including A protein-drug conjugate according to claim 111, comprising:

113. The protein-drug conjugate according to claim 111, wherein the anti-hTfR antigen-binding protein comprises an HCVR having the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 262 and an LCVR having the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

267.

114. The protein-drug conjugate according to claim 111, wherein the anti-hTfR antigen-binding protein comprises a heavy chain region comprising the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 381, and a light chain comprising the amino acid sequence (or a variant thereof) shown in SEQ ID NO:

380.

115. The protein-drug conjugate according to claim 111, wherein the anti-hTfR antigen-binding protein comprises a heavy chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 569 and a light chain containing the amino acid sequence (or a variant thereof) shown in SEQ ID NO: 380.

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