Blood-brain barrier penetrating antibodies

Abstract

The disclosure related to antibodies that bind to transferrin receptor 1 (TfR1) and conjugates and fusion proteins comprising the antibodies. The disclosure also relates to methods of using the antibodies, conjugates, and fusion proteins.

Description

[0001] Attorney Docket No. H0498.70819WO00

[0002] BLOOD-BRAIN BARRIER PENETRATING ANTIBODIES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 692,379, filed September 9, 2024, which is hereby incorporated by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (H049870819WO00-SEQ-KVC.xml; Size: 51,637 bytes; and Date of Creation: September 4, 2025) is herein incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] The blood-brain barrier (BBB) is a highly specialized neurovascular unit, commonly described as an intact barrier to prevent toxins, pathogens, and potentially harmful substances from entering the brain. The restrictive nature of the intact BBB presents a major hurdle for delivery of therapeutics and other agents into the brain. Sufficient delivery across the BBB has been a key challenge in the development of drugs, such as monoclonal antibodies and enzyme replacement therapies, to treat central nervous system (CNS) disorders. These biopharmaceuticals are largely excluded from the brain following systemic administration. Recently, increasing research efforts by pharmaceutical and biotechnology companies, academic institutions and public-private consortia have resulted in the evaluation of various strategies and technologies developed to deliver therapeutics to the CNS, some of which have entered clinical testing.

[0009] One such strategy uses the physiological process of receptor-mediated transcytosis (RMT) to transport cargo through the brain endothelial cells toward and into the brain parenchyma. In general, strategies using RMT for drug delivery to the brain involve the generation of a complex between the drug of interest and a receptor-targeting entity. This targeting entity can be, for example, the endogenous receptor ligand, an antibody targeting the receptor or a mimetic peptide ligand. The drug and the targeting entity can be genetically fused to generate a fusion protein, can be chemically linked (conjugated), or the drug can be incorporated in liposomes or nanoparticles decorated with the receptor-targeting ligand. One well-studied target for RMT in brain endothelial cells is the transferrin receptor.

[0010] Iron delivery to the brain is accomplished via binding and intracellular trafficking of

[0011] 1

[0012] #14369922vl Attorney Docket No. H0498.70819WO00 the iron binding protein transferrin (Tf). The Tf receptor (TfR) has been the target of numerous in vitro and in vivo studies aiming to deliver drugs to the brain. Anti-TfR antibodies have emerged as a widely studied targeting entity. Nonetheless, challenges using anti-TfR antibodies to deliver drugs to the brain via RMT include binding to transferrin expressed on many tissues other than brain endothelial cells resulting in rapid clearance from the blood and possible side effects such as reticulocyte depletion, rapid clearance from the brain due to uptake by neurons and other brain cells resulting from their expression of TfR, cross-reactivity to cynomolgus TfRl protein, and sub-optimal Cmax and area under the curve for brain exposure achieved by this delivery route. Together, these challenges indicate that a significant unmet need remains for effective therapeutic carriers across the BBB.

[0013] SUMMARY

[0014] The present disclosure is based, at least in part, on the development of antibodies (e.g., single-chain antibodies) specific to transferrin receptor 1 (TfRl) that effectively cross the blood-brain barrier (BBB). These antibodies have exhibited a broad range of binding affinities each useful for different applications, rapid cellular internalization upon binding to the extracellular domain of TfRl, and high brain uptake and brain exposure. Additionally, these antibodies do not compete with transferrin for binding to TfRl, indicating that these antibodies bind to a surface-exposed epitope of TfRl that is conserved and distinct from the transferrin binding site. In some embodiments, the disclosure provides anti-TfRl antibodies (e.g., heavy-chain variable domain (VHH) antibodies) that provide improved stability relative to other anti-TfRl antibodies known in the art. In some embodiments, an anti-TfRl antibody of the disclosure is capable of incorporating changes in backbone scaffold of the antibody such that the properties of the antibody are not impacted. Furthermore, anti-TfRl antibodies of the disclosure can bind to TfRl belonging to multiple species, such as human TfRl and cynomolgus (monkey) TfRl (e.g., antibodies are cross -reactive).

[0015] Accordingly, the antibodies provided herein are useful compounds for delivering a diagnostic or therapeutic agent (e.g., a therapeutic polypeptide, for example, a therapeutic antibody, or a therapeutic polynucleotide) to cells expressing TfRl by linking one or more of the anti-TfRl antibodies of the disclosure to the diagnostic or therapeutic agent. In some embodiments, an anti-TfRl antibody functions (in part due to its relatively smaller size compared to a conventional full-length antibody) to effectively shuttle a diagnostic or therapeutic agent across the BBB to the brain parenchyma or interstitial space. In some embodiments, an anti-TfRl antibody functions to shuttle a diagnostic or therapeutic agent

[0016] 2

[0017] #14369922vl Attorney Docket No. H0498.70819WO00 into the brain (e.g., into target brain cells). For example, in some embodiments, an anti-TfRl antibody functions to shuttle a diagnostic or therapeutic agent to (and / or into) neurons expressing TfRl (e.g., diseased neurons expressing TfRl). In some embodiments, anti-TfRl antibodies of the disclosure provide a therapeutic (e.g., as a therapeutic antibody), for example, for treating diseases of the central nervous system.

[0018] Some aspects of the disclosure relate to an antibody comprising a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR), such as TfRl.

[0019] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2; and / or a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0020] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4.

[0021] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0022] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

[0023] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary 3

[0024] #14369922vl Attorney Docket No. H0498.70819WO00 determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 9 and / or a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 9; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0025] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10.

[0026] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0027] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11.

[0028] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0029] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13.

[0030] 4

[0031] #14369922vl Attorney Docket No. H0498.70819WO00

[0032] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0033] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14.

[0034] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0035] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33.

[0036] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 29; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 29; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0037] 5

[0038] #14369922vl Attorney Docket No. H0498.70819WO00

[0039] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34.

[0040] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0041] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35.

[0042] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 30; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 30; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0043] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36.

[0044] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28; and 6

[0045] #14369922vl Attorney Docket No. H0498.70819WO00 complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ

[0046] ID NO: 3.

[0047] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37.

[0048] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 29; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 29; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0049] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38.

[0050] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0051] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39.

[0052] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining 7

[0053] #14369922vl Attorney Docket No. H0498.70819WO00 region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0054] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40.

[0055] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0056] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41.

[0057] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0058] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42.

[0059] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 25;

[0060] 8

[0061] #14369922vl Attorney Docket No. H0498.70819WO00 and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 25; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0062] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43.

[0063] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 26; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 26; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0064] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44.

[0065] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 27; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 27; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0066] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45.

[0067] 9

[0068] #14369922vl Attorney Docket No. H0498.70819WO00

[0069] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0070] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46.

[0071] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 25; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 25; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0072] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47.

[0073] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 31; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 31; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0074] 10

[0075] #14369922vl Attorney Docket No. H0498.70819WO00

[0076] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48.

[0077] In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 32; and / or complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody comprises a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 32; and complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3.

[0078] In some embodiments, an antibody comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49.

[0079] In some embodiments, the equilibrium dissociation constant (KD) of binding of the antibody to the TfR is in a range from IO0M to 10'5M.

[0080] In some embodiments, the equilibrium dissociation constant (KD) of binding of the antibody to the TfR is in a range from 0.5 nM to 5000 nM.

[0081] In some embodiments, the antibody selectively binds to human TfR relative to a nonhuman TfR.

[0082] In some embodiments, the antibody selectively binds to non-human primate TfR relative to human TfR.

[0083] In some embodiments, the TfR is a transferrin receptor 1 (TfRl).

[0084] In some embodiments, the antibody is internalized into a cell, optionally wherein the cell is a brain endothelial cell.

[0085] In some embodiments, the antibody is a single chain antibody.

[0086] In some embodiments, the single chain antibody is a heavy-chain variable domain (VHH) antibody or a single-chain variable fragment (scFv) antibody.

[0087] In some embodiments, the antibody is a humanized antibody.

[0088] In some embodiments, the antibody is a chimeric antibody.

[0089] Some aspects relate to a cell comprising the antibody of any one of the preceding paragraphs, optionally wherein the cell is a brain endothelial cell.

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[0091] #14369922vl Attorney Docket No. H0498.70819WO00

[0092] Some aspects relate to a fusion protein in which an anti-TfR antibody (e.g., anti-TfRl antibody) is fused to a heavy chain or light chain of a payload antibody or a therapeutic protein.

[0093] Other aspects relate to a conjugate comprising the antibody of any one of the preceding paragraphs.

[0094] In some embodiments, an antibody is linked directly to (e.g., fused to) or indirectly to a diagnostic agent. In some embodiments, an antibody is linked directly to (e.g., fused to) or indirectly to a therapeutic agent. In some embodiments, a therapeutic agent is a therapeutic polypeptide, for example, a therapeutic recombinant protein selected from antibodies, cytokines, and growth factors. In some embodiments, a therapeutic agent is a therapeutic polynucleotide, optionally an antisense oligonucleotide, a RNA interference molecule (e.g., siRNA, shRNA, miRNA), a CRISPR RNA (crRNA), or a double stranded RNA (dsRNA).

[0095] In some embodiments, an antibody is linked to (e.g., fused to) a nanoparticle, for example, a lipid nanoparticle (LNP), a polymer nanoparticle, a DNA nanoparticle, a magnetic nanoparticle, metal (e.g., gold or silver) nanoparticle, a liposome, or a nanoemulsion.

[0096] In some embodiments, an antibody is linked to (e.g., fused to) an imaging agent, for example, a radiopharmaceutical, a magnetic particle, or a light-emitting reporter (e.g., luciferase, green fluorescent protein (GFP), or near-infrared (IR) labels).

[0097] In some embodiments, an antibody is linked to (e.g., fused to) a viral vector, for example, an adeno-associated virus (AAV) vector, a lentiviral vector.

[0098] Further aspects relate to a therapeutic fusion protein comprising: a heavy chain comprising a heavy chain constant domain and a heavy chain variable domain; a light chain comprising a light chain constant domain and a light chain variable domain; and at least one anti-TfR antibody of any one of the preceding paragraphs.

[0099] In some embodiments, an anti-TfR antibody (e.g., anti-TfRl antibody) is linked to the heavy chain constant domain or the heavy chain variable domain. In some embodiments, an anti-TfR antibody is linked to the light chain constant domain or the light chain variable domain.

[0100] In some embodiments, a therapeutic fusion protein comprises two anti-TfR antibodies (e.g., anti-TfRl antibodies), wherein (a) each of the two anti-TfR antibodies is linked to the heavy chain constant domain or (b) each of the two anti-TfR antibodies is linked to the heavy chain variable domain.

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[0102] #14369922vl Attorney Docket No. H0498.70819WO00

[0103] In some embodiments, a therapeutic fusion protein comprises two anti-TfR antibodies (e.g., anti-TfRl antibodies), wherein (a) each of the two anti-TfR antibodies is linked to the light chain constant domain or (b) each of the two anti-TfR antibodies is linked to the light chain variable domain.

[0104] In some embodiments, a therapeutic fusion protein comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 17 and / or the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the therapeutic fusion protein comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 17 and the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 18.

[0105] Some aspects relate to a therapeutic fusion protein comprising at least one anti-TfR antibody (e.g., anti-TfRl antibody) of any one of the preceding paragraphs fused to a polypeptide selected from: Fc-containing antibodies, Ab fragments, Fc fusion proteins, Fc domains; and non-antibody proteins.

[0106] Some aspects relate to a pharmaceutical composition comprising an anti-TfR antibody (e.g., anti-TfRl antibody) of any one of the preceding paragraphs and a pharmaceutically acceptable excipient.

[0107] Other aspects relate to a method comprising administering to a subject an anti-TfR antibody of any one of the preceding paragraphs, a fusion protein of any one of the preceding paragraphs, a therapeutic fusion protein of any one of the preceding paragraphs, or a pharmaceutical composition of any one of the preceding paragraphs.

[0108] Some aspects relate to a nucleic acid or set of nucleic acids encoding an anti-TfR antibody (e.g., anti-TfRl antibody) of any one of the preceding paragraphs.

[0109] Other aspects relate to a vector or set of vectors comprising one or more nucleic acid encoding an anti-TfR antibody (e.g., anti-TfRl antibody) of any one of the preceding paragraphs.

[0110] Further aspects relate to a method of producing an anti-TfR antibody (e.g., anti-TfRl antibody) of any one of the preceding paragraphs, the method comprising culturing a cell comprising any one of the anti-TfR antibodies of the preceding paragraphs in cell culture and obtaining the anti-TfR antibody from the cell or cell culture.

[0111] Still other aspects relate to an engineered polypeptide comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein the peptide binds to a transferrin receptor (e.g., TfRl). In some embodiments, an engineered polypeptide further comprises: a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8,

[0112] 13

[0113] #14369922vl Attorney Docket No. H0498.70819WO00 or 25-27; and / or a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0114] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 2; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0115] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 5; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0116] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 8; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 9; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0117] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 8; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0118] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 8; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 12; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0119] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 5; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 12; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0120] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 28; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0121] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 29; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0122] 14

[0123] #14369922vl Attorney Docket No. H0498.70819WO00

[0124] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 30; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0125] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0126] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 25; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0127] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 26; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0128] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 27; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0129] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0130] Some aspects relate to an engineered polypeptide comprising: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising the amino acid sequence of SEQ ID NO: 32; and (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, a peptide of (c) binds to a transferrin receptor (e.g., TfRl).

[0131] In some embodiments, an engineered polypeptide has a length of about 110-115 amino acids.

[0132] In some embodiments, an engineered polypeptide has a molecule weight of about 10- 20 kilodaltons (kDa).

[0133] In some embodiments, an engineered polypeptide comprises an amino acid sequence having at least 75% identity to the amino acid sequence of any one of SEQ ID NO: 4, 7, 10,

[0134] 15

[0135] #14369922vl Attorney Docket No. H0498.70819WO00

[0136] 11, 13, 14, or 33-49.

[0137] In some embodiments, an engineered polypeptide is linked directly to (e.g., fused to) or indirectly to a diagnostic agent.

[0138] In some embodiments, an engineered polypeptide is linked directly to (e.g., fused to) or indirectly to a therapeutic agent.

[0139] In some embodiments, an therapeutic agent is a therapeutic polypeptide, optionally a therapeutic recombinant protein selected from antibodies, cytokines, enzymes, and growth factors.

[0140] In some embodiments, a therapeutic agent is a therapeutic polynucleotide, for example, an antisense oligonucleotide, an RNA interference molecule (e.g., siRNA, shRNA, miRNA), a CRISPR RNA (crRNA), or a double stranded RNA (dsRNA).

[0141] In some embodiments, an engineered polypeptide is linked to (e.g., conjugated to) a nanoparticle, for example, a lipid nanoparticle (LNP), a polymer nanoparticle, a DNA nanoparticle, a magnetic nanoparticle, metal (e.g., gold or silver) nanoparticle, a liposome, or a nanoemulsion.

[0142] In some embodiments, an engineered polypeptide is linked to (e.g., conjugated to) a virus, e.g. an adeno-associated virus (AAV) or other virus.

[0143] In some embodiments, an engineered polypeptide is linked to (e.g., conjugated to or fused to) an imaging agent, for example, a radiopharmaceutical, a magnetic particle, or a light-emitting reporter (e.g., luciferase, green fluorescent protein (GFP), or near-infrared (IR) labels.

[0144] In some embodiments, an engineered polypeptide is linked to (e.g., conjugated to) a viral vector, for example, an adeno-associated virus (AAV) vector, a lentiviral vector.

[0145] Some aspects relate to a dual shuttle comprising any antibody (e.g., anti-TfR antibody) disclosed herein and a binding moiety (e.g., antibody) that binds to a second target (e.g., a second target capable of crossing the BBB).

[0146] Some aspects relate to a dual shuttle comprising an antibody comprising: a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR), and a binding moiety that binds to a second target (e.g., a second target capable of crossing the BBB). In some embodiments, the antibody further comprises: a complementary determining region 1 (CDR1) comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0147] 16

[0148] #14369922vl Attorney Docket No. H0498.70819WO00

[0149] Some aspects relate to a dual shuttle, comprising an antibody and a binding moiety, wherein the antibody comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 11, 13, 14, or 33-49. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 11, 13, 14, or 33-49.

[0150] In some embodiments, the binding moiety (e.g., antibody) enhances transport of the dual shuttle across the blood-brain barrier.

[0151] In some embodiments, the binding moiety (e.g., antibody) enhances retention of the dual shuttle in the brain parenchyma.

[0152] In some embodiments, the binding moiety (e.g., antibody) mediates receptor-mediated transcytosis across the blood-brain barrier.

[0153] In some embodiments, the binding moiety (e.g., antibody) binds to Basigin (CD 147), the glucose transporter 1 (Glutl or SLC2A1), the human insulin receptor (HIR), the insulinlike growth factor receptor- 1 (IGF1-R), low-density lipoprotein receptor family members (e.g., LDLR, LRP1, and LRP-8), CD98 heavy chain, leptin receptor, or a myelin oligodendrocyte glycoprotein (MOG) (e.g. MOG-1). In some embodiments, the binding moiety binds to CD98 heavy chain.

[0154] In some embodiments, the dual shuttle is linked directly to (e.g., fused to) or indirectly to a therapeutic agent.

[0155] BRIEF DESCRIPTION OF THE DRAWINGS

[0156] FIG. 1 provides exemplary configurations of fusion constructs comprising a single domain antibody (sdAb) shuttle with an IgG antibody (e.g., an IgG therapeutic antibody).

[0157] FIG. 2A provides a graph showing the ability of an exemplary anti-transferrin receptor 1 (anti-TfRl) antibody of the disclosure to bind to human TfRl (huTfR) and cynomolgus monkey TfRl (cyTfR).

[0158] FIG. 2B provides a graph showing the ability of an exemplary anti-TfRl antibody of the disclosure to bind to a human endothelial cell line (hCMEC / D3).

[0159] FIG. 2C provides a graph showing the surface and total binding of exemplary anti- TfRl antibodies to hCMEC / D3 cells, indicating that these antibodies are capable of being internalize into the cells.

[0160] FIG. 2D provides a graph showing that an exemplary anti-TfRl antibody does not compete with transferrin (Tf) for TfR binding.

[0161] FIG. 3 provides the binding affinities of an exemplary anti-TfRl antibody of the

[0162] 17

[0163] #14369922vl Attorney Docket No. H0498.70819WO00 disclosure for human TfRl and cynomolgus monkey TfRl.

[0164] FIGs. 4A-4B provide graphs showing that exemplary anti-TfRl antibodies of the disclosure are capable of increased uptake in the brain relative to an anti-BACEl antibody (FIG. 4A) and decreased residual concentrations in plasma (FIG. 4B).

[0165] FIGs. 4C-4D provide graphs showing brain parenchyma (FIG. 4C) and plasma (FIG. 4C) pharmacokinetics (PK) of an exemplary anti-TfRl antibody in a one- week dose-response and time-course study in mice.

[0166] FIGs. 5A-5B provide graphs showing that exemplary anti-TfRl antibodies of the disclosure exhibit significantly higher uptake in mouse brain tissue than control anti-HIV-1 and anti-BACEl antibodies.

[0167] FIG. 6 provides a Western blot showing that exemplary anti-TfRl antibodies of the disclosure do not deplete TfR expression in mouse brain tissues.

[0168] FIGs. 7A-7B provide graphs showing that a monovalent configuration of anti-TfRl antibodies (IgG-monoVHH) demonstrated increased uptake in the brain relative to a bivalent configuration of anti-TfRl antibodies (IgG-biVHH) (FIG. 7A) and similar residual concentrations in plasma (FIG. 7B).

[0169] FIGs. 8A-8B provide graphs showing the ability of exemplary anti-TfRl antibodies of the disclosure to bind to human TfRl (FIG. 8A) and cynomolgus monkey TfRl (FIG. 8B).

[0170] FIG. 9 provides the binding affinities of exemplary anti-TfRl antibodies of the disclosure for human TfRl and cynomolgus monkey TfRl.

[0171] FIGs. 10A-10B provide graphs showing that exemplary anti-TfRl antibodies of the disclosure are capable of increased uptake in the brain relative to trastuzumab (an anti-HER2 antibody) (FIG. 10A) and decreased residual concentrations in plasma (FIG. 10B).

[0172] FIGs. 10C-10D provide graphs showing brain parenchyma (FIG. 10C) and plasma (FIG. 10C) PK of exemplary anti-TfRl antibodies in a one-week dose-response and timecourse study in mice.

[0173] FIG. 11 shows that anti-TfRl antibodies of the disclosure provide broad antibody distribution in whole mouse brain regions (at 20 mg / kg dose administration via tail- vein intravenous injection). An equimolar dose of trastuzumab was used as negative control.

[0174] FIG. 12A shows concentrations of anti-TfRl antibodies of the disclosure in mouse spinal cords at day 1, 7 and 14 after a single dose injection of 20 mg / kg through tail- vein intravenous injection. An equimolar dose of trastuzumab was used as control.

[0175] FIG. 12B shows that an anti-TfR antibody of the disclosure colocalized with neuronal cells (marked by NeuN) in the ventral horn region of lumbar spinal cord (arrows, top panel)

[0176] 18

[0177] #14369922vl Attorney Docket No. H0498.70819WO00 while trastuzumab showed no uptake in the same region.

[0178] FIGs. 13A-13C show that additional humanized anti-TfR variants bind to human (FIG. 13A) and cynomolgus (FIG. 13B) TfR proteins immobilized on plates. FIG. 13C shows the data plotted in FIG. 13A and FIG. 13B.

[0179] FIGs. 14A-14C show brain uptake of dual versus single shuttles. The CD98hc / TfR dual shuttle exhibited a 2-fold or more higher Cmax and AUC than single anti-CD98hc or anti- TfR shuttles in parenchymal (FIG. 14A) and total (FIG. 14B) lysates. The dual shuttle serum clearance was also faster than either of the single shuttles (FIG. 14C).

[0180] DETAILED DESCRIPTION

[0181] Disclosed herein are antibodies that bind to (e.g., specifically or preferentially bind to) transferrin receptor 1 (TfRl). In some embodiments, these antibodies specifically or preferentially bind to TfRl with increased binding affinity relative to transferrin receptor 2 (TfR2). In some embodiments, these antibodies do not detectably bind to TfR2 (e.g., an anti- TfRl antibody of the disclosure does not bind to TfR2, or binding affinity of an anti-TfRl antibody of the disclosure to TfR2 is so low that it cannot be detected using convention detection methods). The anti-TfRl antibodies provided herein demonstrate a broad range of binding affinities to TfRl and subsequent cellular internalization of the antibodies upon binding to an extracellular domain of TfRl.

[0182] Antibodies binding to Transferrin Receptor 1 (TfRl)

[0183] The present disclosure provides antibodies that bind to transferrin receptor (e.g., transferrin receptor 1 (TfRl)) and are capable of targeting cells having transferrin receptor on their surface (e.g., brain cells, such as brain endothelial cells, expressing transferrin receptor). Antibodies that bind to transferrin receptor are referred to herein as anti-TfR antibodies, and antibodies that bind specifically or preferentially to transferrin receptor 1 are referred to herein as anti-TfRl antibodies. Anti-TfR antibodies, in some embodiments, can be fused to or conjugated to payload antibodies, which can bind specifically to a molecule other than a transferrin receptor. In some embodiments, a payload antibody is a therapeutic antibody or a diagnostic antibody. Payload antibodies are discussed in more detail elsewhere herein.

[0184] Transferrin receptors include internalizing cell surface receptors that transport transferrin across the cellular membrane and are involved in the regulation of intracellular iron levels. In some embodiments, an anti-TfR antibody binds to a TfR and is internalized by a cell (e.g., via receptor-mediated endocytosis), along with any molecule (e.g., a payload

[0185] 19

[0186] #14369922vl Attorney Docket No. H0498.70819WO00 antibody, such as a therapeutic or diagnostic payload antibody) to which the anti-TfR antibody is fused or conjugated. In some embodiments, an anti-TfR antibody (e.g., an anti- TfR 1 antibody) is an antibody that binds a transferrin receptor and is capable of transporting a therapeutic or diagnostic payload across the blood brain barrier into the brain. In some embodiments, an anti-TfR antibody (e.g., an anti-TfRl antibody) is an antibody that binds a transferrin receptor and is capable of transporting a therapeutic or diagnostic payload into brain microvascular endothelial cells.

[0187] An anti-TfR antibody (interchangeably used in plural form) includes an immunoglobulin molecule or fragment thereof capable of specific binding to a transferrin receptor (e.g., transferrin receptor 1) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule or fragment thereof. An “antibody” encompasses intact (i.e., full-length) polyclonal or monoclonal antibodies as well as antigenbinding fragments thereof (such as Fab, Fab', F(ab')2, Fv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies (e.g., heavy-chain variable domain (VHH) antibody or a single-chain variable fragment (scFv) antibody), multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An “antibody” includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0188] In some embodiments, an anti-TfR antibody is paired with a binding moiety to a second target that enhances BBB transport or retention in the brain parenchyma, in order to generate a dual shuttle. A dual shuttle is a bispecific construct designed to facilitate drug delivery across the blood-brain barrier (BBB). In some embodiments, a dual shuttle comprises an anti-TfR antibody linked to a binding moiety for a second target. The second

[0189] 20

[0190] #14369922vl Attorney Docket No. H0498.70819WO00 target, in some embodiments, is selected to enhance BBB transport or promote retention in the brain parenchyma — such as a molecule highly expressed on the luminal surface of brain microvascular endothelial cells. By engaging both TfR and the second target, the dual shuttle increases local BBB concentration, enhances TfR-mediated transcytosis, and in some embodiments, leverages receptor-mediated transcytosis via the second target as well.

[0191] Non-limiting examples of such second BBB receptors include Basigin (CD 147), the glucose transporter 1 (Glutl or SLC2A1), the human insulin receptor (HIR), the insulin-like growth factor receptor-1 (IGF1-R), low-density lipoprotein receptor family members (e.g., LDLR, LRP1, and LRP-8), CD98 heavy chain, and leptin receptor. In some embodiments, a dual shuttle comprises an anti-TfR antibody of the disclosure and an antibody that binds an insulin receptor. In some embodiments, a dual shuttle comprises an anti-TfR antibody of the disclosure and an antibody that binds an IGF1 receptor. In some embodiments, a dual shuttle comprises an anti-TfR antibody of the disclosure and an antibody that binds a low-density lipoprotein receptor family member. In some embodiments, a dual shuttle comprises an anti- TfR antibody of the disclosure and an antibody that binds a CD98 heavy chain. In some embodiments, a dual shuttle comprises an anti-TfR antibody of the disclosure and an antibody that binds a leptin receptor.

[0192] In other embodiments, an anti-TfR antibody is paired with a binding moiety to a second target that enhances retention of the dual shuttle fusion drug in the brain parenchyma, in order to increase brain exposure. A dual shuttle can bind to a transferrin receptor and another molecule such as, for example, another molecule that is highly expressed on neurons, microglia, astrocytes, pericytes, basal lamina or other components of the brain parenchyma, or any combination of these, such as myelin oligodendrocyte glycoprotein- 1 (MOG-1). In some embodiments, a dual shuttle comprises an anti-TfR antibody of the disclosure and an antibody that binds myelin oligodendrocyte glycoprotein- 1 (MOG-1).

[0193] An antibody molecule can comprise a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art,

[0194] 21

[0195] #14369922vl Attorney Docket No. H0498.70819WO00 for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art.

[0196] In some embodiments, anti-TfRl antibodies as described herein can bind and inhibit the activity of TfRl by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or greater). The apparent inhibition constant (Kiappor Ki,app), which provides a measure of inhibitor potency, is related to the concentration of inhibitor required to reduce enzyme activity and is not dependent on enzyme concentrations. The inhibitory activity of an anti-TfRl antibody described herein can be determined by routine methods known in the art.

[0197] The anti-TfRl antibodies described herein can be murine, rat, human, camelid (including from camels, alpacas, and llamas), or any other origin (including chimeric or humanized antibodies). Such anti-TfRl antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof).

[0198] Any of the anti-TfRl antibodies described herein can be monoclonal or polyclonal. A “monoclonal antibody” includes an antibody from (e.g., obtained from or derived from) a homogenous antibody population and a “polyclonal antibody” includes an antibody from (e.g., obtained from or derived from) a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

[0199] An anti-TfRl antibody described herein can be in any antibody form, including, but not limited to, single-chain antibodies (e.g., heavy-chain variable domain (VHH) antibody or a single-chain variable fragment (scFv) antibody), intact (e.g., full-length) antibodies, antigen-binding fragments thereof (such as Fab, F(ab'), F(ab')2, Fv), bi-specific antibodies, or nanobodies. In some embodiments, an antibody described herein is an Fc polypeptide (e.g., an Fc polypeptide dimer). In some embodiments, an anti-TfRl antibody described herein is an Fc-fusion polypeptide (e.g., an Fc dimer fused to a Fab).

[0200] Anti-TfRl antibodies of the disclosure, in some embodiments, are humanized. Humanized antibodies include forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin.

[0201] In some embodiments, humanized antibodies are heavy-chain variable domain (VHH) antibodies in which camelid- specific amino acids in the framework region(s) have been replaced with corresponding human- specific amino acids. A humanized VHH antibody has been changed such that immunogenicity upon administration of the VHH in human patients is reduced, minor or nonexistent. Humanizing a VHH antibody can comprise a step of

[0202] 22

[0203] #14369922vl Attorney Docket No. H0498.70819WO00 replacing one or more of the Camelidae amino acids by their human counterpart as found in the human consensus sequence, without that VHH losing its typical character (e.g., the humanization does not significantly affect the antigen binding capacity of the resulting VHH). In some embodiments, humanization of single domain antibodies (e.g., VHH antibodies) requires the introduction and mutagenesis of a limited number of amino acids in a single VHH chain. This is in contrast to humanization of scFv, Fab, (Fab)2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

[0204] Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and / or six), which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanization of antibodies can also involve affinity maturation.

[0205] In some embodiments, anti-TfRl antibodies have been deimmunized. Deimmunization involves scanning the primary amino acid sequence of an anti-TfRl antibody for known peptides that can be processed and presented by antigen presenting cells in a way that they can be recognized by helper T lymphocytes (T cell epitopes). Such recognition increases the likelihood of an immune response to the anti-TfR antibody upon administration to a subject, potentially resulting in anti-drug antibodies. Deimmunization may further involve selective replacement of some or all amino acids in one of more of the predicted T cell epitopes in the shuttle amino acid sequence with other amino acids not predicted to result in T cell epitopes. Optionally, as part of deimmunization, the parent and deimmunized clones may be tested in in vitro assays of T cell proliferation, or in vivo for anti-drug antibody formation.

[0206] Anti-TfRl antibodies of the disclosure, in some embodiments, are chimeric. Chimeric antibodies include antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and / or the constant region.

[0207] In some embodiments, the anti-TfRl antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater

[0208] 23

[0209] #14369922vl Attorney Docket No. H0498.70819WO00 affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. An antibody that specifically (or preferentially) binds to a TfRl antigen or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens or other epitopes in the same antigen. For example, an anti-TfRl antibody may specifically bind to transferrin receptor 1 relative to transferrin receptor 2 (TfR2). In other words, an anti-TfRl antibody may have a higher binding affinity for TfRl than its binding affinity for TfR2. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that “specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen. In some embodiments, the anti-TfRl antibodies described herein specifically binds human TfRl. In some examples, its binding activity to a non-human TFR1 antigen is not detectable in a conventional assay or is very low such that it would have no significant biological significance as known to those skilled in the art. In other examples, the anti-TfRl antibodies described herein may cross-react with TFR1 from different species, for example, between human TFR1 and a non-human TFR1 (e.g., TFR1 from an experimental animal such as a non-human primate, mouse, or rat).

[0210] In some embodiments, an anti-TfRl antibody specifically binds to an extracellular epitope of a transferrin receptor 1 or an epitope that becomes exposed to an antibody, including the apical domain, the transferrin binding domain, and / or the protease-like domain. In some embodiments, anti-TfRl antibodies as described herein can bind and inhibit the activity of transferrin receptors from multiple species. For example, the anti-TfRl antibodies as described herein can bind and inhibit the activity of human TfRl and cynomolgus (monkey) TfRl. In some embodiments, an anti-TfRl antibody specifically binds to an amino acid segment of a human and / or non-human primate TfRl. In some embodiments, an anti- TfRl antibody specifically binds with binding affinity of at least about 10'4M, 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, IO'10M, 10'11M, 10'12M, 10’13M, or less.

[0211] An example human TfRl amino acid sequence, corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:

[0212] 24

[0213] #14369922vl Attorney Docket No. H0498.70819WO00

[0214] MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKAN VTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGE DFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVE NQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAA TVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYM DQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAA EKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEP DHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWS AGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQ NVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGT TMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDL NQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMR VEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLAL ATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 15).

[0215] An example non-human primate TfRl amino acid sequence, corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKAN GTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEE DFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIEN QFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAAT VTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMD QTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAE KLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPD HYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSA GDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQD VKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTT MDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLN QYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMR VEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALA TWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 16).

[0216] TfRl molecules from other species were well known in the art and the amino acid sequences thereof can be retrieved from a publicly available database, for example, GenBank or NCBI.

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[0219] In some embodiments, an anti-TfRl antibody as described herein has a suitable binding affinity for a target antigen (e.g., human TfRl) or antigenic epitopes thereof. “Binding affinity” refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The anti-TfRl antibodies described herein may have a binding affinity (KD) of at least 10'5, 10'6, 10'7, 10'8, 10'9, IO0M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen. In such cases, an antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). In some embodiments, the anti-TfRl antibodies described herein have a higher binding affinity (a higher KA or smaller KD) to human TfRl as compared to the binding affinity to TfRl of a different species. Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 105fold. In some embodiments, any of the anti-TfRl antibodies can be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0220] Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v / v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:

[0221] [Bound] = [Free] / (KD+[Free])

[0222] It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to

[0223] 26

[0224] #14369922vl Attorney Docket No. H0498.70819WO00 obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

[0225] The anti-TfRl antibodies described herein may comprise a complementary determining region 1 (CDR1), a complementary determining region 2 (CDR2), and a complementary determining region 3 (CDR3). In some embodiments, a CDR1 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 5, 8, or 25-27. In some embodiments, a CDR1 comprises an amino acid sequence having at least 85%, 90%, or 95% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 5, 8, or 25-27. In some embodiments, a CDR2 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32. In some embodiments, a CDR2 comprises an amino acid sequence having at least 85%, 90%, or 95% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32. In some embodiments, a CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, a CDR3 comprises an amino acid sequence having at least 85%, 90%, or 95% identity to the amino acid sequence as set forth in SEQ ID NO: 3.

[0226] In some embodiments, an anti-TfRl antibody is a single-chain antibody (e.g., heavychain variable domain (VHH) antibody or a single-chain variable fragment (scFv) antibody). In some embodiments, an anti-TfRl antibody is a heavy-chain variable domain (VHH) antibody.

[0227] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4.

[0228] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 2 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0231] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4.

[0232] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

[0233] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 5 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

[0234] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10.

[0235] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 9 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0238] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10.

[0239] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11.

[0240] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11.

[0241] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13.

[0242] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 12 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0245] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13.

[0246] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14.

[0247] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 5 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 12 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14.

[0248] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 28, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33.

[0249] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 28 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0252] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33.

[0253] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34.

[0254] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 29 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34.

[0255] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35.

[0256] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 2 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0259] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35.

[0260] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36.

[0261] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 30 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36.

[0262] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 28, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37.

[0263] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 28 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0266] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37.

[0267] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38.

[0268] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 29 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38.

[0269] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 28, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39.

[0270] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 28 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0273] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39.

[0274] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40.

[0275] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40.

[0276] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41.

[0277] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0280] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41.

[0281] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42.

[0282] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 8 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42.

[0283] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 25, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43.

[0284] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 25 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0287] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43.

[0288] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 26, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44.

[0289] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 26 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44.

[0290] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 27, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45.

[0291] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 27 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0294] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45.

[0295] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46.

[0296] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46.

[0297] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 25, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47.

[0298] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 25 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 6 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0301] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47.

[0302] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 31, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48.

[0303] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 31 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48.

[0304] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 32, and a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, an anti-TfRl antibody comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, an anti-TfRl antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49.

[0305] In some embodiments, an anti-TfRl antibody comprises a CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, an anti-TfRl antibody comprises a CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 32 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, an anti-TfRl antibody comprises a CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:

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[0308] 3 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49.

[0309] An anti-TfRl antibody comprises any one of the amino acid sequences described in Table 1. In some embodiments, an anti-TfRl antibody of the present disclosure includes one or more of the CDR amino acid sequences from any one of the anti-TfRl antibodies selected from Table 1. In some embodiments, transferrin receptor antibodies include the CDR1, CDR2, and CDR3 as provided for any one of the anti-TfRl antibodies selected from Table 1. In some embodiments, transferrin receptor antibodies include the CDR3 as provided for each of the anti-TfRl antibodies selected from Table 1 (i.e., CDR3 comprising the amino acid sequence of SEQ ID NO: 3). In some embodiments, antibody heavy chain CDR3 domains may play a particularly important role in the binding specificity / affinity of an antibody for an antigen. Accordingly, anti-TfRl antibodies of the disclosure may include at least the heavy chain CDR3s of any one of the anti-TfRl antibodies selected from Table 1. Table 1. Exemplary Anti-TfRl antibody sequences

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[0320] Also within the scope of the present disclosure are functional variants of any of the reference anti-TfRl antibodies as disclosed herein. In some embodiments, a functional variant can comprise up to 5 (e.g., 4, 3, 2, 1, or 0) amino acid residue variations per CDR in one or more of the heavy chain and light chain CDR regions of the reference antibody and binds the same epitope of the TfRl antigen with substantially similar affinity (e.g., having a KD value in the same order). In some embodiments, a functional variant can comprise amino acid residue variations in one or more of the heavy chain and light chain CDR regions of the reference antibody and retains brain uptake (e.g., within 5-fold that of the reference antibody, between 20% and 500% of the brain uptake of the reference antibody). In some instances, each of the heavy chain and / or light chain CDRs in a functional variant contains no more than 2 amino acid residue variations as relative to the counterpart CDRs in the reference antibody. In some examples, each of the heavy chain and / or light chain CDRs in a functional variant contains no more than 1 amino acid residue variations as relative to the counterpart CDRs in the reference antibody.

[0321] In some embodiments, the amino acid residue variations are conservative amino acid residue substitutions. A “conservative amino acid substitution” includes an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art Conservative substitutions of amino acids include substitutions made amongst amino acids within the

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[0323] #14369922vl Attorney Docket No. H0498.70819WO00 following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0324] In some embodiments, an anti-TfRl antibody comprises heavy chain CDRs that, collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs of a reference antibody, and / or light chain CDRs that, collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain CDRs of the reference antibody. In some embodiments, an anti-TfRl antibody comprises a heavy chain variable region (VH) that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain variable region of any of the reference antibody and / or a light chain variable region (VL) that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain variable region of the reference antibody.

[0325] The “percent identity” of two amino acid sequences is determined, in some embodiments, using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873- 77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0326] In some embodiments, the percent identity of two amino acid sequences is performed by aligning the two amino acid sequences and performing a distance measurement between the aligned amino acid sequences. In some embodiments, a distance measurement is a Levenshtein distance or a Hamming distance.

[0327] Percent identity can be determined using a global alignment (e.g., an alignment across the entire length of two proteins). Lor example, a global alignment can be used to determine the percent identity between an anti-TfRl antibody and a reference antibody (e.g., comprising the amino acid sequence of SEQ ID NO: 4). Percent identity can also be determined using a local alignment (e.g., an alignment across a set length of amino acids). Lor example, a local alignment can be used to determine the percent identity between a CDR of an anti-TfRl antibody and a corresponding CDR of a reference antibody (e.g., a CDR3, e.g., comprising the amino acid sequence of SEQ ID NO: 3).

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[0330] The present disclosure also provides germline variants of any of the reference anti- TfRl antibodies disclosed herein. A germline variant contains one or more mutations in the framework regions as relative to its parent antibody towards the corresponding germline sequence. In some embodiments, an anti-TfRl antibody is encoded by a nucleic acid having at least 70%, 80%, 85%, 90%, or 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 19-24. In some embodiments, an anti-TfRl antibody is encoded by a nucleic acid having at least 70%, 80%, 85%, 90%, or 95% identity to the nucleic acid sequence of any one of the sequences provided in Table 2.

[0331] Table 2. Nucleic acid sequences encoding exemplary anti-TfRl antibodies

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[0333] Also provided herein are engineered polypeptides comprising a peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein the peptide binds to a transferrin receptor (TfR). In some embodiments, these engineered polypeptides further comprise a peptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25- 27. In some embodiments, these engineered polypeptides further comprise a peptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0334] In some embodiments, an engineered polypeptide comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 3; a second peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27; and a third peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0335] In some embodiments, an engineered polypeptide comprises a length of 100-150 amino acids, 100-140 amino acids, 100-130 amino acids, 110-150 amino acids, 110-130 amino acids, 100-120 amino acids, 110-120 amino acids, or 110-115 amino acids.

[0336] In some embodiments, an engineered polypeptide comprises a molecule weight of about 10-20 kilodaltons (kDa), about 10-30 kDa, about 10-25 kDa, about 10-15 kDa, about 15-20 kDa, or about 15-30 kDa.

[0337] The anti-TfRl antibodies of the disclosure can be prepared by any one or more method known to one of skill in the art. Generally, antibody preparation is tailored to the

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[0339] #14369922vl Attorney Docket No. H0498.70819WO00 specific type and application of the antibody. One common approach is hybridoma technology, which entails the fusion of a specific antibody-producing B cell with a myeloma cell to create a hybrid cell line capable of continuous antibody production. Polyclonal antibodies can be produced by immunizing an animal, such as a rabbit or goat, with the target antigen and subsequently collecting and purifying the serum containing the antibodies. Monoclonal antibodies, on the other hand, are typically generated using recombinant DNA technology, where the gene encoding the desired antibody is inserted into an expression vector and introduced into a host cell line for large-scale production. Another method is phage display, which involves the expression of antibody fragments on the surface of bacteriophages, allowing for the selection and amplification of high-affinity antibodies.

[0340] In some embodiments, single-domain VHH antibodies (e.g., anti-TfRl VHH antibodies) are obtained from llamas (e.g., llamas immunized with a mixture of human and mouse TfRl antigens). Animals can be, in certain embodiments, immunized with multiple (e.g., six) weekly subcutaneous injections of human and mouse TfRl recombinant proteins (e.g., extracellular domains of TfRl). Additionally, animals may receive boost injections of cell lines transiently overexpressing human or mouse TfRl (HEK293 cell background) with a 2-week interval between injections. Following the final protein or the final cell boost, blood samples can be collected and PBMCs isolated. RNA may then be then extracted and stored. RNA can be converted to cDNA using reverse transcriptase and random primers. The VHH encoding gene fragments may then be amplified using multiple primers and cloned into a phagemid vector, resulting in the generation of two phage display libraries per llama. Specific antibody fragments are then selected via phage display from the generated libraries, using previously biotinylated recombinant human and mouse TfRl recombinant proteins as bait. The biotinylated antigens can be incubated with phage at various concentrations over up to two rounds of panning. One final phage display selection round on cell lines transiently overexpressing human or mouse TfRl (HEK293 cell background) may also be performed, for most selection strategies used. E. coli bacteria may then be infected with output phage after each selection round to use for subsequent selections or screening by binding of individual clones.

[0341] Some aspects of the application provide nucleic acids encoding an anti-TfR antibody (e.g., anti-TfRl antibody) described herein. Any one of the chains or domains of an anti-TfR antibody can be encoded on one or multiple nucleic acids. The nucleic acids encoding chains or domains of an anti-TfR antibody (or a full-length anti-TfR antibody) can be in operable linkage with (e.g., operably linked to) one or more promoters. In some embodiments, an 48

[0342] #14369922vl Attorney Docket No. H0498.70819WO00 internal ribosomal entry site (IRES) can be inserted between the nucleic acid(s) encoding an anti-TfR antibody and a promoter.

[0343] Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors / promoter would depend on the type of host cells for use in producing the antibodies.

[0344] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

[0345] Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP 16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0346] Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR- VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetRmammalian cell transcription factor fusion derivatives can function as potent trans-modulator

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[0348] #14369922vl Attorney Docket No. H0498.70819WO00 to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shocked et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

[0349] Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0350] Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

[0351] One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of an antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of an antibody can be incubated under suitable conditions for a suitable period of time allowing for production of an antibody.

[0352] In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti-TfRl antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form an antibody, which can be recovered from the cells or from the culture medium.

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[0355] When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of an antibody.

[0356] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-TfRl antibody and the other encoding the light chain of an anti-TfRl antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of an antibody. When the two expression vectors are introduced into the same host cells, an antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of an antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of an antibody.

[0357] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

[0358] Any of the nucleic acids encoding the heavy chain, the light chain, a single chain, a single domain of an anti-TfRl antibody as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.

[0359] Payload Molecules

[0360] An anti-TfR antibody of the disclosure can be fused to or conjugated to a payload molecule. A payload molecule can be a payload antibody, a payload protein, a therapeutic agent (e.g., a therapeutic protein, enzyme, small molecule, etc.), or a diagnostic agent. In some embodiments, a payload antibody is a therapeutic antibody. In some embodiments, a pay load antibody is a diagnostic antibody (e.g., used for imaging).

[0361] In some embodiments, a payload antibody is a therapeutic antibody that targets a cancer antigen. In some embodiments, a payload antibody is a therapeutic antibody that binds (e.g., specifically binds) to a cancer antigen. In some embodiments, a payload antibody is an 51

[0362] #14369922vl Attorney Docket No. H0498.70819WO00 immune checkpoint inhibitor antibody. In some embodiments, a therapeutic antibody is an antibody that binds to cytotoxic T lymphocyte associated protein 4 (CTLA-4). In some embodiments, a therapeutic antibody is an antibody that binds to programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1).

[0363] In some embodiments, a therapeutic antibody is an antibody that binds to human epidermal growth factor receptor 2 (HER2). An anti-HER2 pay load antibody, in some embodiments, is trastuzumab. An anti-HER2 antibody (trastuzumab) comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, a payload antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, a payload antibody comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, a pay load antibody comprises a GGGGS (SEQ ID NO: 50) linker. In some embodiments, a payload antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17 linked to a VL domain comprising the amino acid sequence of SEQ ID NO: 18 via a GGGGS (SEQ ID NO: 50) linker.

[0364] Table 3. Anti-HER2 antibody sequences

[0365] A therapeutic agent can be a biomolecule or a small molecule drug, for example. In some embodiments, a therapeutic agent is a biomolecule. A biomolecule includes an organic molecule that can by produced by a living cell. Although, biomolecules used for therapeutic and diagnostic applications can be obtained from a living cell, synthesized in vitro, or recombinantly engineered, for example. Non-limiting examples of biomolecules that can be used as therapeutic agents include polynucleotides, polypeptides, lipids, and carbohydrates. In some embodiments, a therapeutic agent is a polynucleotide. Non-limiting examples of polynucleotides include double-stranded DNA or RNA, and single-stranded DNA or RNA. Single-stranded RNA interference molecules, such as antisense RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA) can be used in some embodiments. In some embodiments, a single stranded RNA is a messenger RNA (mRNA).

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[0368] In some embodiments, a single stranded RNA is a guide RNA (gRNA) (e.g., used in combination with a naturally-occurring or engineered Cas enzyme). A gRNA can be made up of two parts crispr RNA (crRNA) and tracr RNA. In some embodiments, a crRNA is used as a therapeutic agent. In some embodiments, a therapeutic agent is a small molecule drug. A small molecule drug includes a drug (e.g., organic compound) that has a molecular weight of less than 10 kilodaltons (kDa).

[0369] Other therapeutic agents are also contemplated herein. Non-limiting examples of other therapeutic agents include toxins, chemotherapeutic agents, antibiotics, ADP-ribosyl transferase, radioactive isotopes and nucleolytic enzymes. In some embodiments, a therapeutic agent is a cytotoxic agent. Examples of cytotoxic agents include, but are not limited to, anthracycline, an auristatin, a camptothecin, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and calicheamicin.

[0370] A diagnostic agent is an agent used in the process of diagnosing or otherwise evaluating a disease or condition in a subject (e.g., a human or non-human subject). In some embodiments, the diagnostic agent is an imaging agent. An imaging agent is an agent that emits signal directly or indirectly thereby enabling its detection in vitro or in vivo. Imaging agents, such as contrast agents and radioactive agents, can be detected using medical imaging techniques such as nuclear medicine scans and magnetic resonance imaging (MRI). Imaging agents for magnetic resonance imaging (MRI) include Gd(DOTA), for example, iron oxide or gold nanoparticles; imaging agents for nuclear medicine include, for example, 201 Tl, gamma-emitting radionuclide 99 mTc; imaging agents for positron-emission tomography (PET) include, for example, positron-emitting isotopes, (18)F-fluorodeoxy glucose ((18)FDG), (18)F-fluoride, copper-64, gadoamide, and radioisotopes of Pb(II) such as 203Pb, and 1 lln; imaging agents for fluorescence imaging include, for example, fluorescent dyes or dye-conjugated nanoparticles. In some embodiments, an imaging agent is conjugated to, fused to, or combined with another agent, such as a therapeutic agent. In some embodiments, the imaging agent comprises a radiopharmaceutical, a magnetic particle, and / or a lightemitting reporter (e.g., luciferase, green fluorescent protein (GFP), enhanced GFP (EGFP), or near-infrared (IR) labels). Imaging agents may also be used for non-diagnostic purposes.

[0371] Fusion Proteins Comprising Anti-TfR Antibodies

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[0373] #14369922vl Attorney Docket No. H0498.70819WO00

[0374] The present disclosure also provides fusion proteins comprising any one of the anti- TfR antibodies described herein. Fusion proteins include single, contiguous polypeptide chains produced by combining the DNA of two or more polypeptides (e.g., proteins). Fusion proteins, in some embodiments, include proteins linked by peptide bonds. In some embodiments, an anti-TfRl fusion protein comprises an anti-TfRl antibody fused to a heavy chain or light chain of a payload antibody or other therapeutic protein. In some embodiments, an anti-TfRl fusion protein comprises an anti-TfRl antibody fused to a pay load antibody. A therapeutic fusion protein may comprise, for example, a heavy chain comprising a heavy chain constant domain and a heavy chain variable domain, a light chain comprising a light chain constant domain and a light chain variable domain, and at least one anti-TfRl antibody.

[0375] A fusion protein can comprise, for example, a heavy chain comprising a constant domain and a variable domain, and / or a light chain comprising a constant domain and a variable domain, e.g., wherein the heavy chain and / or the light chain do not bind to TfRl. In some embodiments, a fusion protein is as described in FIG. 1. FIG. 1 shows that a singledomain antibody (e.g., an anti-TfRl single-chain antibody) can be fused to a variety of different non-TfRl antibody components (e.g., Therapeutic IgG antibody). In some embodiments, a fusion protein comprises an anti-TfRl single-chain antibody fused to the constant domain of a therapeutic antibody (e.g., heavy chain constant domain or light chain constant domain). In some embodiments, a fusion protein comprises an anti-TfRl singlechain antibody fused to the variable domain of a therapeutic antibody (e.g., heavy chain variable domain or light chain variable domain).

[0376] In some embodiments, a fusion protein comprises two anti-TfRl single-chain antibodies linked to the constant domain of a therapeutic antibody (e.g., heavy chain constant domain or light chain constant domain). In some embodiments, a fusion protein comprises two anti-TfRl single-chain antibodies, each of which is linked to the heavy chain constant domain. In some embodiments, a fusion protein comprises two anti-TfRl single-chain antibodies, each of which is linked to the light chain constant domain.

[0377] In some embodiments, a fusion protein comprises two anti-TfRl single-chain antibodies linked to the variable domain of a therapeutic antibody (e.g., heavy chain variable domain or light chain variable domain). In some embodiments, a fusion protein comprises two anti-TfRl single-chain antibodies, each of which is linked to the heavy chain variable domain. In some embodiments, a fusion protein comprises two anti-TfRl single-chain antibodies, each of which is linked to the light chain variable domain.

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[0380] Fusion proteins, in some embodiments, comprise a cell permeability (CP) moiety. Cell permeability moieties include a chemical group or molecule that enhances the ability of a compound (e.g., a fusion protein or antibody) to cross cellular membranes. These moieties are often attached to drugs, peptides, or other biomolecules to improve their delivery into cells, especially when the primary compound has poor membrane permeability. Non-limiting examples of CP moieties include peptide-based moieties (e.g., TAT peptide, penetratin, or poly arginine), lipophilic moieties (e.g., alkyl chains or cholesterol), small molecule moieties (e.g., arginine-rich molecules or guanidine-containing compounds), certain prodrugs strategies, and cationic polymers (e.g., polylysine or polyethyleneimine). In some embodiments, the CP moiety is a peptide comprising the following sequence or a variant thereof that retains CP properties: 3E10 scFv DIQMTQSPSSLSASVGDRVTISCRASKSVSTSSYSYMHWYQQKPEKAPKLLIKYASYL QSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHSREFPWTFGAGTKLELKGGGGS GGGGSGGGGSEVQLQESGGGVVQPGGSLRLSCAASGFTFSNYGMHWIRQAPGKGLE WVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARRGLLL DYWGQGTLVTVSS (SEQ ID NO: 51)

[0381] Conjugates Comprising Anti-TfR Antibodies

[0382] The present disclosure also provides conjugates comprising any one of the anti-TfR antibodies described herein and one or more additional agents (e.g., therapeutic agents and / or diagnostic agents). Conjugates comprising an anti-TfR antibody can be formed by (a) chemically or enzymatically linking the anti-TfR antibody to another molecule (e.g., a therapeutic or diagnostic agent); (b) by chemically or enzymatically linking the anti-TfR antibody to one or more polypeptides (e.g., proteins); or (c) chemically or enzymatically linking a fusion protein comprising the anti-TfR antibody to another molecule (e.g., a therapeutic or diagnostic agent (e.g., after the anti-TfR antibody or fusion protein has been produced). In some embodiments, a conjugate comprises an anti-TfR (e.g., TfRl) antibody linked to a payload antibody. In some embodiments, an anti-TfRl antibody is directly linked to the payload antibody. In some embodiments, a conjugate comprises an anti-TfR (e.g., TfRl) antibody linked to one or more therapeutic agents. In some embodiments, an anti-TfRl antibody is directly linked to the one or more therapeutic agents. In some embodiments, the TfRl antibody is indirectly attached to the one or more therapeutic agents. In some

[0383] 55

[0384] #14369922vl Attorney Docket No. H0498.70819WO00 embodiments, a conjugate comprises (a) a fusion protein comprising an anti-TfR (e.g., TfRl) antibody, that is conjugated to (b) one or more therapeutic or diagnostic agents. In some embodiments, a conjugate comprises an anti-TfR (e.g., TfRl) antibody linked to one or more diagnostic agents. In some embodiments, an anti-TfRl antibody is directly linked to the one or more diagnostic agents. In some embodiments, the TfRl antibody is indirectly attached to the one or more diagnostic agents.

[0385] The linkage in protein conjugates can be through different types of chemical bonds (e.g., amide bonds, disulfide bonds, or through click chemistry). In some embodiments, an anti-TfRl antibody is directly (e.g., covalently) linked to the one or more additional agents. In some embodiments, the TfRl antibody is indirectly (e.g., non-covalently) linked to the one or more additional agents. In some embodiments, the conjugate functions to increase therapeutic effects by delivering the components of therapeutic agent to a TfRl+cell, which is targeted by an antibody, for example, a TfRl+brain cell.

[0386] In some embodiments, a conjugate comprises at least one anti-TfRl single-chain antibody linked to a polypeptide selected from: Fc-containing antibodies, Ab fragments, Fc fusion proteins, Fc domains; and non-antibody proteins (optionally wherein the polypeptide is then conjugated to a therapeutic or diagnostic agent). For other exemplary antibody formats, see website: absoluteantibody.com / periodic-table-of-antibodies (accessed December 8, 2022), incorporated herein by reference.

[0387] In some embodiments, an anti-TfRl antibody and an additional agent (e.g., a payload antibody, polypeptide such as an Fc region, therapeutic agent and / or diagnostic agent) are connected to each other via a linker. Such a linker can be a cleavable linker, for example, cleavable under a certain pH condition (a pH-sensitive linker), cleavable by a protease (a protease-sensitive linker), or cleavable in the presence of glutathione (a glutathione- sensitive linker). In some examples, the linker comprises a protease cleavage site, which may contain 2-5 amino acid residues that are recognizable and / or cleavable by a suitable protease. Such a peptide may comprise naturally-occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof. In one example, the peptide linker can be a dipeptide linker. Examples include a valine-citrulline (val-cit) linker, a phenylalanine-lysine (phe-lys) linker, or maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (vc) linker. In some embodiments, the peptide linker comprises or consists of glycine and serine residues. In some embodiments, the peptide linker comprises a -GGGGS (SEQ ID NO: 50)-; -GSS-; -GGS-; or -GSG- sequence (wherein G is the one-letter abbreviation for glycine; and

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[0390] S is the one-letter abbreviation for serine). Alternatively, the linker can be non-cleavable, e.g., a linker comprising optionally substituted alkane or thioether.

[0391] In some examples, the linker may comprise a functional group that can form a covalent bond with an antibody. Exemplary functional groups include, but are not limited to, a maleimide group, an iodoacetamide group, a vinyl sulfone group, an acrylate group, an acrylamide group, an acrylonitrile group, or a methacrylate group. In some instances, the linker can contain one or more reactive amines include, but are not limited to, acetyl-lysine- valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC) or amino PEG6-propionyl. See, e.g., WO2012 / 059882. Other exemplary linkers include Sulfosuccinimidyl-4- [Nmaleimidomethyl] cyclohexane- 1 -carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, — SH), while its Sulfo-NHS ester is reactive toward primary amines (as found in Lysine and the protein or peptide N terminus).

[0392] Methods for conjugating therapeutic agents or other agents (e.g., imaging molecules, cytotoxic agents, biomolecules, polynucleotides, or small molecules) to antibodies or fusion proteins (e.g., fusion proteins comprising an antibody and an Fc region) were known in the art and have been described in various publications. For example, chemical modification can be made in the antibodies or fusion proteins either through lysine side chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds for the conjugation reaction to occur. See, e.g., Tanaka et al., FEBS Letters 579:2092-2096, (2005), and Gentle et al., Bioconjug. Chem. 15:658-663, (2004). Reactive cysteine residues engineered at specific sites of antibodies for specific drug conjugation with defined stoichiometry have also been described. See, e.g., Junutula et al., Nature Biotechnology, 26:925-932, (2008). Conjugation using an acyl donor glutamine-containing tag and / or an endogenous glutamine made reactive by polypeptide engineering in the presence of transglutaminase and an amine, for example, a cytotoxic agent modified with a reactive amine, is also described in WO2012 / 059882, Strop et al., Chem. Biol. 20(2): 161- 167 (2013), and Farias et al., Bioconjug. Chem. 25(2):245-250 (2014). The relevant disclosures of such publications are herein incorporated by reference for the purpose and subject matter referenced therein.

[0393] Pharmaceutical Compositions and Kits

[0394] The anti-TfRl antibodies, the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, as described herein, as well as the conjugates (e.g., therapeutic conjugates) or fusion proteins comprising the anti-TfRl

[0395] 57

[0396] #14369922vl Attorney Docket No. H0498.70819WO00 antibodies can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art.

[0397] The present disclosure also provides kits for use in targeting TfRl+disease cells. Such kits can include one or more containers comprising an anti-TfRl antibody, a conjugate, a fusion protein, or pharmaceutical composition e.g., any of those described herein. In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of a therapeutic conjugate comprising an anti-TfRl antibody or an anti-TfRl antibody itself to treat, delay the onset, or alleviate a target disease. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease. In still other embodiments, the instructions comprise a description of administering an antibody or a therapeutic conjugate to an individual at risk of the target disease.

[0398] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.

[0399] Also provided herein are kits for use in detecting TfRl+cells in a sample. Such a kit may comprise any one of the anti-TfRl antibodies described herein. In some instances, an anti-TfRl antibody can be conjugated with a detectable label or a diagnostic agent as those described herein. Conjugation between the two entities can be either direct (no linker) or indirect via a linker, such as a polymer linker. Conjugation can include covalent or noncovalent bonding as well as other forms of association, such as entrapment, e.g., of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.

[0400] Methods of Administration

[0401] Some aspects of the disclosure provide methods of administering an anti-TfRl antibody, a conjugate (e.g., a therapeutic conjugate), a fusion protein, or a pharmaceutical composition as described herein to a subject. In some embodiments, such methods result in 58

[0402] #14369922vl Attorney Docket No. H0498.70819WO00 therapeutic benefit to the subject. In some embodiments, such methods result in delivering of an antibody across the blood-brain barrier (e.g., to deliver an antibody to brain tissue and cells). In some embodiments, methods of administering an anti-TfRl antibody, a conjugate (e.g., a therapeutic conjugate), fusion protein, or a pharmaceutical composition to a subject results in targeting the antibody, conjugate, fusion protein, or pharmaceutical composition to the brain. In some embodiments, methods of administration involve the delivery of the anti- TfRl antibody across the blood-brain barrier. The use of a therapeutic conjugate comprising an anti-TfRl antibody of the disclosure can enable delivery of a therapeutic agent across the blood-brain barrier. In some embodiments, the therapeutic agent itself may be incapable of successfully transporting across the blood-brain barrier as a standalone molecule (e.g., without the assistance of the anti-TfRl antibody). In such embodiments, the anti-TfRl antibody functions as a shuttle to transport the therapeutic agent across the blood-brain barrier and to brain parenchyma, brain tissue, and brain cells (e.g., brain endothelial cells, neurons, microglia).

[0403] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment can be a human patient having, at risk for, or suspected of having a target disease / disorder associated with TfRl+disease cells. In some embodiments, the TfRl+disease cells are diseased brain cells, for example, diseased neurons. A subject having a target disease or disorder can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. A subject suspected of having any of such target disease / disorder might show one or more symptoms of the disease / disorder. A subject at risk for the disease / disorder can be a subject having one or more of the risk factors for that disease / disorder.

[0404] To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route. An “effective amount” includes the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is reduced TfRl activity or the activity of TfRl+cells. Determination of whether an amount of an antibody or other therapeutic agents comprising such (e.g., therapeutic conjugates) achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the

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[0406] #14369922vl Attorney Docket No. H0498.70819WO00 severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.

[0407] “Treating” includes the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results.

[0408] Additional Embodiments

[0409] Additional embodiments are provided in the following numbered paragraphs:

[0410] 1. An antibody comprising a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR).

[0411] 2. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2.

[0412] 3. The antibody of paragraph 2, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4.

[0413] 4. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0414] 5. The antibody of paragraph 4, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

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[0417] 6. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 9.

[0418] 7. The antibody of paragraph 6, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10.

[0419] 8. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0420] 9. The antibody of paragraph 8, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11.

[0421] 10. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12.

[0422] 11. The antibody of paragraph 10, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13.

[0423] 12. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12.

[0424] 13. The antibody of paragraph 12, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14.

[0425] 14. The antibody of paragraph 1 further comprising:

[0426] 61

[0427] #14369922vl Attorney Docket No. H0498.70819WO00 a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28.

[0428] 15. The antibody of paragraph 14, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33.

[0429] 16. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 29.

[0430] 17. The antibody of paragraph 16, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34.

[0431] 18. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2.

[0432] 19. The antibody of paragraph 18, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35.

[0433] 20. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 30.

[0434] 21. The antibody of paragraph 20, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36.

[0435] 22. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or

[0436] 62

[0437] #14369922vl Attorney Docket No. H0498.70819WO00 a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28.

[0438] 23. The antibody of paragraph 22, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37.

[0439] 24. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 29.

[0440] 25. The antibody of paragraph 24, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38.

[0441] 26. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 28.

[0442] 27. The antibody of paragraph 26, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39.

[0443] 28. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0444] 29. The antibody of paragraph 28, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40.

[0445] 30. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

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[0448] 31. The antibody of paragraph 30, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41.

[0449] 32. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0450] 33. The antibody of paragraph 32, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42.

[0451] 34. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 25; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0452] 35. The antibody of paragraph 34, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43.

[0453] 36. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 26; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0454] 37. The antibody of paragraph 36, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44.

[0455] 38. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 27; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

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[0458] 39. The antibody of paragraph 38, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45.

[0459] 40. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0460] 41. The antibody of paragraph 40, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46.

[0461] 42. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 25; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

[0462] 43. The antibody of paragraph 42, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47.

[0463] 44. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 31.

[0464] 45. The antibody of paragraph 44, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48.

[0465] 46. The antibody of paragraph 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 32.

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[0468] 47. The antibody of paragraph 46, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49.

[0469] 48. An antibody comprising a complementary determining region 3 (CDR3) having at least 70% identity to the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR).

[0470] 49. The antibody of paragraph 48, wherein the CDR3 has at least 80%, 90%, 95%, or 98% identity to the amino acid sequence of SEQ ID NO: 3.

[0471] 50. The antibody of paragraph 48 or 49 further comprising a complementary determining region 1 (CDR1) having at least 70% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27.

[0472] 51. The antibody of paragraph 50, wherein the CDR1 has at least 80%, 90%, 95%, or 98% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27.

[0473] 52. The antibody of any one of paragraphs 48-51 further comprising a complementary determining region 2 (CDR2) having at least 70% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0474] 53. The antibody of paragraph 52, wherein the CDR2 has at least 80%, 90%, 95%, or 98% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0475] 54. The antibody of any one of the preceding paragraphs, wherein the equilibrium dissociation constant (KD) of binding of the antibody to the TfR is in a range from IO0M to 10'5M.

[0476] 55. The antibody of any one of the preceding paragraphs, wherein the equilibrium dissociation constant (KD) of binding of the antibody to the TfR is in a range from 0.5 nM to 5000 nM.

[0477] 56. The antibody of any one of the preceding paragraphs, wherein the antibody binds human TfR and a non-human TfR, optionally wherein the antibody selectively binds to human TfR relative to a non-human TfR.

[0478] 57. The antibody of any one of the preceding paragraphs, wherein the TfR is a transferrin receptor 1 (TfRl).

[0479] 58. The antibody of any one of the preceding paragraphs, wherein the antibody is internalized into a cell, optionally wherein the cell is a brain endothelial cell.

[0480] 59. The antibody of any one of the preceding paragraphs, wherein the antibody is a single chain antibody.

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[0483] 60. The antibody of paragraph 59, wherein the single chain antibody is a heavy-chain variable domain (VHH) antibody or a single-chain variable fragment (scFv) antibody.

[0484] 61. The antibody of any one of paragraphs 1-58, wherein the antibody is a Fc-containing antibody, antibody fragment, or Fc-fusion protein.

[0485] 62. The antibody of paragraph 61, wherein the Fc-containing antibody or Fc-fusion protein comprises one or more mutations, optionally knob-in-hole mutations, effector function mutation or FcRN mutations.

[0486] 63. The antibody of any one of the preceding paragraphs, wherein the antibody is a humanized antibody and / or a chimeric antibody.

[0487] 64. The antibody of any one of the preceding paragraphs, wherein the antibody is a deimmunized antibody.

[0488] 65. A cell comprising the antibody of any one of the preceding paragraphs, optionally wherein the cell is a brain endothelial cell.

[0489] 66. A conjugate comprising the antibody of any one of the preceding paragraphs.

[0490] 67. The conjugate of paragraph 66, wherein the antibody is linked directly to (e.g., fused to) or indirectly to a diagnostic agent.

[0491] 68. The conjugate of paragraph 66, wherein the antibody is linked directly to (e.g., fused to) or indirectly to a therapeutic agent.

[0492] 69. The conjugate of paragraph 66, wherein the antibody is fused to a polypeptide that is conjugated to a therapeutic agent.

[0493] 70. The conjugate of paragraph 69, wherein the antibody is fused to a second antibody that is conjugated to a therapeutic agent.

[0494] 71. The conjugate of paragraph 70, wherein the second antibody is an antibody heavy chain Fc region.

[0495] 72. The conjugate of paragraph 69, wherein the therapeutic agent is a therapeutic polypeptide, optionally a therapeutic recombinant protein selected from antibodies, cytokines, enzymes, and growth factors.

[0496] 73. The conjugate of paragraph 69 or 70, wherein the therapeutic agent is a therapeutic polynucleotide, optionally an antisense oligonucleotide, a RNA interference molecule (e.g., siRNA, shRNA, miRNA), a CRISPR RNA (crRNA), or a double stranded RNA (dsRNA).

[0497] 74. The conjugate of any one of paragraphs 66-73, wherein the antibody is linked to (e.g., fused to) a nanoparticle, optionally a lipid nanoparticle (LNP), a polymer nanoparticle, a DNA nanoparticle, a magnetic nanoparticle, metal (e.g., gold or silver) nanoparticle, a liposome, or a nanoemulsion.

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[0500] 75. The conjugate of any one of paragraphs 66-73, wherein the antibody is linked to (e.g., fused to) an imaging agent, optionally a radiopharmaceutical, a magnetic particle, or a lightemitting reporter e.g., luciferase, green fluorescent protein (GFP), or near- infrared (IR) labels).

[0501] 76. The conjugate of any one of paragraphs 66-73, wherein the antibody is linked to (e.g., fused to) a viral vector, optionally wherein the viral vector is an adeno-associated virus (AAV) vector, a lentiviral vector.

[0502] 77. A therapeutic fusion protein comprising: a heavy chain comprising a heavy chain constant domain and a heavy chain variable domain; a light chain comprising a light chain constant domain and a light chain variable domain; and at least one antibody of any one of paragraphs 1-64.

[0503] 78. The therapeutic fusion protein of paragraph 77, wherein the at least one antibody is linked to the heavy chain constant domain or the heavy chain variable domain.

[0504] 79. The therapeutic fusion protein of paragraph 78 further comprising an additional antibody, wherein:

[0505] (a) each of the two antibodies is linked to one of the heavy chain constant domains or (b) each of the two antibodies is linked to one of the heavy chain variable domains; or

[0506] (a) each of the two antibodies is linked to the light chain constant domain or (b) each of the two antibodies is linked to the light chain variable domain; or the two antibodies are linked to each other and one of the two antibodies is linked to one of the heavy chain constant domains; or the at least one antibody is linked to the light chain constant domain or the light chain variable domain.

[0507] 80. The therapeutic fusion protein of any one of paragraphs 77-79, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 17

[0508] 81. The therapeutic fusion protein of any one of paragraphs 77-80, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 18.

[0509] 82. A therapeutic fusion protein comprising at least one antibody of any one of the preceding paragraphs fused to a polypeptide selected from: Fc-containing antibodies, Ab fragments, Fc fusion proteins, Fc domains; and non-antibody proteins.

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[0512] 83. A pharmaceutical composition comprising the antibody of any one of the preceding paragraphs and a pharmaceutically acceptable excipient.

[0513] 84. A method comprising administering to a subject the antibody of any one of the preceding paragraphs, the conjugate of any one of the preceding, the therapeutic fusion protein of any one of any one of the preceding, or the pharmaceutical composition of paragraph 83.

[0514] 85. A nucleic acid or set of nucleic acids encoding the antibody of any one of the preceding paragraphs.

[0515] 86. A vector or set of vectors comprising one or more nucleic acid encoding the antibody of any one of the preceding paragraphs.

[0516] 87. A method of producing the antibody of any one of the preceding paragraphs, the method comprising culturing the cell of paragraph 65 in cell culture and obtaining the antibody from the cell or cell culture.

[0517] 88. A method of producing a polypeptide, the method comprising: introducing a nucleic acid into a cell, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 11, 13, 14, or 33-49; culturing the cell to produce the polypeptide; and isolating the polypeptide from the cell.

[0518] 89. A method of producing a polypeptide, the method comprising: introducing a nucleic acid encoding a polypeptide into a cell, wherein the nucleic acid comprises a sequence having at least 70%, 80%, 85%, 90%, or 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 19-24; culturing the cell to produce the polypeptide; and isolating the polypeptide from the cell.

[0519] 90. An engineered polypeptide comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0520] 91. The engineered polypeptide of paragraph 90 further comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27.

[0521] 92. The engineered polypeptide of paragraph 90 or 91 further comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

[0522] 93. An engineered polypeptide comprising:

[0523] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0524] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 2; and

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[0527] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0528] 94. An engineered polypeptide comprising:

[0529] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 5;

[0530] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and

[0531] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0532] 95. An engineered polypeptide comprising:

[0533] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 8;

[0534] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 9; and

[0535] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0536] 96. An engineered polypeptide comprising:

[0537] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 8;

[0538] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and

[0539] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0540] 97. An engineered polypeptide comprising:

[0541] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 8;

[0542] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 12; and

[0543] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0544] 98. An engineered polypeptide comprising:

[0545] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 5;

[0546] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 12; and

[0547] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0548] 99. An engineered polypeptide comprising:

[0549] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0550] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 28; and

[0551] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0552] 100. An engineered polypeptide comprising:

[0553] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0554] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 29; and

[0555] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0556] 101. An engineered polypeptide comprising:

[0557] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0558] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 30; and

[0559] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0560] 102. An engineered polypeptide comprising:

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[0563] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0564] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and

[0565] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0566] 103. An engineered polypeptide comprising:

[0567] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 25;

[0568] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and

[0569] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0570] 104. An engineered polypeptide comprising:

[0571] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 26;

[0572] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and

[0573] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0574] 105. An engineered polypeptide comprising:

[0575] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 27;

[0576] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 6; and

[0577] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0578] 106. An engineered polypeptide comprising:

[0579] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0580] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 31; and

[0581] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0582] 107. An engineered polypeptide comprising:

[0583] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1;

[0584] (b) a peptide comprising the amino acid sequence of SEQ ID NO: 32; and

[0585] (c) a peptide comprising the amino acid sequence of SEQ ID NO: 3.

[0586] 108. The engineered polypeptide of any one of the preceding paragraphs having a length of about 110-115 amino acids.

[0587] 109. The engineered polypeptide of any one of the preceding paragraphs having a molecule weight of about 10-20 kilodaltons (kDa).

[0588] 110. The engineered polypeptide of any one of the preceding paragraphs comprising an amino acid sequence having at least 75% identity to the amino acid sequence of any one of SEQ ID NO: 4, 7, 10, 11, 13, 14, or 33-49.

[0589] 111. The engineered polypeptide of any one of the preceding paragraphs linked directly to (e.g., fused to) or indirectly to a diagnostic agent.

[0590] 112. The engineered polypeptide of any one of the preceding paragraphs linked directly to (e.g., fused to) to a therapeutic agent.

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[0593] 113. The engineered polypeptide of any one of the preceding paragraphs linked indirectly to a therapeutic agent.

[0594] 114. The engineered polypeptide of paragraph 113, wherein the therapeutic agent is a therapeutic polypeptide, optionally a therapeutic recombinant protein selected from antibodies, cytokines, enzymes, and growth factors.

[0595] 115. The engineered polypeptide of paragraph 113 or 114, wherein the therapeutic agent is a therapeutic polynucleotide, optionally an antisense oligonucleotide, a RNA interference molecule (e.g., siRNA, shRNA, miRNA), a CRISPR RNA (crRNA), or a double stranded RNA (dsRNA).

[0596] 116. The engineered polypeptide of any one of the preceding paragraphs linked to (e.g., fused to) a nanoparticle, optionally a lipid nanoparticle (LNP), a polymer nanoparticle, a DNA nanoparticle, a magnetic nanoparticle, metal (e.g., gold or silver) nanoparticle, a liposome, or a nanoemulsion.

[0597] 117. The engineered polypeptide of any one of the preceding paragraphs linked to (e.g., fused to) an imaging agent, optionally a radiopharmaceutical, a magnetic particle, or a lightemitting reporter (e.g., luciferase, green fluorescent protein (GFP), or near- infrared (IR) labels).

[0598] 118. The engineered polypeptide of any one of the preceding paragraphs linked to (e.g., fused to) a viral vector, optionally wherein the viral vector is an adeno-associated virus (AAV) vector, a lentiviral vector.

[0599] 119. A dual shuttle, comprising the antibody of any one of paragraphs 1-64 and a binding moiety that binds to a second target.

[0600] 120. A dual shuttle, comprising an antibody comprising:

[0601] (i) a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR), and

[0602] (ii) a binding moiety that binds to a second target.

[0603] 121. The dual shuttle of paragraph 120, wherein the antibody further comprises:

[0604] (i) a complementary determining region 1 (CDR1) comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27; and / or

[0605] (ii) a complementary determining region 2 (CDR2) comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

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[0608] 122. A dual shuttle, comprising an antibody and a binding moiety, wherein the antibody comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 11, 13, 14, or 33-49.

[0609] 123. The dual shuttle of paragraph 122, wherein the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 11, 13, 14, or 33-49.

[0610] 124. The dual shuttle of any one of paragraphs 119-123, wherein the binding moiety enhances transport of the dual shuttle across the blood-brain barrier.

[0611] 125. The dual shuttle of any one of paragraphs 119-124, wherein the binding moiety enhances retention of the dual shuttle in the brain parenchyma.

[0612] 126. The dual shuttle of any one of paragraphs 119-125, wherein the binding moiety mediates receptor-mediated transcytosis across the blood-brain barrier.

[0613] 127. The dual shuttle of any one of paragraphs 119-126, wherein the binding moiety binds to Basigin (CD147), the glucose transporter 1 (Glutl or SLC2A1), the human insulin receptor (HIR), the insulin-like growth factor receptor- 1 (IGF1-R), low-density lipoprotein receptor family members (e.g., LDLR, LRP1, and LRP-8), CD98 heavy chain, leptin receptor, or a myelin oligodendrocyte glycoprotein (MOG) (e.g. MOG-1).

[0614] 128. The dual shuttle of any one of paragraphs 119-127, wherein the binding moiety binds to CD98 heavy chain.

[0615] 129. The dual shuttle of any one of paragraphs 119-128, wherein the dual shuttle is linked directly to (e.g., fused to) or indirectly to a therapeutic agent.

[0616] 130. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0617] 131. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4.

[0618] 132. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6.

[0619] 133. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

[0620] 134. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 9.

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[0623] 135. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10.

[0624] 136. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 6.

[0625] 137. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11.

[0626] 138. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 12.

[0627] 139. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13.

[0628] 140. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 12.

[0629] 141. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 14.

[0630] 142. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 28, and SEQ ID NO: 3.

[0631] 143. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 33.

[0632] 144. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 29, and SEQ ID NO: 3.

[0633] 145. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 34.

[0634] 146. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0635] 147. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 35.

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[0638] 148. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 30, and SEQ ID NO: 3.

[0639] 149. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 36.

[0640] 150. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 37.

[0641] 151. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 38.

[0642] 152. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 39.

[0643] 153. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 3.

[0644] 154. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 40.

[0645] 155. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 6, and SEQ ID NO: 3.

[0646] 156. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 41.

[0647] 157. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 42.

[0648] 158. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 6, and SEQ ID NO: 3.

[0649] 159. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 43.

[0650] 160. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 6, and SEQ ID NO: 3.

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[0653] 161. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 44.

[0654] 162. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 6, and SEQ ID NO: 3.

[0655] 163. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 45.

[0656] 164. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 46.

[0657] 165. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 47.

[0658] 166. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 31, and SEQ ID NO: 3.

[0659] 167. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 48.

[0660] 168. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 32, and SEQ ID NO: 3.

[0661] 169. An engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 49.

[0662] 170. The engineered polypeptide of any one of paragraphs 130-169 that selectively binds to human TfR relative to a non-human TfR.

[0663] 171. The engineered polypeptide of paragraph 170, wherein the TfR is a transferrin receptor 1 (TfRl).

[0664] 172. A fusion protein comprising the engineered polypeptide of any one of paragraphs 130- 171.

[0665] 173. A polynucleotide (one or more polynucleotide) encoding the engineered polypeptide of any one of paragraphs 130-171 or the fusion protein of paragraph 172.

[0666] 174. A vector comprising the polynucleotide of paragraph 173, optionally a viral vector.

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[0669] 175. A cell comprising the engineered polypeptide of any one of paragraphs 130-171, the fusion protein of paragraph 172, the polynucleotide of paragraph 173, or the vector of paragraph 174.

[0670] 176. The cell of paragraph 175 selected from bacterial cells (e.g., Escherichia coli), yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris), mammalian cells (e.g., CHO cells, HEK293 cells), insect cells (e.g., Sf9 cells, Sf21 cells), and plant cells.

[0671] 177. A conjugate comprising the engineered polypeptide of any one of paragraphs 130-171 or the fusion protein of paragraph 172.

[0672] 178. A method comprising administering to a subject the engineered polypeptide of any one of paragraphs 130-171, the fusion protein of paragraph 172, the polynucleotide of paragraph 173, the vector of paragraph 174, or the cell of paragraph 175 or 176.

[0673] 179. A method comprising culturing the cell of paragraph 175 or 176 and isolating the engineered polypeptide.

[0674] 180. A method comprising transfecting a cell with the polynucleotide of paragraph 173 or the vector of paragraph 174, culturing the cell, and isolating the engineered polypeptide.

[0675] EXAMPLES

[0676] Example 1: Generation and evaluation of an exemplary anti-TfRl antibody

[0677] Heavy-chain variable (VHH) antibodies that specifically bind to transferrin receptor 1 (TfRl) were identified. This Example describes in vitro and in cellulo characterization of a first anti-TfRl antibody, referred to as “Antibody A,” which comprises an amino acid sequence of SEQ ID NO: 4. This first anti-TfRl antibody was evaluated for its abilities to bind transferrin receptor, to be internalized by cells, and to compete with transferrin for binding to the transferrin receptor.

[0678] Identification of Antibody A

[0679] Single-domain VHH antibodies were obtained from two llamas immunized with a mixture of human and mouse TfRl antigens. Animals were immunized with six subcutaneous injections, weekly, of human and mouse TfRl extracellular domain (ECD) recombinant proteins. Additionally, both animals received two final boost injections of cell lines transiently overexpressing human or mouse TfRl (HEK293 cell background) with a 2- week interval between injections. Following the final protein or the final cell boost, blood samples were collected and PBMCs were isolated. RNA was then extracted and stored. RNA was

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[0681] #14369922vl Attorney Docket No. H0498.70819WO00 converted to cDNA using reverse transcriptase and random primers. The VHH encoding gene fragments were amplified using multiple primers and cloned into a phagemid vector, resulting in the generation of two phage display libraries per llama. Phage were prepared.

[0682] Specific antibody fragments were selected via phage display from the generated libraries, using previously biotinylated recombinant human and mouse TfRl ECD proteins as bait. The biotinylated antigens were incubated with phage at various concentrations over up to two rounds of panning. One final phage display selection round on cell lines transiently overexpressing human or mouse TfRl (HEK293 cell background) was also performed, for most selection strategies used. E. coli bacteria were infected with output phage after each selection round to use for subsequent selections or screening by binding of individual clones.

[0683] Individual clones from outputs from different selection rounds and conditions were picked into 96-well master plates, tested as periplasmic extract (P.E.) for binding to cell lines transiently overexpressing human TfRl (CHO-K1 cell background) and positive hits were sequenced. Transiently transfected cells were aliquoted into a 96 well plate at a density of 150,000 cells per well. WT, not-transfected CHO-K1 cells were used as a control cell line. Specific binding to transiently transfected cells was normalized to the fold over background. Representative VHH antibodies from individual sequences were re-screened as P.E. in a similar FACS set-up on CHO-K1 cells transiently transfected with human and mouse TfRl, to determine mouse cross -reactivity. Additionally, a chimera construct was designed, bearing the human TfRl apical domain and remaining mouse TfRl sequence. VHH antibodies were screened by FACS on CHO-K1 cells transiently expressing this chimera sequence as P.E. to assess human apical domain binding. From this screening effort, Antibody A (among other anti-TfRl antibodies) was identified.

[0684] Human Fc sandwich ELISA

[0685] To determine antibody concentrations in tissue lysates and plasma samples, a human Fc sandwich ELISA was used. White 96-well MaxiSorp Nunc plates were coated with 3 ug / mL of capture antibody, goat anti-human IgG (Jackson ImmunoResearch, 109-006-098), and were then blocked in 1% BSA / PBS. Tissues samples were diluted in blocking buffer and added to the microplate (75uL / well) and incubated for 1.5 hours at room temperature (RT). After washing four times with IX PBS Tween, an HRP-conjugated anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-035-088) was added to each well and incubated for an additional 1.5 hours at RT. After washing five times with IX PBS Tween, ECL substrate was added to the microplate and the plate was immediately read on a BioTek 78

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[0687] Synergy Hl plate reader. Antibody concentrations of samples were interpolated from standard curves. All data analysis was performed using PRISM software.

[0688] In vitro protein binding

[0689] White 96-well plates were coated with 3-5 pg / mE of human TfR (R&D systems, 2474-TR-050) or cyno TfR proteins and blocked in 1% BSA / PBS. Anti-TfR antibodies were diluted in a 1:4 titration. Diluted antibodies were added to microplates (75 pL / well) and incubated for 1.5 hours at room temperature. After washing four times with IX PBS Tween, an HRP-conjugated anti-human IgG secondary antibody was added to each well and incubated for an additional 1.5 hours at RT. After washing five times with IX PBS Tween, ECL substrate was added to the microplate and the plate was immediately read on a BioTek Synergy Hl plate reader. All data analysis was performed using PRISM software.

[0690] FIG. 2A shows binding of an anti-TfR antibody (Antibody A) for transferrin receptor proteins. An exemplary antibody (Antibody A) demonstrated low nanomolar binding affinities to human TfRl and binding to cyno TfRl. A negative control antibody (anti- BACE1) did not demonstrate any binding to TfRl.

[0691] In vitro cell binding hCMEC / D3 cells (Millipore, SCC066) were cultured following manufactures’ instruction. To test cell binding, hCMEC / D3 cells were seeded at 150K cells per well. TfR antibodies were diluted in 1% BSA / PBS in 1:4 titration. Diluted antibodies were added to the microplate and incubated with cells for 2 hours on ice to prevent internalization. After incubation, cells were fixed and stained with an Alexa Fluor® 488 conjugated anti-human IgG secondary antibody for 30 min at RT. Following secondary Ab staining and washing with PBS, the binding of anti-TfR antibodies to cells was detected on a CytoFEEX flow cytometer. All data analysis was performed using FlowJo and PRISM software.

[0692] FIG. 2B shows that an exemplary anti-TfR antibody (Antibody A) demonstrated binding to hCMEC-D3 cells (i.e., TfRl -expressing human endothelial cells). A negative control antibody (anti-BACEl antibody) did not demonstrate significant binding to hCMEC- D3 cells.

[0693] Internalization assay

[0694] The internalization assay was performed using hCMEC / D3 cells (Millipore, SCC066). Cells were cultured following the manufacturer’s instructions. Cells were seeded at 79 #14369922vl Attorney Docket No. H0498.70819WO00

[0695] 1 million cells per well and allowed to grow for 24 hours before the assay. Anti-TfR antibodies were diluted to 1-3 g / mL in culture medium and were allowed to bind with hCMEC / D3 cells for 30 min before harvesting and fixation. After fixation, the cells for each antibody were separated into 2 groups, the surface binding group and the total binding group. For the surface binding group, an Alexa Fluor® 488 conjugated anti-human IgG secondary antibody was added to the cells and incubated for 1 hour at RT. For the total binding group, cells were permeabilized with 0.25% Triton before secondary antibody staining with an Alexa Fluor® 488 conjugated anti-human IgG secondary antibody. After secondary Ab staining, all groups were washed with PBS and binding of anti-TfR antibodies to cells was detected on a CytoFFEX flow cytometer. All data analysis was performed using FlowJo and PRISM software.

[0696] FIG. 2C shows that an exemplary anti-TfR antibody (Antibody A) has significantly higher total binding than surface binding on hCMEC / D3 cells, indicating that the antibodies internalize into the cells. Antibody 1, a reference antibody, also showed positive internalization in hCMEC / D3 cells.

[0697] Transferrin competition assay

[0698] Human transferrin protein was coated at 10 pg / ml on the plates and blocked with 1% casein. Anti-TfR VHH antibodies (IgG-VHH, 100 nM) incubated with Biotin-hTfRl (2.5 ug / mE) for 30 min at RT to form anti-TfR antibodies / Biotin-hTfRl complexes, which were then added to human Tf plates and incubated for 1 hour at RT to assess competition. ExtrAvidin-HRP reagent was used to detect captured Biotin-hTfRl. Excess human Tf was used as positive control for competition, which showed 88-93% inhibition. Irrelevant VHH was used as a negative control. The competition threshold was set at 25% inhibition.

[0699] FIG. 2D shows that an exemplary anti-TfR antibody (Antibody A) does not compete with transferrin (Tf) for TfR binding. An IgG-VHH antibody (Irr-VHH) was used as a negative control, and excess transferrin was used as a positive control for competition. Antibody A showed less than 25% inhibition, and thus is not considered to compete with transferrin for binding to the transferrin receptor (TfRl).

[0700] Human TfR and cyno TfR affinity determination

[0701] Affinities to human and cyno TfR were determined by surface plasmon resonance using a Biacore™ 8 K instrument. Antibodies were immobilized on a CM5 Series S sensor chips (Cytiva, 29104992) using an anti-human Fc molecule. 2-fold serial dilutions of 80

[0702] #14369922vl Attorney Docket No. H0498.70819WO00 recombinant human CD98hc from 1.56-100 nM for Antibody A and from 23-1500 nM for Antibodies B-F were injected at a flow rate of 30 pL / min in a IX HBS-EP+ pH7.4 running buffer (Cytiva, BR100669). 2-fold serial dilutions of recombinant cyno CD98hc from 23- 1500 nM for all antibodies were injected at a flow rate of 30 pL / min. Data analysis was conducted using Biacore Insight Evaluation software. Kinetic parameters were calculated using the 1:1 Langmuir binding model, and the Steady-state binding model.

[0703] FIG. 3 shows the Biacore-derived binding affinities of Antibody A to human and cyno TfR proteins. These data further validated that these antibodies specifically bind to TfRl.

[0704] Example 2. Administration of antibodies to mice

[0705] This Example describes in vivo characterization of Antibody A (comprising an amino acid sequence of SEQ ID NO: 4). Antibody A was evaluated for its abilities to be trafficked into the brain (e.g., brain uptake), which, in some embodiments, results from an ability to cross the blood-brain barrier and be internalized into brain cells and tissues following binding to a surface-exposed transferrin receptor.

[0706] Mouse model, Ab compound injection and tissue harvesting

[0707] The extracellular domain (ECD) of TfRl belonging to humanized TfRC knock-in (KI) mice was replaced with the human TfR ECD. Homozygous humanized KI mice (5-6 weeks) were used in the study.

[0708] Antibody compounds were injected by tail-vein intravenous injection at indicated doses. After 24-hours post injection, mice were perfused with cold phosphate-buffered saline, and brain and blood samples were then collected for further analysis. In selected studies, additional time points were evaluated and additional peripheral tissues (lung, liver, kidney, heart and quadriceps) were also collected.

[0709] Blood samples were collected in EDTA tubes via submandibular venipuncture. Blood samples were spun down at 5,000 g for 10 min and the supernatant was saved as plasma.

[0710] Parenchymal lysate preparation (capillary depletion)

[0711] To quantify antibody concentrations in the parenchymal fraction, capillary depletion was performed following a standard protocol. Briefly, one brain hemisphere was homogenized in ice-cold Hanks’ Balanced Salt solution (3 pl per mg of tissue) with 20 strokes in a Dounce homogenizer. Brain homogenate was then transferred to a clean Eppendorf tube and an equal volume of 26% Dextran was added to the homogenate. The 81

[0712] #14369922vl Attorney Docket No. H0498.70819WO00 brain homogenate / Dextran mixture was mixed well on a sharker in the cold room and then centrifuged at 5,400 g for 15 min to pellet the capillary fraction from the parenchymal fraction. The supernatant (parenchyma) was transferred to a clean tube and incubated with RIPA buffer for lysis in the cold room for 20 mins. Samples were then spun down at 20,000 g for 15 mins at 4°C and the supernatants were saved as parenchymal brain lysates.

[0713] Total tissue lysate preparation

[0714] For total lysate preparation, RIPA buffer was added to each tissue (10 pl per mg of tissue). Tissues were homogenized in a Dounce homogenizer for 20-30 strokes. Homogenates were transferred to clean Eppendorf tubes and rotated on a shaker in the cold room for 20 mins. Samples were spun down at 20,000 g for 15 mins at 4°C and the supernatants were saved as total tissue lysates.

[0715] It was found that Antibody A had significant brain uptake relative to (i) control antibodies that do not target brain tissues (anti-BACEl antibody and trastuzumab) and (ii) control anti-TfRl antibodies (Antibody 2 and Antibody 3). Specifically, mice that were administered Antibody A had more than a 20-fold increase in antibody concentration in their brain tissue relative to mice that were administered a control antibody.

[0716] FIGs. 4A-4B show concentrations of three anti-TfR antibodies (Antibody A, Antibody 2, and Antibody 3) found in mouse brain parenchyma (Par) and plasma 24 hours post injection. 1.8 mg / kg of each anti-TfR antibody or an equimolar amount (1.7 mg / kg) of anti-BACEl were injected into humanized TfRC knock-in mice (hTfRC KI) via tail- vein IV injection. FIG. 4A shows that anti-TfR antibodies (Antibody A, Antibody 2, and Antibody 3) exhibit more than 10-fold to 20-fold higher brain uptake compared to anti-BACEl antibody in the parenchymal fraction. FIG. 4B shows that the anti-TfR antibody concentrations in the plasma are about 10% of the anti-BACEl concentration in the plasma after 24 hours. Each data point represents data from 1 mouse. These data demonstrate that Antibody A is capable of crossing the blood-brain barrier and traversing to the brain following systemic administration to mammalian subjects.

[0717] FIGs. 4C-4D show brain parenchyma and plasma pharmacokinetics (PK) of Antibody A in a one-week dose-response and time-course study. 16 mg / kg or 50 mg / kg of Antibody A were administered through tail- vein IV injection in hTfRC KI mice. Equimolar doses of the control antibody trastuzumab were used in control subjects. As shown in FIG. 4C, Antibody A is found in the brain parenchyma within a single day following injection, while little-to-no trastuzumab is found in the brain parenchyma.

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[0720] FIG. 5 shows that anti-TfR antibodies (Antibody A and Antibody 2) have significantly higher uptake in mouse brain tissue than control antibodies (anti-HIV-1 and anti-BACEl antibodies). In liver, kidney and heart, the anti-TfR antibodies show slightly higher uptake than the control antibodies; in lung and quadriceps, the anti-TfR antibodies show slightly lower uptake than the control antibodies. FIG. 5B shows the uptake of individual antibodies in each tissue based on the mean uptake from FIG. 5A.

[0721] FIG. 6, which is a western blot analysis of brain tissues, shows that Antibody A (and Antibody 2) does not deplete expression of human transferrin receptor protein in mouse brain tissues. GAPDH was used a loading control.

[0722] Example 3. Monovalent and bivalent configurations of anti-TfRl antibodies

[0723] This Example describes the generation and evaluation of monovalent and bivalent configurations of anti-TfRl antibodies. Antibody configurations are as described in FIG. 1. Specifically, this Example involved generation of monovalent configurations (IgG- monoVHH) and bivalent configurations (IgG-biVHH) of anti-TfRl antibodies (TfRl l, TfR16, TfR19, and TfR22).

[0724] Mice were injected with anti-TfRl antibodies using the mouse model and procedures described in Example 2. FIG.7 shows that the monovalent configuration (IgG-monoVHH) produced higher brain uptake than the bivalent configuration (IgG-biVHH) of the same anti- TfRl antibody. The IgG-monoVHH antibodies were tested at 5.5 mg / kg and the IgG-biVHH antibodies were tested at 6 mg / kg to provide equimolar doses. FIG. 7A shows brain uptake in the parenchymal fraction after capillary depletion. FIG. 7B shows Ab concentrations in plasma 24 hours post injection.

[0725] Example 4. Evaluation of anti-TfRl antibodies

[0726] Anti-TfRl antibodies of the disclosure (Antibody A, Antibody B, Antibody C, Antibody D, Antibody E, and Antibody F), each of which comprise a CDR3 having an amino acid sequence of SEQ ID NO: 3, were evaluated for their abilities to bind transferrin receptor in vitro and to be trafficked into the brain (e.g., brain uptake).

[0727] Binding affinity experiments were performed as described in Example 1. FIGs. 8A- 8B show that each of the tested anti-TfRl antibodies bind to human and cyno TfR proteins immobilized on the plates. FIG. 9 shows binding affinities of anti-TfRl antibodies for human and cyno TfR apical domain proteins (using SPR binding methods).

[0728] The ability to anti-TfRl antibodies to be trafficked into brain tissues (and cross the 83

[0729] #14369922vl Attorney Docket No. H0498.70819WO00 blood-brain barrier) following systemic administration was evaluated using the method as described in Example 2. FIGs. 10A-10B show concentrations of anti-TfRl antibodies in mouse brain parenchyma and plasma 24 hours following injection. 2 mg / kg of each anti-TfR antibody or an equimolar dose of trastuzumab were injected into hTfRC KI mice via tail-vein IV injection. FIGs. 10C and D show brain parenchyma and plasma PK of selected anti-TfRl antibodies in a two-week time-course study. 20 mg / kg of anti-TfR antibodies were administered through tail- vein IV injection. An equimolar dose of trastuzumab was used as control.

[0730] These data demonstrate that the anti-TfRl antibodies of the disclosure (e.g., comprising a CDR3 having an amino acid sequence of SEQ ID NO: 3) are capable of binding to transferrin receptor and being trafficked across the blood-brain barrier. Accordingly, these data suggest that the anti-TfRl antibodies of the disclosure are useful shuttle agents (e.g., to transport therapeutic antibodies across the blood-brain barrier).

[0731] Example 5.

[0732] FIG. 11 shows that anti-TfRl antibodies of the disclosure provide broad antibody distribution in whole mouse brain regions (at 20 mg / kg dose administration via tail- vein intravenous injection). An equimolar dose of trastuzumab was used as negative control.

[0733] FIG. 12A shows concentrations of anti-TfRl antibodies of the disclosure in mouse spinal cords at day 1, 7 and 14 after a single dose injection of 20 mg / kg through tail- vein intravenous injection. An equimolar dose of trastuzumab was used as control. FIG. 12B shows that an anti-TfR antibody of the disclosure colocalized with neuronal cells (marked by NeuN) in the ventral horn region of lumbar spinal cord (arrows, top panel) while trastuzumab showed no uptake in the same region.

[0734] FIGs. 13A-13C show that additional humanized anti-TfR variants bind to human (FIG. 13A) and cynomolgus (FIG. 13B) TfR proteins immobilized on plates. FIG. 13C shows the data plotted in FIG. 13A and FIG. 13B.

[0735] FIGs. 14A-14C show results of a head-to-head comparison of an optimized dual shuttle format and anti-CD98hc and anti-TfR single shuttles in the same study. The CD98hc / TfR dual shuttle achieved a two-fold or greater increase in both Cmax and AUC compared to single anti-CD98hc or anti-TfR shuttles in parenchymal (FIG. 14A) and total brain lysates (FIG. 14B). The dual shuttle also exhibited faster serum clearance than either of the single shuttles (FIG. 14C).

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[0738] EQUIVALENTS

[0739] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0740] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0741] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0742] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0743] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0744] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

[0745] 85

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Claims

Attorney Docket No. H0498.70819WO00CLAIMSWhat is claimed is:

1. An antibody comprising a complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR).

2. The antibody of claim 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2.

3. The antibody of claim 2, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 4.

4. The antibody of claim 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 5; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

5. The antibody of claim 4, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 7.

6. The antibody of claim 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 9.86#14369922vlAttorney Docket No. H0498.70819WO007. The antibody of claim 6, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 10.

8. The antibody of claim 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6.

9. The antibody of claim 8, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11.

10. The antibody of claim 1 further comprising: a complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; and / or a complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 12.

11. The antibody of claim 10, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 13.

12. An antibody comprising a complementary determining region 3 (CDR3) having at least 70% identity to the amino acid sequence of SEQ ID NO: 3, wherein the antibody binds to a transferrin receptor (TfR).

13. The antibody of claim 12, wherein the CDR3 has at least 80%, 90%, 95%, or 98% identity to the amino acid sequence of SEQ ID NO: 3.

14. The antibody of claim 12 further comprising a complementary determining region 1 (CDR1) having at least 70% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27.87#14369922vlAttorney Docket No. H0498.70819WO0015. The antibody of claim 14, wherein the CDR1 has at least 80%, 90%, 95%, or 98% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27.

16. The antibody of claim 12 further comprising a complementary determining region 2 (CDR2) having at least 70% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

17. The antibody of claim 16, wherein the CDR2 has at least 80%, 90%, 95%, or 98% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

18. The antibody of claim 1, wherein: the equilibrium dissociation constant (KD) of binding of the antibody to the TfR is in a range from I O’10M to 10'5M; and / or the equilibrium dissociation constant (KD) of binding of the antibody to the TfR is in a range from 0.5 nM to 5000 nM.

19. The antibody of claim 1, wherein the antibody binds human TfR and a non-human TfR, optionally wherein the antibody selectively binds to human TfR relative to a non-human TfR.

20. The antibody of claim 1, wherein the TfR is a transferrin receptor 1 (TfRl).

21. The antibody of claim 1, wherein the antibody is internalized into a cell, optionally wherein the cell is a brain endothelial cell.

22. The antibody of claim 1, wherein the antibody is a single chain antibody.

23. The antibody of claim 22, wherein the single chain antibody is a heavy-chain variable domain (VHH) antibody or a single-chain variable fragment (scFv) antibody.

24. The antibody of claim 1, wherein the antibody is a Fc-containing antibody, antibody fragment, or Fc-fusion protein.88#14369922vlAttorney Docket No. H0498.70819WO0025. The antibody of claim 24, wherein the Fc-containing antibody or Fc-fusion protein comprises one or more mutations, optionally knob-in-hole mutations, effector function mutation or FcRN mutations.

26. The antibody of claim 1, wherein: the antibody is a humanized antibody and / or a chimeric antibody; and / or the antibody is a deimmunized antibody.

27. A cell comprising the antibody of claim 1, optionally wherein the cell is a brain endothelial cell.

28. A conjugate comprising the antibody of claim 1.

29. The conjugate of claim 28, wherein the antibody is linked directly to (e.g., fused to) or indirectly to a diagnostic agent or a therapeutic agent.

30. A therapeutic fusion protein comprising: a heavy chain comprising a heavy chain constant domain and a heavy chain variable domain; a light chain comprising a light chain constant domain and a light chain variable domain; and at least one antibody of claim 1.

31. A therapeutic fusion protein comprising at least one antibody of claim 1 fused to a polypeptide selected from: Fc-containing antibodies, Ab fragments, Fc fusion proteins, Fc domains; and non-antibody proteins.

32. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable excipient.

33. A method comprising administering to a subject the antibody of claim 1.

34. A nucleic acid or set of nucleic acids encoding the antibody of claim 189#14369922vlAttorney Docket No. H0498.70819WO0035. A vector or set of vectors comprising one or more nucleic acid encoding the antibody of claim 1.

36. A method of producing the antibody of claim 1, the method comprising culturing the cell of claim 27 in cell culture and obtaining the antibody from the cell or cell culture.

37. A method of producing a polypeptide, the method comprising: introducing a nucleic acid into a cell, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 11, 13, 14, or 33-49; culturing the cell to produce the polypeptide; and isolating the polypeptide from the cell.

38. An engineered polypeptide comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 3, optionally further comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5, 8, or 25-27, and optionally further comprising a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 9, 12, or 28-32.

39. A dual shuttle, comprising the antibody of claim 1 and a binding moiety that binds to a second target.90#14369922vl

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