Hematopoietic cell targeting conjugates and related methods

Abstract

Provided herein are, inter alia, conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); operably connected to (b) at least one oligonucleotide that modulates the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a target cell; and methods of manufacturing and pharmaceutical compositions comprising the same. Further provided herein are methods of utilizing the conjugates, including, e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or a thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

Description

HEMATOPOIETIC CELL TARGETING CONJUGATES AND RELATED METHODS RELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No.: 63 / 514,956, filed July 21, 2023, the entire contents of which is incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 16, 2024, is named 62992_6WO01_SL.xml and is 2,241 ,941 bytes in size.1. FIELD

[0003] This disclosure relates to, inter alia, conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); and an oligonucleotide that modulates the expression and / or activity of a target gene expressed by a target cell. The disclosure further relates to pharmaceutical compositions comprising the same; and methods of utilizing the same, including, e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or a thalassemia (e.g., a-thalassemia, 0- thalassemia, 5-thalassemia, or y-thalassemia)).2. BACKGROUND

[0004] The bone marrow is a soft gelatinous tissue that fills the cavities of the bones. In adults bone marrow is either red or yellow, depending upon the preponderance of hematopoietic (red) or fatty (yellow) tissue. Genetic perturbations, including, e.g., gene mutations, overexpression of a gene, deficiency of a gene (and in turn the encoded product (e.g., protein)) in subsets of cells within the bone marrow are associated with various genetic disorders, including, e.g., genetic blood disorders (e.g., hemoglobinopathies and inherited bone marrow failure syndromes).

[0005] In humans the red bone marrow forms all of the blood cells, including red blood cells, with the exception of the lymphocytes, which are produced in the marrow and reach their mature form in the lymphoid organs. Normal red blood cells contain the protein hemoglobin, which functions to transport oxygen (O2) from the lungs to peripheral tissues and carbon dioxide (CO2)from the tissues to the lungs. Hemoglobin is a heterotetramer composed of a-like and (3-like globin subunits, each bound to a heme prosthetic group. Hemoglobin is synthesized from separate a-likc and P-like globin gene clusters, with different types of hemoglobin produced through different subunit combinations. Fetal hemoglobin is the primary hemoglobin produced by the fetus. In healthy humans, a shift from y-globin to p-globin gene expression around birth underlies a switch from fetal hemoglobin to adult hemoglobin production, such that by 6 months of age the major hemoglobin adult hemoglobin. The hemoglobin switch, is not total or irreversible; as adults retain the ability to produce residual levels of fetal hemoglobin (<1% of total hemoglobin). The switch from fetal to adult hemoglobin relies on repression or silencing of the upstream y-globin gene, through a network of repressor proteins. Hemoglobinopathies are a class of genetic diseases associated with the abnormal production and / or structure of hemoglobin and are the most common inherited blood diseases.3. SUMMARY

[0006] Provided herein are, inter alia, conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) that comprises a protein (e.g., an antibody) that specifically binds to TFR (e.g., hTFR (e.g., hTFRl)); and an oligonucleotide that modulates the expression and / or activity of a target gene expressed by a target cell; and methods of manufacturing and pharmaceutical compositions comprising the same. Further provided herein are methods of utilizing the conjugates including, e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or a thalassemia (e.g., a-thalassemia, P-thalassemia, 5-thalassemia, or y-thalassemia (e.g., P-thalassemia)).

[0007] Accordingly, in one aspect provided herein are conjugates comprising: (a) a hematopoietic cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to (b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by the hematopoietic cell.

[0008] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell.

[0009] In some embodiments, the conjugate exhibits one or more of the followingproperties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR 1)) expressed on the surface of a hematopoietic cell, the conjugate docs not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (c) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and / or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0010] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0011] In some embodiments, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the hematopoietic cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0012] In some embodiments, the protein that specifically binds TFR (e.g., hTFR (e.g., TFR1))is an anti-TFR (e.g., hTFR (e.g., TFR1)) antibody. Tn some embodiments, the antibody does not (or docs not substantially) block binding of TF (e.g., hTF) to the TFR (e.g., hTFRl). In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fab-Fc, a scFv, a scFv-Fc, a (scFv) -Fc, an Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), a (sdAb)z (e.g., a (VHH)2), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the antibody is an IgG (e.g., a human IgG (hlgG)) antibody. In some embodiments, the antibody is a hlgGl, hIgG2, hIgG3, or hIgG4 antibody (e.g., a hlgGl or hIgG4 antibody).

[0013] In some embodiments, the antibody comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region. In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab-Fc, a scFv-Fc, a (scFv)2-Fc, a sdAb-Fc (e.g., a VHH-Fc), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is a hlg. In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl or hIgG4.

[0014] In some embodiments, the Ig (e.g., hlg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference hlg Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))). In some embodiments, the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))).

[0015] In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence ofthe Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, a serine at amino acid position L235, and / or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

[0016] In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is h!gG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position L235, numbering according to EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

[0017] In some embodiments, the antibody comprises a first Fc region and a second Fc region that associate via at least one covalent (e.g., disulfide) bond. In some embodiments, the antibody the amino acid sequence of the first Fc region and / or the amino acid sequence of the second Fc region comprise one or more amino acid substitution that promotes the association (e.g., heterodimerization) of the first and second Fc regions.

[0018] In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering accordingto the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises a cysteine at amino acid position S354, numbering according to the EU index of Kabat.

[0019] In some embodiments, the protein that specifically binds TFR (e.g., hTFR (e.g., hTFRl)) is a TFR ligand (or a functional fragment or functional variant thereof). In some embodiments, the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

[0020] In some embodiments, the oligonucleotide enhances the expression and / or activity of the target gene, nucleic acid e.g., mRNA), and / or protein. In some embodiments, the oligonucleotide inhibits the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein. In some embodiments, the oligonucleotide modulates (e.g., enhances or inhibits) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein through binding to a target nucleic acid molecule encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein (e.g., target mRNA molecule (e.g., a portion of a target mRNA molecule)). In some embodiments, the target nucleic acid molecule is a target mRNA molecule (e.g., a portion of a target mRNA molecule). In some embodiments, the oligonucleotide mediates one or more of the following degradation of the target nucleic acid molecule (e.g., mRNA), disabling of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration in the splicing of the target nucleic acid molecule (e.g., mRNA), alteration (e.g., a decrease) in the stability of the target nucleic acid molecule (e.g., mRNA), or a block in the translation of the target nucleic acid molecule (e.g., mRNA), or any combination of the foregoing.

[0021] In some embodiments, the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the oligonucleotide comprises or consists of an antisense strand comprising a region of complementarity to a target sequence (e.g., an mRNA sequence encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein). In some embodiments, the oligonucleotide is single stranded or double stranded. In some embodiments,the oligonucleotide is a DNA, RNA, or RNA and RNA hybrid molecule. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand forming a double stranded region. In some embodiments, the sense strand and the antisense strand are part of a single nucleic acid molecule (e.g., wherein a hairpin loop is between the sense strand and the antisense strand of the single nucleic acid molecule. In some embodiments, the sense strand and the antisense strand are separate nucleic acid molecules (i.e., connected only through the double stranded region). In some embodiments, the double stranded region is from about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide pairs in length.

[0022] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the oligonucleotide are modified. In some embodiments, substantially all (or all) of the nucleotides in the oligonucleotide are modified. In some embodiments, at least one of the modified nucleotides comprises a modified sugar (e.g., ribose moiety). In some embodiments, at least one of the modified nucleotides comprises a modified nucleobase. In some embodiments, the oligonucleotide comprises at least one modified intemucleoside linkage (e.g., at least one phosphoro thioate intemucleoside linkage). In some embodiments, the at least one modified nucleotide is a 2’ modified nucleotide (e.g., a 2'-fluoro (2'- F), 2'-O-methyl (2'-0-Me), 2'-O-methoxyethyl (2'-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)- constrained ethyl-bridged nucleic acid (cEt) (e.g., a 2' modified nucleotide is 2'- O-methyl or 2'-fluoro (2'-F))).

[0023] In some embodiments, the protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with one or more hemoglobinopathy. In some embodiments, inhibition of or a reduction in expression and / or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with an increase in the level of fetal hemoglobin, the induction of expression of fetal hemoglobin, and / or an increase in the ratio of fetal hemoglobin to adult hemoglobin. In some embodiments, expression and / or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with a repression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the levelof adult hemoglobin, and / or an increase in ratio of adult hemoglobin to fetal hemoglobin. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein is a transcription factor. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein is highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

[0024] In some embodiments, the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11 A (e.g., hBCLl 1 A)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pseudouridine Synthase 1 (DKC1) (e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11), Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S 17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed (ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1) (e.g., human HAX1), Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) (e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPL Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Like (SAMD9L) (e.g., human SAMD9L), or MDS1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

[0025] In some embodiments, the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11 A (e.g., hBCLl 1 A)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), or KLF Transcription Factor 1 (KLF1) (e.g., hKLFl).

[0026] In some embodiments, (a) the protein that specifically binds TFR is non-covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein thatspecifically binds TFR is covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is directly conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is indirectly conjugated to (b) the at least one oligonucleotide through (c) a linker. In some embodiments, the linker is cleavable or non-cleavable.

[0027] In some embodiments, the wherein (b) comprises at least 2, 3, 4, 5, 6, or more oligonucleotides. In some embodiments, each of the at least 2, 3, 4, 5, 6, or more oligonucleotides are individually conjugated to (a) the protein (e.g., antibody) that specifically binds TFR (e.g., as described herein).

[0028] In one aspect, provided herein are conjugates comprising: (a) an erythroid precursor cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to (b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by the erythroid precursor cell.

[0029] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

[0030] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (c) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and / or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0031] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remainsviable; (d) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and / or (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0032] In some embodiments, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the erythroid precursor cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0033] In some embodiments, the protein that specifically binds TFR (e.g., hTFR (e.g., TFR1)) is an anti-TFR (e.g., hTFR (e.g., TFR1)) antibody. In some embodiments, the antibody does not (or does not substantially) block binding of TF (e.g., hTF) to the TFR (e.g., hTFRl). In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fab-Fc, a scFv, a scFv-Fc, a (scFv)i-Fc, an Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), a (sdAb)2 (e.g., a (VHH)2), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the antibody is an IgG (e.g., a human IgG (hlgG)) antibody. In some embodiments, the antibody is a hlgGl, hIgG2, hIgG3, or hIgG4 antibody (e.g., a hlgGl or hIgG4 antibody).

[0034] In some embodiments, the antibody comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region. In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab-Fc, a scFv-Fc, a (scFv)2-Fc, a sdAb-Fc (e.g., a VHH-Fc), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the Ig (e.g., hlg) Fc region comprises at least a portion of ahinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is a hlg. In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl or hIgG4.

[0035] In some embodiments, the Ig (e.g., hlg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference hlg Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fey I Ila))). In some embodiments, the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))).

[0036] In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, a serine at amino acid position L235, and / or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

[0037] In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanineat amino acid position F234, and / or an alanine at amino acid position L235, numbering according to EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

[0038] In some embodiments, the antibody comprises a first Fc region and a second Fc region that associate via at least one covalent (e.g., disulfide) bond. In some embodiments, the antibody the amino acid sequence of the first Fc region and / or the amino acid sequence of the second Fc region comprise one or more amino acid substitution that promotes the association (e.g., heterodimerization) of the first and second Fc regions.

[0039] In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises a cysteine at amino acid position S354, numbering according to the EU index of Kabat.

[0040] In some embodiments, the protein that specifically binds TFR (e.g., hTFR e.g., hTFRl)) is a TFR ligand (or a functional fragment or functional variant thereof / ). In some embodiments, the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

[0041] In some embodiments, the oligonucleotide enhances the expression and / or activity ofthe target gene, nucleic acid (e.g., mRNA), and / or protein. In some embodiments, the oligonucleotide inhibits the expression and / or activity of the target gene, nucleic acid e.g., mRNA), and / or protein. In some embodiments, the oligonucleotide modulates (e.g., enhances or inhibits) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein through binding to a target nucleic acid molecule encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein (e.g., target mRNA molecule (e.g., a portion of a target mRNA molecule)). In some embodiments, the target nucleic acid molecule is a target mRNA molecule (e.g., a portion of a target mRNA molecule). In some embodiments, the oligonucleotide mediates one or more of the following degradation of the target nucleic acid molecule (e.g., mRNA), disabling of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration in the splicing of the target nucleic acid molecule (e.g., mRNA), alteration (e.g., a decrease) in the stability of the target nucleic acid molecule (e.g., mRNA), or a block in the translation of the target nucleic acid molecule (e.g., mRNA), or any combination of the foregoing.

[0042] In some embodiments, the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the oligonucleotide comprises or consists of an antisense strand comprising a region of complementarity to a target sequence (e.g., an mRNA sequence encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein). In some embodiments, the oligonucleotide is single stranded or double stranded. In some embodiments, the oligonucleotide is a DNA, RNA, or RNA and RNA hybrid molecule. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand forming a double stranded region. In some embodiments, the sense strand and the antisense strand are part of a single nucleic acid molecule (e.g., wherein a hairpin loop is between the sense strand and the antisense strand of the single nucleic acid molecule. In some embodiments, the sense strand and the antisense strand are separate nucleic acid molecules (i.e., connected only through the double stranded region). In some embodiments, the double stranded region is from about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide pairs in length.

[0043] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the oligonucleotide are modified. In someembodiments, substantially all (or all) of the nucleotides in the oligonucleotide are modified. In some embodiments, at least one of the modified nucleotides comprises a modified sugar- (e.g., ribose moiety). In some embodiments, at least one of the modified nucleotides comprises a modified nucleobase. In some embodiments, the oligonucleotide comprises at least one modified intemucleoside linkage (e.g., at least one phosphoro thioate intemucleoside linkage). In some embodiments, the at least one modified nucleotide is a 2’ modified nucleotide (e.g., a 2'-fluoro (2'- F), 2'-O-methyl (2'-0-Me), 2'-O-methoxyethyl (2'-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-0-DMAE0E), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)- constrained ethyl-bridged nucleic acid (cEt) (e.g., a 2' modified nucleotide is 2'- O-methyl or 2'-fluoro (2'-F))).

[0044] In some embodiments, the protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with one or more hemoglobinopathy. In some embodiments, inhibition of or a reduction in expression and / or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with an increase in the level of fetal hemoglobin, the induction of expression of fetal hemoglobin, and / or an increase in the ratio of fetal hemoglobin to adult hemoglobin. In some embodiments, expression and / or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with a repression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the level of adult hemoglobin, and / or an increase in ratio of adult hemoglobin to fetal hemoglobin. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein is a transcription factor.

[0045] In some embodiments, the target gene and / or protein is highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

[0046] In some embodiments, the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11 A (e.g., hBCLl 1 A)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pseudouridine Synthase 1 (DKC1) (e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11),Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed (ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1) (e.g., human HAX1), Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) (e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPL Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Like (SAMD9L) (e.g., human SAMD9L), or MDS1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

[0047] In some embodiments, the target gene is BCL11A (e.g., hBCLUA), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

[0048] In some embodiments, (a) the protein that specifically binds TFR is non-covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is directly conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is indirectly conjugated to (b) the at least one oligonucleotide through (c) a linker. In some embodiments, the linker is cleavable or non-cleavable.

[0049] In some embodiments, the wherein (b) comprises at least 2, 3, 4, 5, 6, or more oligonucleotides. In some embodiments, each of the at least 2, 3, 4, 5, 6, or more oligonucleotides are individually conjugated to (a) the protein (e.g., antibody) that specifically binds TFR (e.g., as described herein).

[0050] In one aspect, provided herein are cells comprising a conjugate described herein. In some embodiments, the cell is in vitro, ex vivo, or in vivo.

[0051] In one aspect, provided herein are pharmaceutical compositions comprising a conjugate described herein and a pharmaceutically acceptable excipient.

[0052] In one aspect, provided herein are kits comprising a conjugate described herein or a pharmaceutical composition described herein.

[0053] In one aspect, provided herein are methods of delivering a conjugate or pharmaceutical composition to a cell, the method comprising introducing into a cell a conjugate described herein, or a pharmaceutical composition described herein, to thereby deliver the conjugate or pharmaceutical composition into the cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0054] In one aspect, provided herein are methods of delivering a conjugate, cell, or pharmaceutical composition to a subject, the method comprising administering to the subject a conjugate described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby deliver the conjugate, cell, or pharmaceutical composition to the subject.

[0055] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0056] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0057] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

[0058] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

[0059] In one aspect, provided herein are methods of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0060] In one aspect, provided herein are methods of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject). In some embodiments, the target is BCL11A (e.g., hBCLHA), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

[0061] In one aspect, provided herein are methods of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

[0062] In one aspect, provided herein are methods of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby reduceor inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

[0063] In one aspect, provided herein are methods of inducing expression of fetal hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby induce expression of fetal hemoglobin the subject.

[0064] In one aspect, provided herein are methods of increasing the level of fetal hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby increase the level of fetal hemoglobin the subject.

[0065] In one aspect, provided herein are methods of increasing the ratio of fetal hemoglobin to adult hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby increase the ratio of fetal hemoglobin to adult hemoglobin in the subject.

[0066] In one aspect, provided herein are methods of treating, ameliorating, or preventing an inherited blood disorder in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the inherited blood disorder in the subject. In some embodiments, the inherited blood disorder is a hemoglobinopathy or an inherited bone marrow failure syndrome.

[0067] In one aspect, provided herein are methods of treating, ameliorating, or preventing a hemoglobinopathy in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the hemoglobinopathy in the subject. In some embodiments, the subject is a human.

[0068] In some embodiments, the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

[0069] In some embodiments, the hemoglobinopathy is sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or ' / -thalassemia).

[0070] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a- thalassemia, P- thalassemia, 8-thalassemia, or ^-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

[0071] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia).

[0072] In one aspect, provided herein are methods of treating, ameliorating, or preventing an inherited bone marrow failure syndrome in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the inherited bone marrow failure syndrome in the subject.

[0073] In some embodiments, the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, or thrombocytopenia absent radii (TAR).

[0074] In one aspect, provided herein are conjugates described herein, cells described herein, or pharmaceutical compositions described herein for use in the treatment of a disease in a subject in need thereof.

[0075] In one aspect, provided herein are conjugates described herein, cells described herein, or pharmaceutical compositions described herein for use as a medicament.

[0076] In one aspect, provided herein are uses of a conjugate described herein, a cell described herein, or a pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a disease in a subject in need thereof.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIGS. 1A-1D are line graphs showing the binding of recombinantly expressed anti- TFR1 monoclonal antibodies (mAbs) (set forth in Example 1) (or isotype control) to soluble TFR1 (using Octet BLI). FIG. 1A is a line graph showing the binding (showing a lack of binding) ofisotype control antibody to soluble TFR1. FIG. IB is a line graph showing the binding of anti- TFR1 mAb2 to soluble TFR1. FIG. 1C is a line graph showing the binding of anti-TFRl mAb3 to soluble TFR1. FIG. ID is a line graph showing the binding of anti-TFRl Fab to soluble TFR1.

[0078] FIGS. 2A-2D are line graphs showing the binding of anti-TFRl mAbs (set forth in Example 1) to the surface of erythroid-progenitor cells. FIG. 2A is a line graph showing the percent of positive cells treated with anti-TFRl mAb2 or anti-TFRl mAb3 (at the indicated concentration). FIG. 2B is a line graph showing the MFI of cells treated with anti-TFRl mAb2 or anti-TFRl mAb3 (at the indicated concentration). FIG. 2C is a line graph showing the percent of positive cells treated with anti-TFRl Fab (at the indicated concentration). FIG. 2D is a line graph showing the MFI of cells treated with anti-TFRl Fab (at the indicated concentration).

[0079] FIGS. 3A-3D are graphs showing the internalization of anti-TFRl mAbs (set forth in Example 1) (or isotype control) into erythroid-progenitor cells. FIG. 3A is a graph showing the percent positive cells treated with anti-TFRl mAb2 or anti-TFRl mAb3 (at the indicated concentration). FIG. 3B is a graph showing the MFI of cells treated with anti-TFRl mAb2 or anti- TFRl mAb3 (at the indicated concentration). FIG. 3C is a graph showing the percent positive cells treated with anti-TFRl Fab (at the indicated concentration). FIG. 3D is a graph showing the MFI of cells treated with anti-TFRl Fab (at the indicated concentration).

[0080] FIGS. 4A-B are line graphs showing the extent of mRNA knock-down of the indicated target gene by the indicated siRNA in erythroid-progenitor cells. FIG. 4A is a line graph showing HPRT1-2 siRNA mediated HPRT1 mRNA knockdown (at the indicated concentration of siRNA). FIG. 4B is a line graph showing BCL11A-11 siRNA or BCL11A-5 siRNA mediated BCL11A mRNA knockdown (at the indicated concentration of siRNA).

[0081] FIGS. 5A-5B are bar graphs showing the extent of the decrease in protein levels of the indicated target by the indicated siRNA in erythroid-progenitor cells. FIG. 5A is a bar graph showing the BCL11A-11 siRNA or BCA11A-5 siRNA mediated decrease in the level of BCL11A protein (at the indicated concentration of siRNA). FIG. SB is a bar graph showing the HPRT1-2 siRNA mediated decrease in the level of HPRT1 protein (at the indicated concentration of siRNA).

[0082] FIGS. 6A-6K are line graphs showing the binding of each of the anti-TFRl antibodysiRNA conjugates (AOCs#l-l l) binding to recombinant TFR1 protein (using Octet BLI). FIG. 6A is a line graph showing the binding of AOC#1 to recombinant TFR1. FIG. 6B is a line graph showing the binding of AOC#2 to recombinant TFR1. FIG. 6C is a line graph showing the bindingof A0C#3 to recombinant TFR1. FIG. 6D is a line graph showing the binding of A0C#4 to recombinant TFR1. FIG. 6E is a line graph showing the binding of A0C#5 to recombinant TFR1. FIG. 6F is a line graph showing the binding of A0C#6 to recombinant TFR1. FIG. 6G is a line graph showing the binding of A0C#7 to recombinant TFR1. FIG. 6H is a line graph showing the binding of A0C#8 to recombinant TFR1. FIG. 61 is a line graph showing the binding of A0C#9 to recombinant TFR1. FIG. 6 J is a line graph showing the binding of AOC#10 to recombinant TFR1. FIG. 6K is a line graph showing the binding of AOC#11 to recombinant TFR1.

[0083] FIGS. 7A-7B are line graphs showing the % viability of erythroid precursor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIG. 7A is a line graph showing the % viability of erythroid precursor cells 48 hours after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIG. 7B is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC at the indicated concentration.

[0084] FIGS. 7C-7D are line graphs showing the knockdown of the HPRT1 transcript in erythroid-progenitor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIGS. 7C is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 48 after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIGS. 7D is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 72 after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration.

[0085] FIGS. 8A-8B are line graphs showing the % viability of erythroid precursor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8A is a line graph showing the % viability of erythroid precursor cells 48 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8B is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration.

[0086] FIGS. 8C-8D are line graphs showing the knockdown of the HPRT 1 mRNA transcript in erythroid-progenitor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8C is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 48 after the first dose with the indicatedAOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8D is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 72 after the first dose with the indicated AOC (A0C#3 or A0C#4) at the indicated concentration.

[0087] FIG. 9 is a line graph showing knockdown of the HPRT1 protein in erythroid- progenitor cells 72 hours after the first dose with the indicated AOC (AOC#2 or AOC#4).

[0088] FIG. 10A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#5) at the indicated concentration. FIG. 10B is a line graph showing the knockdown of the HPRT 1 mRNA transcript in erythroid-progenitor cells 72 after the first dose with the indicated AOC (AOC#5) at the indicated concentration.

[0089] FIG. 11A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#10 or AOC#11) at the indicated concentration. FIG. 11B is a line graph showing the knockdown of the BCL11 A mRNA transcript in erythroid- progenitor cells 72 hours after the first dose with the indicated AOC (AOC#10 or AOC#11) at the indicated concentration.

[0090] FIG. 12A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#9) at the indicated concentration. FIG. 12B is a line graph showing the knockdown of the BCL11 A transcript in erythroid-progenitor cells 72 hours after the first dose with the indicated AOC (AOC#9) at the indicated concentration.

[0091] FIG. 13A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#7 or AOC#8) at the indicated concentration. FIG. 13B is a line graph showing the knockdown of the BCL11A mRNA transcript in erythroid- progenitor cells 72 hours after the first dose with the indicated AOC (AOC#7 or AOC#8) at the indicated concentration.5. DETAILED DESCRIPTION

[0092] The inventors have, inter alia, discovered that molecular payloads (e.g., oligonucleotides) capable of modulating (e.g., inhibiting) expression and / or activity of genes capable of modulating (e.g., repressing) e.g., fetal hemoglobin production, can be specifically targeted to hematopoietic cells (e.g., erythroid precursor cells) through conjugation to a targeting agent (e.g., an anti-TFR antibody). As such, the conjugates described herein are useful, inter alia, for the treatment of hemoglobinopathies (including, e.g., sickle cell disease and thalassemias). Assuch, the current disclosure provides conjugates; and their use in, inter alia, pharmaceutical compositions, and methods of treating diseases (e.g., hemoglobinopathies).TABLE OF CONTENTS5.1 Definitions5.2 Conjugates5.3 Hematopoietic Cell Targeting Agents5.3.1 TFR Targeting Agents5.3.1.1 TF Proteins5.3.1. l(i) Exemplary hTF Variant Proteins5.3.1. l(ii) Heterologous Moieties5.3.1.2 TFR Binding Peptides and Antibody-Like Scaffolds5.3.1.2(i) Exemplary TFR Binding Peptides and Antibody-Like Scaffolds5.3.1.2(ii) Heterologous Moieties5.3.1.3 Anti-TFR (e.g., Anti-TFRl) Antibodies5.3.1.3(i) Exemplary Anti-TFR (e.g., Anti-TFRl) Antibodies5.3.1.4 Ig Effector Function5.3.1.5 Promotion of Heterodimerization5.3.1.6 Ig Constant Region Variations for Site Specific Conjugation5.3.L7 Exemplary Variant Fc Regions5.4 Methods of Making Proteins (e.g., Targeting Agents)5.5 Molecular Payloads5.5.1 Oligonucleotides5.5.1.1 Overall Length5.5.1.2 Targeting Region5.5.1.3 Antisense Oligonucleotides5.5.1.3(i) Overall Length5.5.1.3(H) Targeting Region5.5.1.4 RNAi Agents5.5.1.4(i) Antisense Strand5.5.1.4(i)(a) Overall Length5.5.1.4(i)(b) Targeting Region5.12.2 Methods of Modulating Expression of a Target Gene, Nucleic Acid (e.g., mRNA), and / or Protein5.12.3 Methods of Reducing or Inhibiting Expression of a Target Gene, Nucleic Acid (e.g., mRNA), and / or Protein5.12.4 Methods of Modulating Splicing of a Target mRNA5.12.5 Methods of Inducing Expression of Fetal Hemoglobin5.12.6 Methods of Treating an Inherited Blood Disorder5.12.7 Methods of Treating Hemoglobinopathies5.12.8 Methods of Treating an Inherited Bone Marrow Failure Syndrome5.13 Kits5.1 Definitions

[0093] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0095] In this disclosure, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0096] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and “consisting essentially of’ are also provided herein.

[0097] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0098] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0099] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the ait, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When particular values or compositions are provided in the disclosure, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value or composition.

[0100] Where proteins are described herein, it is understood that nucleic acid molecules (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) encoding the protein are also provided herein.

[0101] Where proteins, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, nucleic acid molecules, vectors, carriers, etc. are also provided herein.

[0102] Where proteins, nucleic acid molecules, etc. are described herein, it is understood that recombinant forms of the proteins, nucleic acid molecules, etc. are also provided herein.

[0103] Where polypeptides or sets of polypeptides are described herein, it is understood that proteins comprising the polypeptides or sets of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa.

[0104] Where proteins are described herein, it is understood that polypeptides comprising the same amino acid sequence either linear or folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein.

[0105] As used herein, the term “administering” refers to the physical introduction of an agent (e.g., a conjugate described herein), e.g., a therapeutic agent (or a precursor of an agent (e.g., a precursor of a therapeutic agent) that is metabolized or altered within the body of the subject to produce the agent (e.g., therapeutic agent) in vivo) to a subject, using any of the various methods and delivery systems known to those skilled in the ait. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. Administeringincludes self-administration.

[0106] The terms “agent” and “moiety” arc used interchangeably herein and arc used generically to describe any macro or micro molecule (and any combination thereof). Exemplary agents include, but are not limited proteins, peptides, nucleic acid molecules (e.g., DNA, RNA), small molecules, carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG), conjugates (e.g., described herein), and any combination of the foregoing. Agents may contain more than one individual agent (wherein the individual agents are the same or different). For example, an agent may comprise an antibody (e.g., a targeting agent described herein) and an oligonucleotide (e.g., an oligonucleotide described herein). Agents as defined herein include e.g., conjugates described herein.

[0107] As used herein, the term “affinity” refers to the strength of the binding of one protein (e.g., an Antibody) to another protein (e.g., an Antigen). The affinity of a protein is measured by the dissociation constant Kd, defined as [Antibody] x [Antigen] / [Antibody-Antigen] where [Antibody-Antigen] is the molar concentration of the Antibody-Antigen complex, [Antibody] is the molar concentration of the unbound Antibody and [Ligand] is the molar concentration of the unbound Antigen. The affinity constant Ka is defined by 1 / Kd. Standard methods of measuring affinity are known to the person of ordinary skill in the art. Exemplary methods of measuring affinity include, surface plasmon resonance (SPR) (e.g., BIAcore®-based assay), a common method known in the ait (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, the full contents of each of which are incorporated by reference herein for all purposes).

[0108] As used herein, the term “antibody” or “antibodies” is used in the broadest sense and encompasses various immunoglobulin (Ig) (e.g., human Ig (hlg), murine Ig (mlg)) structures, including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity (i.e., antigen binding fragments or variants). The term antibody thus includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising antibody CDRs, VH regions, and / or VL regions; and antibody-like scaffolds (e.g., fibronectins). Examples of antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, a variable domainof a new antigen receptor beta-lactamase (VNAR fragments), affybodies, diabodies, tribodies, hctcroconjugatc antibodies, antibody-drug conjugates, single domain antibodies (e.g.,VHH, (VHH)2), single chain antibodies, single-chain Fvs (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab’)2 fragments, disulfide-linked Fvs (sdFv), Fc fusions (e.g., Fab-Fc, scFv- Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-FC), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. Antibodies can be of Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgGi, IgG2, IgG , IgG4, IgAi or IgA2), or any subclass (e.g., IgG2a or IgG2b) of Ig). In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgGi or IgG4) or subclass thereof. In certain embodiments, antibodies described herein are mlgG antibodies, or a class (e.g., mlgGl or mIgG2a) or subclass thereof. In some embodiments, the antibody is a human, humanized, or chimeric IgGi or IgG4 monoclonal antibody. In some embodiments, the term antibodies refers to a monoclonal or polyclonal antibody population. Antibodies described herein can be produced by any standard methods known in the art, e.g., recombinant production in host cells, see, e.g., § 5.4; or synthetic production.

[0109] As used herein, the term “antibody-like scaffold” refers to non-Ig based antigen binding domain. Various antibody-like scaffolds are known in the art. For example, 10th type III domain of fibronectin (e.g., AdNectins®) and designed ankyrin repeat proteins (e.g., DARPins®) have been used as alternative scaffolds for antigen -binding domains, see, e.g., Gebauer and Skcrra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008), the full contents of each of which is incorporated by reference herein for all purposes. Exemplary antibody-like scaffolds include, but are not limited to, lipocalins (see, e.g., US7250297) (e.g., Anticalin®), protein A-derived molecules such as z- domains of protein a (see, e.g., US5831012) (e.g., Affibody®), A domains of membrane receptors stabilized by disulfide bonds and Ca2+ (see, e.g., US7803907) (e.g., Avimer / Maxibody®), a serum transferrin (see, e.g., US2004023334) (e.g., Transbody®); a designed ankyrin repeat protein (see, e.g., US7417130) (e.g., DARPin®), a fibronectin (see, e.g., US6818418) (e.g., AdNectin®), a C- type lectin domain (see, e.g., US2004132094) (e.g., Tetranectin®); a human gamma-crystallin or ubiquitin (see, e.g., US7838629) (e.g., Affilin®); a kunitz type domain of human protease inhibitors (see, e.g., US2004209243), C-Type Lectins (see, e.g., US2004132094) (e.g.,Tetranectins®), cysteine knots or knottins (see, e.g., US7186524) (e.g., Microbodies®), nucleic acid aptamers (see, e.g., US5475096), thioredoxin A scaffold (see, e.g., US6004746) (peptide aptamers), and 10th type III domain of fibronectin (see, e.g., US6818418) (e.g., AdNectins®), and cystine-dense peptides (see, e.g., W02023023031). Additional exemplary antibody-like scaffolds are known in the art and for example described in Storz U. Intellectual property protection: strategies for antibody inventions. MAbs. 2011 ;3(3):310-317. doi:10.4161 / mabs.3.3.15530. The entire contents of each of the foregoing references is incorporated herein by reference for all purposes. Antibody like scaffolds include e.g., naturally occurring antigen binders, variant (e.g., functional variants) of naturally occurring antigen binders, fragments (e.g., functional fragments) of naturally occurring antigen binders, and synthetic antigen binders (i.e., not naturally occurring antigen binders).

[0110] As used herein, the term “antibody dependent cell mediated cytotoxicity” or “ADCC” refers to an immune mechanism leading to the lysis of antibody (or an Fc region containing protein) (e.g., an Ig Fc containing fusion protein described herein)-coated target cells by immune effector cells (e.g., NK cells). As used herein, the term “reduced ADCC” and the like refers to either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody (or an Ig Fc region containing protein) (e.g., an Fc region containing fusion protein described herein) in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or an increase in the concentration of antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC defined above. The reduction in ADCC is relative to the ADCC mediated by the same antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered (e.g., does not comprise one or more amino acid variation, e.g., amino acid substitution, that mediates a decrease in ADCC). For example the reduction in ADCC mediated by an antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) comprising in its Fc region an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) without said amino acid substitution in the Fc region.

[0111] As used herein, the term “antisense oligonucleotide” or “ASO” refer to the standard single stranded oligonucleotides known in the art that arc e.g., capable of modulating expression of a target gene (or protein) by hybridizing to a target nucleic acid (e.g., an mRNA encoded by the target gene and encoding the target protein), in particular to a contiguous sequence on a target nucleic acid. Antisense oligonucleotides include DNA, RNA, and hybrid DNA / RNA oligonucleotides.

[0112] As used herein, the term “antisense strand” with reference to an oligonucleotide described herein (e.g., an RNAi agent (e.g., siRNA agent), an antisense oligonucleotide) refers to an oligonucleotide that comprises a region of complementarity comprising a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to a target nucleic acid sequence (e.g., a target mRNA (e.g., a portion of a target mRNA). In the case of single stranded oligonucleotides (e.g., antisense oligonucleotides) the antisense strand will be the only strand. In the case of double stranded oligonucleotides (e.g., siRNAs) the sense strand will typically be paired with a sense strand (as described herein).

[0113] As used herein, the term “ BCL11 Transcription Factor A” or “BCL11A” refers to the C2H2 type zinc-finger transcription factor that functions, inter alia, in the repression of fetal hemoglobin and the switch from fetal hemoglobin to adult hemoglobin. The amino acid sequence of a reference human BCL11A (hBCLUA) protein is set forth in SEQ ID NO: 291 (NCBI Ref.: NP_075044.2).

[0114] As used herein, the term “bicyclic sugar” refers to a modified sugar (e.g., ribose, deoxyribose) moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In some embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In some embodiments, the furanosyl sugar moiety is a ribosyl moiety.

[0115] As used herein, the term “bicyclic nucleoside” (“BNA”) is a nucleoside comprising a bicyclic sugar.

[0116] As used herein, the term “blunt end” refers to a double stranded oligonucleotide that does not contain any unpaired nucleotides at the end (e.g., 3' terminus, 5' terminus) of the double stranded oligonucleotide (i.e., no nucleotide overhang(s)). The double stranded oligonucleotide can have, for example, a blunt end at the 3' end, 5' end, or both the 3' and 5' end of the molecule.

[0117] As used herein, the term “CDR” or “complementarity determining region” refers to thenoncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chcm. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), the entire contents of each of which is incorporated herein by reference for all purposes. Unless otherwise specified, the term “CDR” is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). A person of ordinary skill in the art would be able to determine the CDRs as defined by another scheme, e.g., Chothia, IMGT, using ordinary methods known in the ail.

[0118] The terms “CHI” and “CHI region” are used interchangeably herein and refer to the first constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CHI region is set forth in SEQ ID NO: 171; and the amino acid sequence of an exemplary reference h!gG4 CHI region is set forth in SEQ ID NO: 184.

[0119] The terms “CH2” and “CH2 region” are used interchangeably herein and refer to the second constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CH2 region is set forth in SEQ ID NO: 173; and the amino acid sequence of an exemplary reference hIgG4 CH2 region is set forth in SEQ ID NO: 186.

[0120] The terms “CH3” and “CH3 region” are used interchangeably herein and refer to the third constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CH3 region is set forth in SEQ ID NO: 174; and the amino acid sequence of an exemplary reference hIgG4 CH3 region is set forth in SEQ ID NO: 187.

[0121] As used herein, the term “complementary” in reference to a first nucleotide sequence (e.g., a sense strand or a target mRNA) in relation to a second nucleotide sequence (e.g., an antisense strand or an antisense oligonucleotide), refers to the ability of a nucleic acid molecule comprising the first nucleotide sequence to hybridize to a nucleic acid molecule comprising the second nucleotide sequence and form a double stranded region (through base pair hydrogen bonds) under suitable in vivo or vitro conditions (e.g., under certain standard conditions, under mammalian (e.g., human) physiological conditions). A person of ordinary skill in the ail would be able to select the set of conditions most appropriate for a hybridization test. Complementary sequences include, e.g., Watson-Crick base pairs. For example, complementary nucleobase pairs include adenine (A) and thymine (T); adenine (A) and uracil (U); and cytosine (C) and guanine (G). Complementary nucleobase pairs include natural and modified nucleotides, and nucleotide mimics, at least to theextent that the above hybridization requirements are fulfilled. As such, determinations of complementarity (as described herein) arc independent of nucleotide chemical modifications (e.g., as described herein). For example, (C) and 5-methyl cytosine (mC) are both complementary to (G).

[0122] As used herein, the term “conjugation” refers to the operable connection (e.g., chemical conjugation) of at least a first agent (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)) with a second agent (e.g., a protein (e.g., a targeting agent (e.g., a targeting agent described herein (e.g., a hematopoietic cell targeting agent (erythroid precursor cell targeting agent described herein (e.g., an anti-TFR (e.g., anti-hTFR (e.g., anti-hTFRi)) antibody described herein))))). The first agent can be directly connected to the second agent or indirectly connected (e.g., through a linker (e.g., as described herein)). Methods of operably connected two agents (e.g., chemical conjugation methods) are well known in the art, as are commercially available conjugation reagents and kits, with detailed instructions for their use readily available from the commercial suppliers. Operable connection (e.g., chemical conjugation) includes both covalent and non- covalent conjugation. In some embodiments, the operable connection (e.g., chemical conjugation) comprises the covalent linkage of the first agent (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)) with the second agent (e.g., a protein (e.g., a targeting agent (e.g., a targeting agent described herein (e.g., a hematopoietic cell targeting agent (erythroid precursor cell targeting agent described herein (e.g., an anti-TFR (e.g., anti-hTFR (e.g., anti-hTFRi)) antibody described herein))))).

[0123] The terms “constant region” and “constant domain” are used interchangeably herein and refer to a carboxyl terminal portion of a light and / or heavy chain of a full-length antibody which is not directly involved in binding of an antibody to antigen, but which can exhibit various effector functions, such as interaction with an Ig Fc receptor (e.g., Fc gamma receptor). The constant region of an Ig molecule generally has a more conserved amino acid sequence relative to an Ig variable domain.

[0124] As used herein, the term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology. The term disease includes infection (e.g., a viral, bacterial, fungal, protozoal infection).

[0125] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple dcoxyribonuclcotidcs that arc polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[0126] As used herein, the term “double stranded oligonucleotide” refers to a complex of two nucleic acid molecules comprising a double stranded region comprising two anti-parallel and at least partially (e.g., substantially, fully) complementary nucleic acid sequences that form the double stranded region. For example, in some embodiments, the double stranded oligonucleotide comprises a sense strand and an antisense strand.

[0127] The term “effector function” when used in reference to an Ig Fc region or a protein comprising an Ig Fc region (e.g., a full-length antibody) refers to those biological activities attributable to the Ig Fc region of a typical full-length antibody, which therefore vary with the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla)), and Clq binding.

[0128] As used herein, the term “erythroid precursor cell” refers to any precursor of a mature erythrocyte (i.e., a mature enucleated red blood cell). As such, erythroid precursor cells include, but are not limited to, megakaryocyte erythroid progenitor cells, proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, late erythroblast cells, and reticulocytes. In some embodiments, erythroid precursor cells include megakaryocyte erythroid progenitor cells, proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, and late erythroblast cells. In some embodiments, erythroid precursor cells include proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, and late erythroblast cells.

[0129] As used herein, the term “erythroid precursor cell targeting agent” refers to an agent that specifically binds to an antigen expressed on an erythroid precursor cell (or a subset thereof). For example, the antigen expressed in or on the erythroid precursor cell may be a membrane protein, for example an integral membrane protein or a peripheral membrane protein. Typically, an erythroid precursor cell targeting agent specifically binds to an antigen on the erythroid precursor cell that facilitates internalization of the erythroid precursor cell targeting agent (and any associated molecular payload) into the erythroid precursor cell. In some embodiments, an ery throid precursor cell targeting agent specifically binds to an internalizing, cell surface receptor on theerythroid precursor cell and is capable of being internalized into the erythroid precursor cell through receptor mediated internalization. In some embodiments, the erythroid precursor cell targeting agent is a protein (e.g., antibody), a peptide, a nucleic acid (e.g., an aptamer), or small molecule. In some embodiments, the erythroid precursor cell targeting agent is linked to a molecular payload.

[0130] As used herein, the term “EU numbering system” refers to the EU numbering convention for the constant regions of an antibody, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, the entire contents of each of which is incorporated herein by reference for all purposes.

[0131] As used herein, the term “Fab” refers to an antigen binding domain that comprises a Fab heavy chain that comprises from N- to C-terminus a VH region and a CHI region; and a light chain comprising from N- to C-terminus a VL region and a CL region; and wherein the Fab heavy chain and the light chain associate to form an antigen binding domain.

[0132] The term “Fab-Fc” as used herein refers to an antibody that comprises a Fab operably linked to an Fc region.

[0133] As used herein, the term “Fc region” refers to the C-terminal region of a Ig (e.g., a human Ig) heavy chain that comprises from N- to C-terminus at least a CH2 region operably connected to a CH3 region. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably connected to the N-terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region (e.g., comprises one or more amino acid modification), see, e.g., §§ 5.3.2.1, 5.3.2.2, 5.3.2.3, 5.3.2.4. Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (K. O. Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” 2019, Frontiers in Immunology, V. 10, Art. 1296, pp. 1-20, the entire contents of which is incorporated herein by reference for all purposes).

[0134] As used herein, the terms “first” and “second” with respect to Fc regions etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation in the protein unless explicitly so stated. For example, an antibody described herein (e.g., in the case of a full-length antibody) may contain two Fc regions that associate e.g., via one or more covalent (e.g., disulfide) bond.

[0135] As used herein, the term “framework region” or “FR region” refers to the amino acid residues that arc part of the variable region of an antibody, but arc not part of the CDRs (e.g., using the Kabat definition of CDRs).

[0136] As used herein, the term “full-length antibody” refers to an antibody having a structure substantially similar’ to a native antibody structure (i) a first Ig light chain comprising from N- to C-terminus a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first Ig heavy chain comprising from N- to C-terminus a heavy chain variable region (VH) region, a CHI region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain comprising from N- to C-terminus a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region; (iv) a second Ig light chain comprising from N- to C-terminus a VL region and a VH region; wherein said first light chain and said first heavy chain associate to form a first antigen binding domain; wherein said second light chain and said second heavy chain associate to form a second antigen binding domain; and wherein said first heavy chain and said second heavy chain associate to form a dimer. In some embodiments, the two heavy chains comprise a substantially identical amino acid sequence; and the two light chains comprise a substantially identical amino acid sequence. In some embodiments, the two heavy chains comprise a substantially identical amino acid sequence except for one or more amino acid modifications that promote heterodimerization of the correct heavy chains (e.g., as described herein); and the two light chains comprise a substantially identical amino acid sequence. Antibody chains may be substantially identical but not entirely identical if they differ due to post-translational modifications, such as C-terminal cleavage of lysine residues, alternative glycosylation patterns, etc.

[0137] As used herein, the term “fully complementary” means that in a hybridized pair of a first nucleic acid molecule and a second nucleic acid molecule, 100% (all), of the bases in a contiguous sequence of the first nucleic acid molecule will hybridize with the same number of bases in a contiguous sequence of the second nucleic acid molecule. The contiguous sequence may comprise all or a part of the first and / or second nucleic acid molecule.

[0138] The term “functional variant” as used herein in reference to a protein refers to a protein that comprises at least one but no more than 20%, not more than 15%, not more than 12%, no more than 10%, no more than 8% amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference protein, wherein the protein retains at least one particularfunction of the reference protein. Not all functions of the reference protein (e.g., wild type) need be retained by the functional variant of the protein. In some instances, one or more functions arc selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein.

[0139] The term “functional fragment” as used herein in reference to a protein refers to a fragment of a reference protein that retains at least one particular function. Not all functions of the reference polypeptide or protein need be retained by a functional fragment of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein.

[0140] As used herein, the term “fuse” and grammatical equivalents thereof refer to the operable connection of at least a one polypeptide derived from a first polypeptide to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are different. The term fuse encompasses both a direct connection of the at least two polypeptides through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker).

[0141] As used herein, the term “fusion protein” and grammatical equivalents thereof refers to a protein that comprises at least one polypeptide derived from a first polypeptide operably connected to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are wherein the first and second polypeptides are not naturally found operably connected together. The at least two polypeptides of the fusion protein can be directly operably connected through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker). Therefore, for example, the term fusion protein encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A - Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A - peptide linker - Polypeptide B). In some embodiments, the first polypeptide and the second polypeptide are different.

[0142] As used herein, the term “half-life extension moiety” refers to a moiety (e.g., small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that when conjugated or otherwise operably connected (e.g., fused) to a polypeptide or protein (the subject polypeptide or protein), increases the half-life of the subject polypeptide or protein in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the polypeptide or protein can be evaluated utilizing in vitro models known in the art.

[0143] As used herein, the term “half-life extension polypeptide” refers to a polypeptide that when operably connected to another polypeptide (the subject polypeptide), increases the half-life of the subject polypeptide in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the polypeptide or protein can be evaluated utilizing in vitro models known in the art.

[0144] As used herein, the term “heavy chain” refers to the portion of an immunoglobulin (e.g., a human Ig) that typically comprises from N- to C-terminus a heavy chain variable region (VH), a CHI region, a hinge region, a CH2 region, and a CH3 region. The constant regions of the heavy chain (i.e., the CHI region, the hinge region, the CH2 region, and the CH3 region) can be any distinct isotype, for example, human alpha (a), delta (5), epsilon (E), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to the hlgA, hlgD, IgE, hlgG, and hlgM classes of human antibodies, respectively, including subclasses of hlgG, e.g., hlgGi, h!gG2, hlgGa, and h!gG4. As used herein, the term “heavy chain” when used in reference to a human antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (E), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to human IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of human IgG, e.g., IgGi, IgG2, IgG?. and IgG4.

[0145] As used herein, the term “hematopoietic cell” refers to any blood cell. As such, the term hematopoietic cell, includes, but is not limited to, hematopoietic pluripotent stem cells (HSPCs), common myeloid progenitor cells, megakaryocyte erythroid progenitor cells, erythroid progenitor cells, proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, late erythroblast cells, reticulocytes, megakaryocytes, platelets, granulocyte monocyte progenitor cells, monoblasts, promonocytes, monocytes, macrophages, myeloblasts, promyelocytes, myelocytes, eosinophils, basophils, neutrophils, common lymphoid progenitor cells, pro-NK lymphoblasts, NK cells, pro-B lymphoblasts, B lymphocytes, pro-T lymphoblasts, T lymphoblasts, and plasma cells.

[0146] As used herein, the term “hematopoietic cell targeting agent” refers to an agent that specifically binds to an antigen expressed on a hematopoietic cell (or a subset thereof). For example, the antigen expressed in or on the hematopoietic cell may be a membrane protein, for example an integral membrane protein or a peripheral membrane protein. Typically, a hematopoietic cell targeting agent specifically binds to an antigen on the hematopoietic cell that facilitates internalization of the hematopoietic cell targeting agent (and any associated molecularpayload) into the hematopoietic cell. In some embodiments, a hematopoietic cell targeting agent specifically binds to an internalizing, cell surface receptor on the hematopoietic cell and is capable of being internalized into the hematopoietic cell through receptor mediated internalization. In some embodiments, the hematopoietic cell targeting agent is a protein (e.g., antibody), a peptide, a nucleic acid (e.g., an aptamer), or small molecule. In some embodiments, the hematopoietic cell targeting agent is linked to a molecular payload.

[0147] As used herein, the term “heterologous,” when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a nucleic acid molecule comprising a “heterologous moiety” means a nucleic acid molecule that is joined to a moiety (e.g., carbohydrate, small molecule, polypeptide, polynucleotide, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the nucleic acid molecule in nature.

[0148] The terms “hinge” or “hinge region” are used interchangeably herein and refer to the hinge region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl hinge region is set forth in SEQ ID NO: 172; and the amino acid sequence of an exemplary reference hIgG4 hinge region is set forth in SEQ ID NO: 185.

[0149] As used herein, the term “isolated” with reference to an agent (e.g., a protein, nucleic acid molecule, etc.) refers to an agent (e.g., a protein, nucleic acid molecule, etc.) that is substantially free of other cellular components with which it is associated in the natural state.

[0150] As used herein, the term “KLF Transcription Factor 1” or “KLF1” refers to the zinc finger transcription factor that functions, inter alia, in the repression of fetal hemoglobin and the switch from fetal hemoglobin to adult hemoglobin. The amino acid sequence of a reference human KLF1 (hKLFl) protein is set forth in SEQ ID NO: 297 (NCBI Ref.: NP_006554.1).

[0151] As used herein, the term “modified nucleotide,” “nucleotide modification,” or use of the term “modification” and the like in reference to a nucleotide or nucleic acid sequence refers to a nucleotide comprising a chemical modification, e.g., a modified sugar moiety, a modified nucleobase, and / or a modified internucleoside linkage, or any combination thereof. Exemplary modifications are provided herein, see, e.g., § 5.5.1.5. In certain embodiments of the instant disclosure, inclusion of a deoxynucleotide - which is acknowledged as a naturally occurring form of nucleotide - if present within an RNA oligonucleotide is considered to constitute a modified nucleotide.

[0152] As used herein, the term “molecular payload” refers to an agent that functions to modulate a biological outcome. In some embodiments, a molecular payload is operably connected to a targeting agent (e. ., a targeting agent described herein (e.g., an anti-TFR antibody)). In some embodiments, the molecular payload is a small molecule, a protein, a peptide, or an oligonucleotide. In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide described herein, see, e.g., § 5.5.1). In some embodiments, the molecular payload functions to modulate (e.g., inhibit) the transcription of a DNA molecule, to modulate (e.g., inhibit) the translation of an RNA (e.g., mRNA) molecule, to modulate (e.g., inhibit) the expression of a protein, or to modulate (e.g., inhibit) the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a target nucleic acid molecule (e.g., an RNA molecule encoded by a target gene (e.g., an mRNA molecule encoded by a target gene)).

[0153] As used herein, the term, “non-complementary nucleotide mismatch” refers to a nucleotide within a region of complementarity (as described herein) that is not complementary to the corresponding nucleotide in the target nucleic acid molecule.

[0154] The terms “nucleic acid molecule,” “polynucleotide,” and “oligonucleotide” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single- stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non-natural, or altered internucleoside linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U).

[0155] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that extends from the double stranded region of a double stranded nucleic acid molecule. For example, when a 3'-end of one strand of a double stranded nucleic acid molecule extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang.

[0156] As used herein, the term “operably connected” refers to the linkage of two agents in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) in frame such that both polypeptides are functional (e.g., a fusion protein described herein). Or for example, a transcription regulatory polynucleotide e.g., a promoter, enhancer, or other expression control element is operably linked to a polynucleotide that encodes a protein if it affects the transcription of the polynucleotide that encodes the protein. The term “operably connected” also refers for example to the conjugation of a first agent (e.g., a protein (e.g., an antibody)) to a second agent (e.g., an oligonucleotide) wherein the first and second agent are both capable of mediating their function.

[0157] As used herein, “partially complementary” means that in a hybridized pair of a first nucleic acid molecule and a second nucleic acid molecule, at least 70%, but not all, of the bases in a contiguous sequence of the first nucleic acid molecule will hybridize with the same number of bases in a contiguous sequence of the second nucleic acid molecule. The contiguous sequence may comprise all or a part of a first or second nucleic acid molecule.

[0158] The determination of “percent identity” between two sequences (e.g., protein (amino acid sequences) or oligonucleotide (nucleic acid sequences)) can be accomplished using a mathematical algorithm. Determinations of identity (as described herein) are independent of nucleotide chemical modifications (e.g., as described herein). For example, (mC) is identical to (C) for the purposes of determining identity. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0159] As used herein, the term “pharmaceutical composition” means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent (e.g., a conjugate described herein) and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier or diluent” means a substance intended for use in contact with the tissues of human beings and / or non-human animals, and without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit / risk ratio.

[0160] As used herein, the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more).

[0161] As used herein, the terms “protein”, “polypeptide”, and peptide refers to a polymer of at least 2 (e.g., at least 5) amino acids linked by a peptide bond. The term “polypeptide” does not denote a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2-50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” and “protein” are used interchangeably herein.In some embodiments, the protein is folded into its three-dimensional structure. Where polypeptides (e.g., in a linear (i.e., primary) structure arc contemplated herein, it should be understood that proteins folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa. Proteins include e.g., naturally occurring proteins, variant (e.g., functional variants) of naturally occurring proteins, fragments (e.g., functional fragments) of naturally occurring proteins, and synthetic proteins (i.e., not naturally occurring proteins).

[0162] As used herein, the term “region of complementarity” refers to a portion of a first nucleic acid molecule comprising a nucleotide sequence that is at least partially complementary to the nucleotide sequence of at least a portion of a second nucleic acid molecule.

[0163] The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar’ is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered intemucleoside linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule.

[0164] As used herein, the term “RNAi agent” refers to an agent that contains one or more RNA molecules which can mediate the targeted cleavage of an RNA molecule (e.g., an mRNA molecule) via an RNA-induced silencing complex (RISC) pathway. The RNAi agent is thereby capable of e.g., modulating, e.g., inhibiting, the expression of a target gene or protein in a cell, e.g., a cell within a subject, such as a mammalian subject. RNAi agents include, for example, siRNAs, miRNAs, and shRNAs.

[0165] The term “scFv” or “single chain variable fragment” refers to an antibody that comprises a VH region operably connected via a peptide linker to a VL region, wherein the VH and VL regions associate to specifically bind an antigen (e.g., form an antigen binding domain). In some embodiments, the scFv comprises from N- to C-terminus an VH region, a peptide linker, and an VL region. In some embodiments, the scFv comprises from N- to C-terminus an VL region, a peptide linker, and an VH region.

[0166] The term “(scFv ” as used herein refers to an antibody that comprises a first and a second scFv operably connected (e.g., via a peptide linker). The first and second scFv can specifically bind the same or different antigens. In some embodiments, the first and second scFvare operably connected by a peptide linker.

[0167] The term “scFv-Fc” as used herein refers to an antibody that comprises a scFv operably linked (e.g., via a peptide linker) to an Fc domain or subunit of an Fc domain. In some embodiments, a scFv is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first scFv is operably connected to a first Fc domain and a second scFv is operably connected to a second Fc domain of a first and second Fc domain pair.

[0168] The term “(scFv)2-Fc” as used herein refers to a (scFv)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (scFv)2 is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (scFv)2 is operably connected to a first Fc domain and a second (scFv)2 is operably connected to a second Fc domain of a first and second Fc domain pair.

[0169] As used herein, the term “sense strand” refers to an RNA molecule (e.g., part of an RNAi agent (e.g., described herein), part of a dsRNA agent (e.g., described herein)) that comprises a region that is at least partially (e.g., substantially, fully) complementary to a region of the antisense strand (as defined herein). The sense strand is often referred to as such with reference to the orientation of the sequence of the sense strand being the same with respect to a target RNA (e.g., mRNA sequence).

[0170] As used herein, the term “single domain antibody” or “sdAb” refers to an antibody having a single monomeric variable antibody domain. A sdAb is able to specifically bind to a specific antigen. A VHH (as defined herein) is an example of a sdAb.

[0171] As used herein, the term “specifically binds” refers to preferential interaction, i.e., significantly higher binding affinity, between a first protein (e.g., an antibody) and a second protein (e.g., an antigen) relative to other amino acid sequences. Herein, when a first protein is said to “specifically bind” to a second protein, it is understood that the first protein specifically binds to an epitope of the second protein. The term “epitope” refers to the portion of the second protein that the first protein specifically recognizes. The term specifically binds includes molecules that are cross reactive with the same epitope of a different species. For example, an antibody that specifically binds human TFR may be cross reactive with TFR of another species (e.g., cynomolgus, murine, etc.), and still be considered herein to specifically bind human TFR. A protein can specifically bind more than one different protein. Specific binding can be measured, e.g., through measuring binding affinity (e.g., using standard methods known in the art anddescribed herein (e.g., surface plasmon resonance (SPR) (e.g., BIAcore®-based assay), a common method known in the art (see, e.g., Wilson, Science 295:2103, 2002; Wolff ct al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, the full contents of each of which are incorporated by reference herein for all purposes).

[0172] As used herein, the term “subject” includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the method subject is a non-human mammal. In some embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).

[0173] As used herein, “substantially complementary” means that in a hybridized pair of a first nucleic acid molecule and a second nucleic acid molecule, at least 85%, but not all, of the bases in a contiguous sequence of the first nucleic acid molecule will hybridize with the same number of bases in a contiguous sequence of the second nucleic acid molecule. The contiguous sequence may comprise all or a part of a first or second nucleic acid molecule.

[0174] As used herein, the term “target nucleic acid sequence” refers to a contiguous portion of the nucleotide sequence of a nucleic acid sequence (e.g., an mRNA molecule formed during the transcription of a target gene). In some embodiments, the target nucleic acid sequence is an mRNA molecule formed during the transcription of a target gene. In some embodiments, the target nucleic acid molecule comprises an mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence (e.g., mRNA) will be at least long enough to serve as a substrate for an oligonucleotide described herein (e.g., an antisense oligonucleotide, an RNAi agent, etc.).

[0175] As used herein, the term “therapeutic agent” refers to an agent (e.g., a conjugate described herein) capable of achieving a desired therapeutic result in a subject or ex vivo (e.g., capable of treating a disease as defined herein) when administered at a therapeutically effective amount.

[0176] As used herein, the term “therapeutically effective amount” of a therapeutic agent refers to any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, improves a disease condition, e.g., protects a subject against the onset of a disease (or infection); improves a symptom of disease or infection, e.g., decreases severity of disease or infection symptoms, decreases frequency or duration of disease or infection symptoms, increases disease or infection symptom- free periods; prevents or reduces impairment or disability due to the disease or infection; or promotes disease (or infection) regression. The ability of a therapeutic agent to improve a disease condition can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0177] As used herein, the term “translatable RNA” refers to any RNA that encodes at least one peptide or protein and can be translated to produce the encoded peptide or protein in vitro, in vitro, in situ or ex vivo. This includes, e.g., messenger RNA (mRNA).

[0178] As used herein, the term “transferrin” or “TF” refers to the plasma glycoprotein transferrin that functions, inter alia, in iron metabolism and the transport of iron through the blood to various tissues, such as the liver, spleen, and bone marrow. The amino acid sequence of a reference human TF (hTF) protein is set forth in SEQ ID NO: 3 (UniProt Accession P02787).

[0179] As used herein, the term “transfenin receptor” or “TFR” refers to the transmembrane homodimeric glycoprotein that functions, inter alia, in the cellular uptake of iron from the plasma glycoprotein transferrin. The term TFR includes, where applicable, multiple isoforms and homologs. For example, human TFR (hTFR) includes homologs hTFRl and hTFR2. The amino acid sequence of a reference hTFRl protein is set forth in SEQ ID NO: 1 (UniProt Accession P02786|). TFR1 is also commonly known in the art as CD71. The terms TFR1 and CD71 are used interchangeably herein. The amino acid sequence of a reference hTFR2 protein is set forth in SEQ ID NO: 2 (UniProt Accession Q9UP52).

[0180] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease and / or symptom(s) associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disease does not require that the disease, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases theintensity of, or cures a disease and / or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of e.g., a conjugate described herein (or a carrier, pharmaceutical composition, etc. comprising the same).

[0181] As used herein, the term “variation” or “variant” or use the like in reference to a nucleotide or nucleic acid sequence refers to a nucleic acid molecule that comprises at least one substitution, addition, deletion, or inversion of one or more nucleotide compared to a reference nucleic acid molecule. Likewise, as used herein, the term “variation” or “variant” or use the like with reference to a peptide or protein refers to a peptide or protein that comprises at least one substitution, addition, deletion, or inversion of an amino acid residue compared to a reference peptide or protein.

[0182] A “variation that promotes heterodimerization of a first Fc region and a second Fc region” (or similar phrasing) is a manipulation of the peptide backbone or the post-translational modifications of an Fc region that reduces or prevents the association of a polypeptide comprising the Fc region with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc regions desired to associate i.e., a first Fc region and a second Fc region), wherein the modifications are complementary to each other so as to promote association of the two Fc regions. For example, a modification promoting association may alter the structure or charge of one or both of the Fc regions so as to make their association sterically or electrostatically favorable, respectively. Thus, heterodimerization occurs between a polypeptide comprising the first Fc region and a polypeptide comprising the second Fc region, which might be non-identical in the sense that further components fused to each of the Fc regions {e.g., antigen binding domains) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc region, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically one or more amino acid substitution, in each of the first Fc region and the second Fc region. See, e.g., § 5.3.2.2.

[0183] As used herein, the term “variable region” refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and arc used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0184] The terms “VL” and “VL region” are used interchangeably to refer to an immunoglobulin light chain variable region. A VL region can be incorporated into an antibody, e.g., a scFv, a Fab, a full-length antibody. For example, a scFv comprises a VL region operably connected via a peptide linker to a VH region.

[0185] The terms “VH” and “VH region” are used interchangeably to refer to an immunoglobulin heavy chain variable region. A VH region can be incorporated into an antibody, e.g., a scFv, a Fab, a full-length antibody. For example, a scFv comprises a VH region operably connected via a peptide linker to a VL region.

[0186] The term “VHH” as used herein refers to a type of single domain antibody (sdAb) that has a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camelid mammals (e.g., camels, llamas) which are naturally devoid of light chains or synthetically produced.

[0187] The term “(VHHh” as used herein refers to an antibody that comprises a first and a second VHH operably connected (e.g., via a peptide linker). The first and the second VHH can specifically bind the same or different antigens. In some embodiments, the first and second VHH are operably connected by a peptide linker.

[0188] The term “VHH-Fc” as used herein refers to an antibody that comprises a VHH operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a VHH is operably connected to only a first Fc domain of a first and a second Fcdomain pair. In some embodiments, a first VHH is operably connected to a first Fc domain and a second VHH is operably connected to a second Fc domain of a first Fc and a second Fc pair.

[0189] The term “(VHH)2-Fc” as used herein refers to (VHH)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (VHH)2 is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (VHH)2 is operably connected to a first Fc domain and a second (VHH)2 is operably connected to a second Fc domain of a first Fc and a second Fc pair.

[0190] As used herein, the term “ZBTB7A” or “Zinc Finger And BTB Domain Containing 7 A” refers to the transcription factor that functions, inter alia, in the repression of fetal hemoglobin and the switch from fetal hemoglobin to adult hemoglobin. The amino acid sequence of a reference human ZBTB7A (hZBTB7A) protein is set forth in SEQ ID NO: 294 (NCBI Ref.: NP_056982.1).5.2 Conjugates

[0191] Provided herein are, inter alia, conjugates (e.g., antibody-oligonucleotide conjugates), useful in, inter alia, modulating (e.g., inhibiting, reducing, enhancing) expression and / or activity of a target gene or protein (e.g., within a cell (e.g., an erythroid precursor cell), e.g., within a cell in a subject, e.g., a mammalian subject, e.g., a human subject) (e.g., through binding to a target nucleic acid molecule (e.g., an mRNA molecule)).

[0192] The conjugates described herein comprise a targeting agent (e.g., a hematopoietic cell targeting agent (e.g., described herein)) and a molecular payload (e.g., an oligonucleotide described herein (e.g., an oligonucleotide that alters (e.g., inhibits or reduces) expression or activity of a target gene or protein (e.g., a target gene or protein expressed by a hematopoietic cell (e.g., an erythroid precursor cell)).

[0193] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell.

[0194] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (c) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and / or (d) upon binding to TFR (e.g., hTFR (e.g., TFRI))expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0195] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

[0196] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (c) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and / or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0197] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0198] In some embodiments, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRI)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRI)) or conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRI)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of anerythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the erythroid precursor cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0199] In some embodiments, the conjugates described herein exhibit one or more of the following properties: (a) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the conjugate is internalized into the target cell; (b) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the conjugate does not induce death of the target cell; (c) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the target cell remains viable; (d) upon internalization into a target cell (e.g., an erythroid precursor cell), the conjugate does not induce death of the target cell; and / or (e) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the conjugate does not induce degradation of the target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))).5.3 Hematopoietic Cell Targeting Agents

[0200] As described above, the conjugates described herein comprise a targeting agent (e.g., for targ eting a molecular pay load (e.g., an oligonucleotide described herein) to a specific cell or cells (e.g., within a subject)). In some embodiments, the targeting agent is a hematopoietic cell (e.g., an erythroid precursor cell) targeting agent. In some embodiments, the targeting agent is capable of targeting a molecular payload (e.g., an oligonucleotide described herein) to a hematopoietic cell (e.g., an erythroid precursor cell). In some embodiments, the hematopoietic cell (e.g., an erythroid precursor cell) is present in the bone marrow (e.g., of a subject). In some embodiments, the targeting agent is capable of targeting a molecular payload to one or more cell within the bone marrow. In some embodiments the targeting agent specifically targets erythroid precursor cells (e.g., within the bone marrow (e.g., within a subject)). In some embodiments the targeting agent specifically targets erythroid precursor cells (e.g., within the bone marrow (e.g.,within a subject)) through specific binding to an antigen expressed on the surface of the erythroid precursor cells (e.g., TFR (e.g., hTFR (e.g., hTFRl))).

[0201] It should be appreciated that various types of targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) may be used in accordance with the disclosure. For example, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) may comprise (or consist of) a small molecule, an oligonucleotide (e.g., DNA, RNA, RNA / DNA hybrid) (e.g., an aptamer), a protein (e.g., an antibody, a peptide), a lipid (e.g., a microvesicle), or a carbohydrate (e.g., a polysaccharide). In some embodiments, the targeting agent is a protein. In some embodiments, the targeting agent is a peptide. In some embodiments, the targeting agent is an antibody. In some embodiments, the targeting agent is an antibody-like scaffold (e.g., as described herein). Exemplary targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) are described in further detail herein, however, it should be appreciated that the exemplary targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) provided herein are not meant to be limiting.

[0202] In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent specifically binds to an antigen expressed on surface of hematopoietic cells (or one or more subset thereof) (e.g., erythroid precursor cells) (e.g., within the bone marrow) (e.g., TFR (e.g., hTFR (e.g., hTFRl))). In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent specifically binds to an antigen expressed on the surface of erythroid precursor cells (e.g., TFR (e.g., hTFR (e.g., hTFRl))). In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent specifically binds to an antigen expressed on the surface of erythroid precursor cells within the bone marrow (e.g., TFR (e.g., hTFR (e.g., hTFRl))).

[0203] By interacting with one or more molecule (e.g., protein) expressed on the surface of a target hematopoietic cell (e.g., erythroid precursor cell) both tissue (e.g., bone marrow) localization and selective or preferred uptake into hematopoietic cells (e.g., erythroid precursor cells) can be achieved. In some embodiments, molecules (e.g., proteins) that are substrates for hematopoietic cell (e.g., erythroid precursor cell) uptake transporters are useful for delivering a molecular- payload (e.g., an oligonucleotide described herein) into hematopoietic cells (e.g., erythroid precursor cells). Binding to molecules (e.g., proteins) expressed on the surface of hematopoietic cells (e.g., erythroid precursor cells) followed by endocytosis can allow large molecules such as antibodies to enter the hematopoietic cells (e.g., erythroid precursor cells). Forexample, as described in detail below, molecular payloads (e.g., an oligonucleotide described herein) conjugated to transferrin (or a functional fragment or functional variant thereof) or anti- TFR (e.g., hTFR (e.g., hTFRl)) antibodies can be taken up by hematopoietic cells (e.g., erythroid precursor cells) via binding to TFR (e.g., hTFR (e.g., hTFRl)), which may then be endocytosed, e.g., via endocytosis, e.g., clathrin-mediated endocytosis.

[0204] The use of hematopoietic cell (e.g., erythroid precursor cell) targeting agents may be useful for concentrating a molecular payload (e.g., an oligonucleotide described herein) in hematopoietic cells (e.g., erythroid precursor cells (e.g., within the bone marrow (e.g., within a subject))) while reducing toxicity associated with effects in other cells or tissues. In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent concentrates a bound molecular payload (e.g., an oligonucleotide described herein) in hematopoietic cells (e.g., erythroid precursor cell (e.g., within the bone marrow (e.g., within a subject))) as compared to another tissue or cell type within a subject. In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent concentrates a bound molecular payload (e.g., an oligonucleotide described herein) in hematopoietic cells (e.g., erythroid precursor cells) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than an amount in non-hematopoietic cells (e.g., non-erythroid precursor cells). In some embodiments, a toxicity of the molecular payload (e.g., an oligonucleotide described herein) in a subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered to the subject when bound to the hematopoietic cell (e.g., erythroid precursor cell) targeting agent.

[0205] In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent exhibits one or more of the following properties: (a) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent is internalized into the target cell; (b) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent does not induce death of the target cell; (c) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the target cell remains viable; (d) upon internalization into a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursorcell) targeting agent does not induce death of the target cell; and / or (e) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent does not induce degradation of the target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))).5.3.1 TFR Targeting Agents

[0206] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to the transferrin receptor (TFR) (e.g., hTFR (e.g., hTFRl)). In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to hTFR. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds hTFRl.

[0207] TFR1 is a transmembrane homodimeric glycoprotein that functions, inter alia, in the cellular uptake of iron from the plasma glycoprotein transferrin (TF). Iron uptake from transferrin involves the binding of TF to TFR (e.g., TFR1), internalization of TF within an endocytic vesicle by receptor-mediated endocytosis and the release of iron induced by a decrease in endosomal pH. TFR1 is expressed by e.g., placental syncytiotrophoblasts, myocytes, basal keratinocytes, hepatocytes, endocrine pancreas, spermatocytes, and erythroid precursor cells. TFR expression, while known to be highly expressed by erythroid precursor cells, is not expressed by mature erythrocytes. TFR2 is a known homolog of TFR1, but TFR1 is considered the major protein responsible for iron uptake owing to its higher affinity and expression pattern. See, e.g., Derek K. Mar.sec, et al., CD71 (Transferrin Receptor): An Effective Marker for Erythroid Precursors in Bone Marrow Biopsy Specimens, American Journal of Clinical Pathology, Volume 134, Issue 3, September 2010, Pages 429-435, https: / / doi.org / 10.1309 / AJCPCRK3MOAOJ6AT; C. Sieff et al., Changes in Cell Surface Antigen Expression During Hemopoietic Differentiation, Blood, Vol 60 (3), 1982, Pages 703-713, https: / / doi.org / 10.1182 / blood.V60.3.703.703; the entire contents of each of which are incorporated herein by reference for all purposes.

[0208] The amino acid sequence of a reference hTFRl protein is set forth in SEQ ID NO: 1. The amino acid sequence of a reference hTFR2 protein is set forth in SEQ ID NO: 2. The amino acid sequence of a reference hTF protein is set forth in SEQ ID NO: 3. See Table 1, herein.Table 1. The Amino Acid Sequence of a Reference hTFRl., hTFR2, and hTF Protein.

[0209] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR2. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1 and TFR2. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1 but does not specifically bind TFR2. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1 and binds to TFR2 with significantly lower affinity.

[0210] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds hTFRl and one or more of murine TFR1, rat TFR1, a non-human primate TFR1 (e.g., cynomolgus TFR1).

[0211] In some embodiments, the TFR (e.g., hTFR (e.g., hTFRl)) targeting agent enhances the distribution and / or uptake (e.g., into a cell, e.g., into a cell in a subject, e.g., a cell that expresses TFR (e.g., hTFR (e.g., hTFRl)) (e.g., an erythroid precursor cell (e.g., in the bone marrow))) of a molecular payload (e.g., an oligonucleotide described herein) (e.g., RNAi agent (e.g., siRNA), ASO, etc.) (e.g., as compared to an oligonucleotide that lacks the targeting moiety). In some embodiments, the TFR (e.g., hTFR (e.g., hTFRl)) targeting agent alters (e.g., extends) the lifetime (e.g., in vivo) of the molecular payload (e.g., an oligonucleotide described herein) (e.g., RNAi agent (e.g., siRNA), ASO, etc.) (e.g., as compared to an oligonucleotide that lacks the targeting moiety). In some embodiments, the TFR (e.g., hTFR (e.g., hTFRl)) targeting agent provides an enhanced affinity for a selected target, e.g., a selected cell type, compartment (e.g., cell type, tissue, organ or region of the body) (e.g., as compared to an oligonucleotide that lacks the targeting moiety) (e.g., erythroid precursor cells (e.g., in the bone marrow)).

[0212] In some embodiments, the TFR targeting agent does not (or does not significantly) interfere with TF binding to TFR. In some embodiments, the TFR targeting agent does not (or does not significantly) compete for binding to TFR with TF.

[0213] In some embodiments, the TFR targeting agent is a protein. In some embodiments, the targeting agent is a transferrin protein. In some embodiments, the targeting agent is an antibody.In some embodiments, the targeting agent comprises an antibody-like scaffold (e.g., a cysteine dense peptide, see, e.g., W02023023031, the entire contents of which is incorporated by reference herein for all purposes).5.3.1.1 TF Proteins

[0214] In some embodiments, the TFR targeting agent comprises TF (e.g., hTF) (or a functional fragment or variant thereof). In some embodiments, the TFR targeting agent comprises the TFR1 binding domain of TF (e.g., hTF) (or a functional fragment or variant thereof). In some embodiments, the TF (e.g., hTF) (or a functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0215] Variant hTF proteins are known in the art, see, e.g., W02009019314A1, W02008152140A2, EP2216341A1, WO2009149393A2, US8158579B2, the entire contents of each of which are incorporated herein by reference for all purposes. In some embodiments, the variant hTF protein exhibits increased stability and / or long plasma half-life (e.g., relative to a reference hTF protein) that does not comprise the one or more variation). In some embodiments, the variant hTF protein comprises a substitution of a non-cysteine amino acid residue with a cysteine (e.g., at position VI, P2, D3, K4 T5, H14, Q20, S21, D24, K27, S28, V29, P31, S32, D33, A43, E89, D104, G106, G1 I4, LI22, G123, P145, SI55, D163, T165, DI66, P168, PI75, GI76, G178, C179, S180, T181, L182, Q184, F187, S189, D197, G198, E212, A215, N216, A218, D221, D229, G257, N268, D277, K278, K280, E281, S287, P288, H289, K291, S298, P307, L326, T33O, P335, T336, N413, S415, D416, D420, K434, S435, A436, S437, D438, D442, N443, G446, N469, N472, G487, K489, D491, S501, G502, L503, N510, T518, P539, Q540, G543, G544, K545, P547, D548, P549, K552, N553, N555, D558, D565, T567, P570, N576, A595, S610, N611, V612, T613, D614, S616, G617, T626, D634, D643, S666, T667 or S669, numbering according to SEQ ID NO: 3) (e.g., as described in W02009019314A1). In some embodiments, the variant hTF protein comprises the addition of a cysteine residue (e.g., as described in W02009019314A1).(i) Exemplary hTF Variant Proteins

[0216] The amino acid sequence of exemplary hTF variants is provided in Table 2.Table 2. The Amino Acid Sequence of Exemplary hTF Variants.

[0217] In some embodiments, the TFR targeting agent comprises a TF valiant (e.g., a hTF variant) (or a functional fragment or variant thereof). In some embodiments, TF variant (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a TF variant set forth in Table 2, wherein the amino acid sequence of the TF variant comprises at least one amino acid variation compared to the amino acid sequence of a reference TF protein (e.g., SEQ ID NO: 3). In some embodiments, TF variant (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 4-12, wherein the amino acid sequence of the TF variant comprises at least one amino acid variation compared to the amino acid sequence of a reference TF protein (e.g., SEQ ID NO: 3).(ii) Heterologous Moieties

[0218] In some embodiments, the TF (e.g., hTF) is operably connected to a heterologous moiety (e.g., an Fc region (e.g., an Fc region described herein (see, e.g., § 5.3.2))). In someembodiments, the heterologous moiety is a half-life extension moiety. Exemplary half-life extension moictics include, but arc not limited to, an immunoglobulin (e.g., human Ig (hlg)), a fragment of an Ig (e.g., hlg), an Ig (e.g., hlg) constant region, a fragment of an Ig (e.g., hlg) constant region, an Ig (e.g., hlg) Fc region, human serum albumin (HSA), an HSA binding protein or peptide, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig, a fragment of an Ig, one or more Ig heavy chain constant region, a fragment of an Ig constant region, an Ig Fc region, a hlg, a fragment of a hlg, one or more hlg heavy chain constant region, a fragment of a hlg constant region, a hlg Fc region, human serum albumin (HSA), and an HSA binding protein or peptide. The immunomodulatory protein or polypeptide described herein fused or conjugated to a half-life extending moiety or a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art.

[0219] In some embodiments, the heterologous moiety is a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises one or more Ig heavy chain constant region (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is IgGl, IgG2, IgG3, or IgG4.

[0220] In some embodiments, the heterologous polypeptide comprises or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0221] In some embodiments, the heterologous polypeptide comprises or consists of an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0222] In some embodiments, the heterologous polypeptide comprises one or more hlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[0223] In some embodiments, the heterologous polypeptide comprises or consists of a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 hinge region, h!gG4 CH2 region,and hIgG4 CH3 region.

[0224] In some embodiments, the heterologous polypeptide comprises or consists of a hlg Fc region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a h!gG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.5.3.1.2 TFR Binding Peptides and Antibody-Like Scaffolds

[0225] In some embodiments, the TFR targeting agent comprises a peptide that specifically binds TFR (e.g., hTFR). TFR (e.g., hTFR (e.g., hTFRl)) binding peptides that can be employed in the conjugates described herein are known in the art. For example, see, e.g., US6743893 and US8399653, the entire contents of each of which is incorporated herein by reference for all purposes.

[0226] In some embodiments, the TFR targeting agent comprises an antibody-like scaffold. Anti-TFR antibody like scaffolds that can be employed in the conjugates described herein are known in the art. See, e.g., W02023023031 (describing anti-TFR cysteine dense peptides) and WO2021076546 (describing TFR binding fibronectin type III domains), the entire contents of each of which is incorporated herein by reference for all purposes.(i) Exemplary TFR Binding Peptides and Antibody-Like Scaffolds

[0227] The amino acid sequence of exemplary TFR specific antibody like scaffolds is provided in Table 3.Table 3. The Amino Acid Sequence of Exemplary TFR Specific Peptides and AntibodyLike Scaffolds.

[0228] In some embodiments, the TFR targeting agent comprises a TFR specific peptide or an antibody like scaffold (or a functional fragment or variant thereof). In some embodiments, the TFR targeting agent comprises a TFR specific peptide (or a functional fragment or valiant thereof). In some embodiments, the TFR specific peptide (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a TFR specific peptide (or the functional fragment or variant thereof) set forth in Table 3. In some embodiments, the TFR specific peptide (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 13-41. In some embodiments, the TFR targeting agent comprises an antibody like scaffold (or a functional fragment or variant thereof). In some embodiments, the antibody like scaffold (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of antibody like scaffold (or the functional fragment or variant thereof) set forth in Table 3. In some embodiments, the antibody like scaffold (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 42-61.(ii) Heterologous Moieties

[0229] In some embodiments, the TFR binding peptide or antibody-like scaffold is operably connected to a heterologous moiety (e.g., an Fc region (e.g., an Fc region described herein (see, e.g., § 5.3.2))). In some embodiments, the heterologous moiety is a half-life extension moiety. Exemplary half-life extension moieties include, but are not limited to, an immunoglobulin (e.g., human Ig (hlg)), a fragment of an Ig (e.g., hlg), an Ig (e.g., hlg) constant region, a fragment of an Ig (e.g., hlg) constant region, an Ig (e.g., hlg) Fc region human transferrin, human serum albumin (HS A), an HSA binding protein or peptide, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig, a fragment of an Ig, one or more Ig heavy chain constant region, a fragment of an Ig constant region, an Ig Fc region, a hlg, a fragment of a hlg, one or more hlg heavy chain constant region, a fragment of a hlg constant region, a hlg Fc region, human serum albumin (HSA), and an HSA binding protein or peptide. The immunomodulatory protein or polypeptide described herein fused or conjugated to a half-life extending moiety or a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art.

[0230] In some embodiments, the heterologous moiety is a heterologous polypeptide. In someembodiments, the heterologous polypeptide comprises one or more Ig heavy chain constant region (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is IgGl, IgG2, IgG3, or IgG4.

[0231] In some embodiments, the heterologous polypeptide comprises or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0232] In some embodiments, the heterologous polypeptide comprises or consists of an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0233] In some embodiments, the heterologous polypeptide comprises one or more hlg heavychain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[0234] In some embodiments, the heterologous polypeptide comprises or consists of a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[0235] In some embodiments, the heterologous polypeptide comprises or consists of a hlg Fc region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.5.3.L3 Anti-TFR (e.g., Anti-TFRl) Antibodies

[0236] In some embodiments, the TFR targeting agent is an anti-TFR antibody (e.g., an anti- hTFR antibody) (e.g., an anti-hTFRl antibody)). In some embodiments, the antibody comprises or consists of a full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), (sdAb)2 (e.g., a (VHH)2, or a (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody comprises or consists of a full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, sdAb-Fc (e.g., a VHH-Fc), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody comprises or consists of a full-length antibody. In some embodiments, the antibody comprises or consists of a Fab. In some embodiments, the antibody comprises or consists of a F(ab')2. In some embodiments, the antibody comprises or consists of a Fab-Fc. In some embodiments, the antibody comprises or consists of a scFv-Fc. In some embodiments, the antibody comprises or consists of a (SCFV)2-FC. In some embodiments, the antibody comprises or consists of a sdAb-Fc (e.g., a VHH- Fc). In some embodiments, the antibody comprises or consists of a o(sdAb)2-Fc (e.g., (VHH)2-Fc)

[0237] In some embodiments, the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is an IgGl or IgG4 antibody. In some embodiments, the antibody is an IgGl antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a hlgGl, h!gG2, h!gG3, or hIgG4 antibody. In some embodiments, the antibody is a hlgGl or hIgG4 antibody. In some embodiments, the antibody is a hlgGl antibody. In some embodiments, the antibody is a hIgG4 antibody.(i) Exemplary Anti-TFR (e.g., Anti-TFRl) Antibodies

[0238] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell)-targeting agent) comprises an anti-TFR antibody. Anti-hTFRl antibodies that can be employed in the conjugates described herein are known in the art.

[0239] Exemplary anti-TFRl antibodies known in the ail that can be employed in the conjugates described herein include, but are not limited to, e.g., OKT9 (see, e.g., US4364934); MH, M23, M27, B84 (see, e.g., WO2015098989 and US9994641); 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4 (see, e.g., WO2016081643 and US9708406); 8D3 (see, e.g., US2010 / 077498 and Lee et al. “Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292: 1048-1052); 0X26 (see, e.g., Haobam, B. et al. 2014. Rabl7-mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion. Cellular microbiology. 16: 1806-21); DF1513 (see, e.g., Ortiz-Zapatcr E et al. Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A. Plant J 48:757-70 (2006)); the following commercially available clones e.g., Novus Biologicals) 1A1B2,661G1, MEM-189, JF0956, 29806, 1A1B2, TFRC / 1818, 1E6, 66Igl0, TFRC / 1059, Ql / 71, 23D10, 13E4, TFRC / 1149, ER-MP21, YTA74.4, BU54, 2B6, RI7 217; BA120g (see, e.g., US20110311544A1 and US7572895); B3 / 25 and T58 / 30 (see, e.g., Trowbridge, I.S. et al. “Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumour cells.” Nature, 1981, volume 294, pages 171-173); the following commercially available clones (e.g., BioXcell) R17 217.1.3, 5E9C11, OKT9 (BE0023 clone), BK19.9, B3 / 25, T56 / 14 and T58 / 1 (see, e.g., Gatter, K.C. et al. “Transferrin receptors in human tissues: their distribution and possible clinical relevance.” J Clin Pathol. 1983 May; 36(5):539-45); 5E9C11; R17 217.1.3 (available from BioXcell), BE0175 (available from BioXcell); the entire contents of each of which is incorporated herein by reference for all purposes.

[0240] Exemplary anti-TFR (e.g., hTFR (e.g., hTFRl)) antibodies that can be employed in the conjugates described herein are described in e.g., WO2023283531; WO2021154477A1;WO2020132584A1; WO2021154476A1; WO2021150382A1; W02023023031A2;WO2021146256A1; WO2021142275A1; US20220017635A1; W02016207240A1;US11267896B2; US20220143206A1, US11028179B2; US11286305B2; W02023087017A1; WO2023086864A1; WO2023044398A1; WO2023039611A2; W02023034409A1;WO2023283623A1; WO2023283624A2; WO2023283619A2; WO2023283620A1;WO2023283615A1; WO2023283613A1; WO2023283614A2; US11672872B2; US11648318B2; WO2022271549A1; WO2022201122A1; WO2022174114A1; WO2022026152A2;W02022020107A1; W02022020106A1; W02022020105A1; W02022020108A;W02022020109A1; WO2021205358A1; US20230174646A1; US20210299266A1;WO2021195469A1; US11446387B2; US20220409735A1; US20210301290A1;US20210369762A1; US11525137B2; US11555190B2; US11111308B2; US10550188B2; US10508151B2; US20160208008A1; US20150291697A1; US20130171061A1; US9562230B2; US7976841B2; US4364934; WO2015098989; US9994641; WO2016081643; US9708406; US2010077498; US20110311544; US7572895; WO2019075417; US20060286030A1;US20190240346A1 ; US20130216476A1 ; WO2023283531 ; US20130177579A1 ; US9598496B2;US20130045206A1; US20060039908A1; US6015555A; US6008326A; US5648469A; EP79696B1; W02023034409A1; US4364934; US8409573; US9708406; US9611323; WO2015098989; Schneider C. et al. “Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody 0KT9.” J Biol Chern. 1982, 257:14, 8516-8522.; Lee et al. “Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292: 1048-1052; Lee et al. “Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292: 1048-1052; Haobam, B. et al. 2014. Rabl7-mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion. Cellular microbiology. 16: 1806-21; Ortiz-Zapater E et al. Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A. Plant J 48:757-70 (2006); Trowbridge, I.S. et al. “Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumour cells.” Nature, 1981, volume 294, pages 171-173; Gatter, K.C. et al. “Transferrin receptors in human tissues: their distribution and possible clinical relevance.” J Clin Pathol. 1983 May; 36(5) :539-4; the entire contents of each of which are incorporated herein by reference for all purposes.

[0241] The amino acid sequence of exemplary anti-hTFR antibodies that can be utilized in the conjugates described herein is provided in Table 4. The CDRs of the anti-hTFR antibodies in Table 4, are denoted according to Kabat. A person of ordinary skill in the art would be able to determine the CDRs as defined by another scheme, e.g., Chothia, IMGT, using ordinary methods known in the art.Table 4. The Amino Acid Sequence of Exemplary Anti-hTFR Antibodies.

[0242] The amino acid sequence of additional exemplary anti-hTFRl antibodies that can be utilized in the conjugates described herein is provided in Table 26. The CDRs of the anti-hTFR antibodies in Table 26, are denoted according to Kabat. A person of ordinary skill in the ail would be able to determine the CDRs as defined by another scheme, e.g., Chothia, IMGT, using ordinary methods known in the art.Table 26. Amino Acid Sequence of Exemplary Anti-TFRl Antibodies.

[0243] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) antibody comprises an anti-TFR (e.g., hTFR, e.g., hTFRl) antibody named and / or incorporated by reference herein).

[0244] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: a VH CDR1, a VH CDR2, and a VH CDR3.

[0245] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and / or incorporated by referenceherein) comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of a VH of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0246] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0247] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and / or incorporated by reference herein),or the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0248] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0249] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[0250] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of a VL of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1,2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.).

[0251] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of a VL of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.).

[0252] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of an anti- TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.).

[0253] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and / or incorporated byreference herein), or the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0254] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[0255] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of a VH of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of a VL of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1 , 2, or3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0256] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of an anti- TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence CDR1 of a VL of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition,etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and / or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and / or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0257] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH of an anti-TFR antibody named and / or incorporated by reference herein). In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL of an anti-TFR antibody named and / or incorporated by reference herein). In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH of an anti-TFR antibody named and / or incorporated by reference herein); and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL of an anti-TFR antibody named and / or incorporated by reference herein).

[0258] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) antibody comprises an anti-TFR e.g., hTFR, e.g., hTFRl) antibody provided in Table 4.

[0259] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: a VH CDR1, a VH CDR2, and a VH CDR3.

[0260] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of theamino acid sequence of a VH CDR3 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0261] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0262] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 4, or the amino acid sequence of a VH CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 4, or the amino acid sequence of a VH CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 4, or the amino acid sequence of a VH CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0263] In some embodiments, the amino acid sequence of VH CDRI comprises or consists of the amino acid sequence of a VH CDRI set forth in Table 4, or the amino acid sequence of a VH CDRI set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 4, or the amino acid sequence of a VH CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises orconsists of the amino acid sequence of a VH CDR3 set forth in Table 4, or the amino acid sequence of a VH CDR3 set forth in Tabic 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0264] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[0265] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0266] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0267] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 4, or the amino acid sequence of a VL CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 4, or the amino acid sequence of a VL CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e. ., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 4, or the amino acid sequence of a VL CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0268] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 4, or the amino acid sequence of a VL CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 4, or the amino acid sequence of a VL CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 4, or the amino acid sequence of a VL CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0269] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[0270] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 4comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0271] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 4, or the amino acid sequence of a VH CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 4, or the amino acid sequence of a VH CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 4, or the amino acid sequence of a VH CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 4, or the amino acid sequence CDR1 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 4, or the amino acid sequence of a VL CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 4, or the amino acid sequence of a VL CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0272] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 4. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acidsequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 4. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 4; and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 4.

[0273] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) antibody comprises an anti-TFR (e.g., hTFR, e.g., hTFRl) antibody provided in Table 26.

[0274] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: a VH CDR1, a VH CDR2, and a VH CDR3.

[0275] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0276] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDRI of a VH set forth in Table 26, or the amino acid sequence of a VH CDRI of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VHset forth in Table 26, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0277] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 26, or the amino acid sequence of a VH CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 26, or the amino acid sequence of a VH CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 26, or the amino acid sequence of a VH CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0278] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 26, or the amino acid sequence of a VH CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 26, or the amino acid sequence of a VH CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 26, or the amino acid sequence of a VH CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0279] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VL that comprises: VL CDRI, VL CDR2, and VL CDR3.

[0280] In some embodiments, the amino acid sequence of VL CDRI comprises or consists of the amino acid sequence of a VL CDRI of a VL set forth in Table 26, or the amino acid sequence of a VL CDRI of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 26comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0281] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0282] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 26, or the amino acid sequence of a VL CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 26, or the amino acid sequence of a VL CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 26, or the amino acid sequence of a VL CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0283] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 26, or the amino acid sequence of a VL CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 26, or the amino acid sequence of aVL CDR2 set forth in Table 26 comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 26, or the amino acid sequence of a VL CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0284] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[0285] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0286] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 26, or the amino acid sequence of a VH CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g.,substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 26, or the amino acid sequence of a VH CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e. ., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 26, or the amino acid sequence of a VH CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 26, or the amino acid sequence CDR1 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 26, or the amino acid sequence of a VL CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 26, or the amino acid sequence of a VL CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0287] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 26. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 26. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 26; and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 26.5.3.2 Ig Constant Regions

[0288] In some embodiments, the antibody (or heterologous polypeptide (e.g., operablyconnected to a protein (e.g., hTF) that specifically binds TFR (see, e.g., § 5.3. 1.3)) comprises an IgG CH2 region and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0289] In some embodiments, the antibody (or heterologous polypeptide) comprises an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0290] In some embodiments, the antibody (or heterologous polypeptide) comprises a first Ig Fc region and a second Ig Fc region. In some embodiments, the first and / or second Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of a hinge region, aCH2 region, and a CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the first and / or second Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0291] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more hlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[0292] In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[0293] In some embodiments, the antibody (or heterologous polypeptide) comprises a hlg Fcregion. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0294] In some embodiments, the antibody (or heterologous polypeptide) comprises a first hlg Fc region and a second hlg Fc region. In some embodiments, the first and / or second hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a h!gG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the first and / or second hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0295] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more Ig (e.g., hlg) light chain constant region (e.g., a hlg light chain kappa constant region (KCL) or a hlg light chain lambda constant region ( ZCL).

[0296] The amino acid sequence of exemplary reference hlgGl and hIgG4 heavy chain andlight chain constant regions, which can be incorporated in one or more of the embodiments described herein (e.g., anti-TFR (e.g., hTFR (e.g., hTFRl)) antibodies and heterologous polypeptides), is provided in Table 5.Table 5. The Amino Acid Sequence of Exemplary hlg Heavy Chain and Light ChainConstant Regions and Components Thereof.

[0297] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more hlg constant region, wherein the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 5. In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5.

[0298] In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0299] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlgconstant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0300] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 171-198. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198.

[0301] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0302] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, theamino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.5.3.2.1 Ig Effector Function

[0303] As described herein, in some embodiments, the antibody (or heterologous polypeptide) comprises an Fc region (see, e.g., § 5.3.2). In some embodiments, the Fc region of an antibody (or heterologous polypeptide) described herein exhibits a decrease in one or more Fc effector function relative to a reference (e.g., wild type) Fc region. Exemplary Fc effector functions include, but are not limited to, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))).

[0304] Standard in vitro and / or in vivo assays known in the art can be conducted to evaluate Fc effector function, including, any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fey receptor) binding affinity, and Clq binding affinity.

[0305] For example, ADCC activity can be assessed utilizing standard (radioactive and nonradioactive) methods known in the art (see, e.g., W02006 / 082515, W02012 / 130831), the entire contents of each of which is incorporated by reference herein for all purposes). For example, ADCC activity can be assessed using a chromium-5 (51Cr) assay. Briefly,51Cr is pre-loaded into target cells, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicative of lysis of the target cells by the NK cells). Similar non-radioactive assays can also be utilized that employ a similar method, but the target cells are pre-loaded with fluorescent dyes, such as calcein-AM, CFSE, BCECF, or lanthanide fhirophore (Europium). See, e.g., Parekh, Bhavin S et al. “Development and validation of an antibody-dependent cell-mediated cytotoxicityreporter gene assay.” mAbs vol. 4,3 (2012): 310-8. Doi: 10.4161 / mabs.19873, the entire contents of which is incorporated by reference herein for all purposes. Exemplary commercially available non-radioactive assays include, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Additional non-limiting examples of in vitro assays that can be used to assess ADCC activity of a fusion protein described herein include those described in US5500362; US5821337; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 83 (1986)7059-7063; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 82 (1985) 1499-1502; and Bruggcmann, M., ct al., J. Exp. Med. 166 (1987) 1351-1361, the entire contents of each of which is incorporated by reference herein. Alternatively, or additionally, ADCC activity of a fusion protein described herein may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, et al., Proc. Nat’l Acad. Sci. USA 95 (1998) 652-656, the entire contents of which is incorporated by reference herein for all purposes.

[0306] C Iq binding assays can be utilized to assess the ability of a an antibody (or heterologous polypeptide) described herein to bind Clq (or bind with less affinity than a reference fusion protein) and hence lack (or have decreased) CDC activity. The binding of an antibody (or heterologous polypeptide) described herein to Clq can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-Clq interactions, including e.g., equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein. For example, see, e.g., Clq and C3c binding ELIS As described in W02006 / 029879 and W02005 / 100402, the entire contents of each of which is incorporated by reference herein for all purposes. Additional CDC activity assays include those described in e.g., Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, M. S., et al., Blood 101 (2003) 1045-1052; and Cragg, M. S., and Glennie, M. J., Blood 103 (2004) 2738-2743), the entire contents of each of which is incorporated by reference herein for all purposes.

[0307] ADCP activity can be measured by in vitro or in vivo methods known in the ail and also commercially available assays (see, e.g., van de Donk NW, Moreau P, Plesner T, et al. “Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma,” Blood, 127(6):681-695 (2016), the entire contents of each of which is incorporated by reference herein for all purposes). For example, a primary cell based ADCP assaycan be used in which fresh human peripheral blood mononuclear cells (PBMCs) are isolated, monocytes isolated and differentiated in culture to macrophages using standard procedures. The macrophages are fluorescently labeled added to cultures containing fluorescently labeled target cells. Phagocytosis events can be analyzed using FACS screening and / or microscopy. A modified reporter version of the above described assay can also be used that employs an engineered cell line that stably expresses FcyRIIa (CD32a) as the effector cell line (e.g., an engineered T cell line, e.g., THP-1), removing the requirement for primary cells. Exemplary ADCP assays are described in e.g., Ackerman, M. E. el al. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J. Immunol. Methods 366, 8-19 (2011); and Mcandrew, E. G. et al. Determining the phagocytic activity of clinical antibody samples. J. Vis. Exp. 3588 (2011). Doi: 10.3791 / 3588; the entire contents of each of which is incorporated by reference herein.

[0308] Binding of an antibody (or heterologous polypeptide) described herein to an Fc receptor can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-Fc receptor interactions, i.e., specific binding of an Fc region to an Fc receptor. Common assays include equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4” Ed., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein for all purposes.

[0309] In some embodiments, the Fc region of an antibody (or heterologous polypeptide) described herein is varied (e.g., comprises one or more amino acid variation (e.g., one or more amino acid substitution, deletion, addition, etc.)) (referred to herein as a “varied Fc region”), relative to the amino acid sequence of a reference Fc region (e.g., a wild type Fc region, e.g., Table 5 herein (e.g., SEQ ID NOS: 178, 180, 191, or 195). In some embodiments, the one or more amino acid variation (e.g., the one or more amino acid substitution, deletion, addition, etc.)) decreases or abolishes one or more Fc effector function, relative to a reference Fc that does not comprise thevariation (e.g., the one or more variation (e.g., the one or more amino acid substitution, deletion, addition, etc.)).

[0310] In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits no detectable or decreased ADCC compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation e.g., the one or more amino acid variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits no detectable or decreased CDC compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more amino acid variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a modified Fc region exhibits no detectable or decreased ADCP compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))) compared to a reference antibody (or heterologous polypeptide)that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to FcyRI, Fcylla, and / or Fey I Ha compared to an antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc exhibits decreased or no detectable specific binding affinity to FcyRI compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc exhibits decreased or no detectable specific binding affinity to Fcylla compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide)comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to Fcyllla compared to an antibody (or heterologous polypeptide) that docs not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to Clq compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0311] In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable ADCC. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable CDC. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable ADCP. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))). In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to FcyRI, Fcylla, and / or. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc exhibits no detectable specific binding affinity to FcyRI. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc exhibits no detectable specific binding affinity to Fcylla. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to Fcyllla. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to Clq.

[0312] Amino acid substitutions that decrease or abolish one or more Fc effector function are known in the ait. See for example, Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, vIO (June 7, 2019) DOI=10.3389 / fimmu.2019.01296, the full contents of which is incorporated by reference herein for all purposes, see more particularly for example, e.g., Table 4 of Saunders.

[0313] In some embodiments, the varied Fc comprises a hlgGl Fc region comprising one or more amino acid variations (e.g., one or more amino acid substitutions). In some embodiments, the hlgGl Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the hlgGl Fc regioncomprises the following amino acid substitutions L234A and / or L235A, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A, L235A, and P329G, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A, L235A, and P329A, EU numbering according to Kabat.

[0314] In some embodiments, the varied Fc region comprises a hlg4 Fc region comprising one or more amino acid variations (e.g., one or more amino acid substitutions). In some embodiments, the hIgG4 Fc region comprises an amino acid substitution at amino acid positions S228, F234, and / or L235, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and / or L235A, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and / or L235E, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc comprises the following amino acid substitutions S228P and / or L235E, EU numbering according to Kabat.

[0315] The amino acid sequence of exemplary varied Fc regions that are known in the art to exhibit a decrease in one more effector function is provided in Table 6.Table 6. The Amino Acid Sequence of Exemplary Varied Fc Regions.

[0316] In some embodiments, the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0317] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0318] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0319] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[0320] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc regionthat comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0321] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0322] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234 and / or an alanine at position E235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234 and an alanine at position E235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises of an alanine, glycine, or serine at position P329, EU numbering according to Kabat.

[0323] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234; an alanine at position E235; and an alanine, glycine, or serine at position P329 EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises of an alanine at position E234; an alanine at position E235; and an alanine at position P329 EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234; an alanine at position E235; and a glycine amino acid at position P329 EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at positionL234; an alanine at position L235; and a serine amino acid at position P329 EU numbering according to Kabat.

[0324] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and / or an alanine, glycine, or serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine, glycine, or serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a glycine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequenceat least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0325] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and / or an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199- 224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a glycine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a serine amino acid at position P329, EU numberingaccording to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[0326] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and / or an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199- 224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a glycine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanineat position L235, and a serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[0327] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and an alanine at position L235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a proline at position S228, EU numbering according to Kabat.

[0328] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234; an alanine at position L235; and a proline at position S228, EU numbering according to Kabat.

[0329] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and / or a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0330] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and / or a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[0331] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and / or a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and / or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.5.3.2.2 Promotion of Heterodimerization

[0332] As described herein, in some embodiments, the antibody (or heterologous polypeptide) comprises a first and second Fc region (see, e.g., § 5.3.2). In some embodiments, the first Ig Fc region and the second Ig Fc region each comprise one or more amino acid modifications relative to each other to promote heterodimerization. IgG derived heterodimeric formats can be generated by methods known in the art, e.g., by forced heavy chain heterodimerization. Forced heavy chain heterodimerization can be obtained using known methods in the art, e.g., knob-in-hole or strandexchange engineered domains (SEED), see, e.g., Ji-Hee et al., “Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins” Frontiers in Immunology, v7(article 394) (2016) DOI=10.3389 / fimmu.2016.00394 (hereinafter “Ji-Hee 2016”), the entire contents of which is incorporated by reference herein for all purposes.

[0333] In some embodiments, an interface of the first and the second Ig Fc regions is varied, e.g., introduction of an amino acid substitution, to increase heterodimerization, e.g., relative to a non-modified interface, e.g., a naturally occurring interface. For example, dimerization of the first and second Ig Fc regions can be enhanced by providing an Ig Fc interface of a first and a second Fc region with one or more of: a paired protuberance-cavity (“knob-in-hole”), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms, e.g., relative to a non-modified interface.

[0334] Knob-in-Hole amino acid pairing modifications are known in the art, and described in e.g., US5731116; US7476724; Ji-Hee 2016; and Ridgway, J. “'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization” etal. Prot. Engineering 9(7): 617-621 (1996), the full contents of each of which is incorporated by reference herein. Generally, Knob-in- Hole comprises 1) introducing one or more amino acid substitutions in the CH3 domain of one or both of the first and second subject Ig Fc regions to promote heterodimerization; and 2) combining the modified Ig Fc regions under conditions that promote heterodimerization. “Knobs” are typically created by substituting a small amino acid in a parental Ig Fc region with a larger amino acid (e.g., T366Y or T366W); “holes” are created by substituting a larger residue in a parental Ig Fc region with a smaller amino acid (e.g., Y407T, T366S, 11368A, or Y407V). Exemplary Knob- in-Hole mutations include S354C, T366W in the “knob” Ig Fc region and Y349C, T366S, E368A, Y407V in the “hole” Ig Fc region. Other exemplary Knob-in-Hole mutations, which can be incorporated into any one or more of the embodiments, are provided in Table 7, with additional exemplary optional stabilizing Ig Fc cysteine mutations.Table 7. Exemplary Knob-in-hole and Stabilizing Cysteine Modifications.

[0335] The amino acid sequence of exemplary Fc regions that are known in the ail to promote heterodimerization is provided in Table 8.Table 8. The Amino Acid Sequence of Exemplary Pairs of Varied Heterodimeric FcRegions.

[0336] As described herein, in some embodiments, the antibody (or heterologous polypeptide) comprises a first Fc region and a second 1g Fc region.

[0337] In some embodiments, the amino acid sequence of the first Fc region comprises a T366W amino acid substitution, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, and Y407V, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5). In some embodiments, the amino acid sequence of the first hlg further comprises a S354C amino acid substitution, EU numbering according to Kabat; and the amino acid sequence of the second Fc region comprises a Y349C amino acid substitution, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[0338] In some embodiments, the amino acid sequence of the first Fc region comprises each of the following amino acid substitutions: T366W and a S354C, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, Y407V, and Y349C, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a referenceIg Fc region set forth in Table 5).

[0339] In some embodiments, the amino acid sequence of the second Fc region comprises a T366W amino acid substitution, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, and Y407V, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5). In some embodiments, the amino acid sequence of the second hlg further comprises a S354C amino acid substitution, EU numbering according to Kabat; and the amino acid sequence of the second Fc region comprises a Y349C amino acid substitution, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[0340] In some embodiments, the amino acid sequence of the second Fc region comprises each of the following amino acid substitutions: T366W and a S354C, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, Y407V, and Y349C, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[0341] In some embodiments, the amino acid sequence of the first Ig Fc region comprises a W amino acid at position T366, EU numbering according to Kabat; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, EU numbering according to Kabat.

[0342] In some embodiments, the amino acid sequence of the first Ig Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat.

[0343] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 225-232; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368,and a V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 241-248.

[0344] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 233-240; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 249-256.

[0345] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 225; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 241.

[0346] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 226; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 242.

[0347] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of any one of SEQ ID NO: 227; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 243.

[0348] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 228; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 244.

[0349] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 229; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 245.

[0350] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 230; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 246.

[0351] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%,88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 231; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 247.

[0352] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 232; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and aV amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 248.

[0353] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 233; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 249.

[0354] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 234; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence ofany one of SEQ ID NO: 250.

[0355] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 235; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 251.

[0356] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 236; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 252.

[0357] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 237; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 253.

[0358] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 238; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 254.

[0359] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 239; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 255.

[0360] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 240; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 256.5.3.2.3 Ig Constant Region Variations for Site Specific Conjugation

[0361] In some embodiments, the molecular payload is conjugated (e.g., directly, or indirectly through a linker) to the Ig constant region. In some embodiments, the molecular payload (or a linker) is conjugated directly to an amino acid (e.g., a naturally occurring amino acid or an engineered (z.e., variant) amino acid) within the Ig constant region.

[0362] In some embodiments, the molecular payload (or the linker) is conjugated directly to an engineered lysine, cysteine, or tyrosine amino acid residue within the Ig constant region. Insome embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more naturally occurring amino acid residue with a lysine, cysteine, or tyrosine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more non-cysteine amino acid residue with a cysteine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more non-lysine amino acid residue with a lysine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more nontyrosine amino acid residue with a tyrosine amino acid residue (e.g., to mediate conjugation).

[0363] In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more lysine, cysteine, or tyrosine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more lysine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more cysteine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more tyrosine amino acid residue (e.g., to mediate conjugation).5.3.2.4 Exemplary Variant Fc Regions

[0364] As described herein, in some embodiments, the antibody (or heterologous moiety) comprises a first and second Ig Fc region (see, e.g., § 5.3.2). In some embodiments, the first Ig Fc region and the second Ig Fc region each comprise multiple amino acid variations described herein, e.g., one or more amino acid variation that decreases or abolishes one or more Ig Fc effector function (e.g., ADCC, ADCP, CDC, and binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRl, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRl, Fcylla, and / or Fcyllla))) (see, e.g., § 5.3.2.1); and one or more amino acid modification that promote heterodimerization of the first and second Fc regions (see, e.g., § 5.3.2.2).

[0365] In some embodiments, the first and second Fc region each comprise one or more amino acid variation that decreases or abolishes one or more Fc effector function (e.g., ADCC, ADCP, CDC, binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRl, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRl, Fcylla, and / or Fcyllla))) (see, e.g., § 5.3.2.1); and one or more amino acid variation that promote heterodimerization of the first andsecond Fc regions (see, e.g., § 5.3.2.2).

[0366] The amino acid sequence of exemplary variant Fc regions is provided in Table 9.Table 9. The Amino Acid Sequence of Exemplary Variant Fc Regions.

[0367] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid residue at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 257-264; and the amino acid sequence of the second Ig Fc region comprises a serine amino acid at position T366, an alanine amino acid at position L368, and a valine amino acid at position Y407, a cysteine amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 265-272.

[0368] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 273-280; and the amino acid sequence of the second Ig Fc region comprises a serine amino acid at position T366, an alanine amino acid at position L368, a valine amino acid at position Y407, and a cysteine amino acid at position Y349, a cysteine amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 281-288.

[0369] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 257; and the amino acid sequence of the second Ig Fc region comprises a serine amino acid at position T366, an alanine amino acid at position L368, a valine amino acid at position Y407, a cysteine amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 265.

[0370] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 258; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 266.

[0371] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 259; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at positionY407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:267.

[0372] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 260; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, an A amino acid at position L368, and a V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:268.

[0373] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 261; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:269.

[0374] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue atposition L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 262; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:270.

[0375] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 263; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:271.

[0376] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 264; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numberingaccording to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 272.

[0377] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 273; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 281.

[0378] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 274; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 282.

[0379] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 275; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, and a C amino acid at position Y349, EU numbering ...

Claims

CLAIMSWhat is claimed is:

1. A conjugate comprising:(a) a hematopoietic cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to(b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by the hematopoietic cell.

2. The conjugate of claim 1, wherein upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell.

3. The conjugate of claim 1 or 2, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (c) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and / or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

4. The conjugate of any one of claims 1-3, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

5. The conjugate of any one of claims 1-4, wherein the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) exhibits one or more of the followingproperties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR 1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the hematopoietic cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

6. A conjugate comprising:(a) an erythroid precursor cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transl'crrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to(b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by the erythroid precursor cell.

7. The conjugate of claim 6, wherein upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

8. The conjugate of claim 6 or 7, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (c) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and / or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

9. The conjugate of any one of claims 6-9, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

10. The conjugate of any one of claims 6-10, wherein the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the erythroid precursor cell; and / or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

11. The conjugate of any one of the preceding claims, wherein the protein that specifically binds TFR (e.g., hTFR (e.g., TFR1)) is an anti-TFR (e.g., hTFR (e.g., TFR1)) antibody.

12. The conjugate of claim 11, wherein the antibody comprises or consists of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fab-Fc, a scFv, a scFv-Fc, a (scFv)2-Fc, an Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), a (sdAb)2 (e.g., a (VHH)2), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc).

13. The conjugate of any one of claims 11 -12, wherein the antibody is an IgG (e.g., a human IgG (hlgG)) antibody.

14. The conjugate of any one of claims 11-13, wherein the antibody is a hlgGl, h!gG2, h!gG3, or h!gG4 antibody (e.g., a hlgGl or h!gG4 antibody).

15. The conjugate of any one of claims 11-14, wherein the antibody comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region.

16. The conjugate of any one of claims 11-15, wherein the antibody comprises or consists of a full-length antibody, a Fab-Fc, a scFv-Fc, a (scFv)2-Fc, a sdAb-Fc (e.g., a VHH-Fc), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc).

17. The conjugate of any one of claims 15-16, wherein the Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

18. The conjugate of any one of claims 15-17, wherein the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

19. The conjugate of any one of claims 15-18, wherein the Ig is a hlg.

20. The conjugate of claim 19, wherein the hlg is a human IgG (hlgG).

21. The conjugate of claim 20, wherein the hlgG is hlgGl or hIgG4.

22. The conjugate of any one of claims 15-21, wherein the Ig (e.g., hlg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference hlg Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))).

23. The conjugate of any one of claims 15-22, wherein the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, Fcylla, and / or Fcyllla))).

24. The conjugate of any one of claims 15-23, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

25. The conjugate of any one of claims 15-24, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat.

26. The conjugate of any one of claims 15-25, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat.

27. The conjugate of any one of claims 15-26, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, a serine at amino acid position L235, and / or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat.

28. The conjugate of any one of claims 15-27, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

29. The conjugate of any one of claims 15-23, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

30. The conjugate of any one of claims 1-23 or 29, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position L235, numbering according to EU index of Kabat.

31. The conjugate of any one of claims 15-23 or 29-30, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

32. The conjugate of any one of claims 11-31, wherein the antibody comprises a first Fc region and a second Fc region that associate via at least one covalent (e.g., disulfide) bond.

33. The conjugate of any one of claims 11-32, wherein the antibody the amino acid sequence of the first Fc region and / or the amino acid sequence of the second Fc region comprise one or more amino acid substitution that promotes the association (e.g., heterodimerization) of the first and second Fc regions.

34. The conjugate of any one of claims 11 -33, wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat.

35. The conjugate of any one of claims 11-34, wherein the amino acid sequence of the first Fc comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat.

36. The conjugate of any one of claims 11-35, wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat.

37. The conjugate of any one of claims 11-36, wherein the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, numbering according to the EU index of Kabat.

38. The conjugate of any one of claims 11-37, wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat.

39. The conjugate of any one of claims 11-38, wherein the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbering according to the EU index of Kabat.

40. The conjugate of any one of claims 11-39, wherein the amino acid sequence of the second Fc region of the antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat.

41. The conjugate of any one of claims 11-40, wherein the amino acid sequence of the second Fc region of the antibody comprises a cysteine at amino acid position S354, numbering according to the EU index of Kabat.

42. The conjugate of any one of claims 11-41, wherein the antibody does not (or does not substantially) block binding of TF (e.g., hTF) to the TFR (e.g., hTFRl).

43. The conjugate of any one of the preceding claims, wherein the protein that specifically binds TFR (e.g., hTFR (e.g., hTFRl)) is a TFR ligand (or a functional fragment or functional variant thereof).

44. The conjugate of claim 43, wherein the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

45. The conjugate of any one of the preceding claims, wherein the oligonucleotide enhances the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein.

46. The conjugate of any one of the preceding claims, wherein the oligonucleotide inhibits the expression and / or activity of the target gene, nucleic acid e.g., mRNA), and / or protein.

47. The conjugate of any one of the preceding claims, wherein the oligonucleotide modulates (e.g., enhances or inhibits) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein through binding to a target nucleic acid molecule encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein (e.g., target mRNA molecule (e.g., a portion of a target mRNA molecule)).

48. The conjugate of any one of the preceding claims, wherein the target nucleic acid molecule is a target mRNA molecule (e.g., a portion of a target mRNA molecule).

49. The conjugate of any one of the preceding claims, wherein the oligonucleotide mediates one or more of the following degradation of the target nucleic acid molecule (e.g., mRNA), disabling of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration in the splicing of the target nucleic acid molecule (e.g., mRNA), alteration (e.g., a decrease) in the stability of the target nucleic acid molecule (e.g., mRNA), or a block in the translation of the target nucleic acid molecule (e.g., mRNA), or any combination of the foregoing.

50. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

51. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises or consists of an antisense strand comprising a region of complementarity to a target sequence (e.g., an mRNA sequence encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein).

52. The conjugate of any one of the preceding claims, wherein the oligonucleotide is single stranded or double stranded.

53. The conjugate of any one of the preceding claims, wherein the oligonucleotide is a DNA, RNA, or RNA and RNA hybrid molecule.

54. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises a sense strand and an antisense strand forming a double stranded region.

55. The conjugate of any one of the preceding claims, wherein the sense strand and the antisense strand arc part of a single nucleic acid molecule (e.g., wherein a hairpin loop is between the sense strand and the antisense strand of the single nucleic acid molecule.

56. The conjugate of any one of the preceding claims, wherein the sense strand and the antisense strand are separate nucleic acid molecules (i.e., connected only through the double stranded region).

57. The conjugate of any one of the preceding claims, wherein the double stranded region is from about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21- 23, or 21-22 nucleotide pairs in length.

58. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide.

59. The conjugate of any one of the preceding claims, wherein at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the oligonucleotide are modified.

60. The conjugate of any one of the preceding claims, wherein substantially all (or all) of the nucleotides in the oligonucleotide are modified.

61. The conjugate of any one of the preceding claims, wherein at least one of the modified nucleotides comprises a modified sugar (e.g., ribose moiety).

62. The conjugate of any one of the preceding claims, wherein at least one of the modified nucleotides comprises a modified nucleobase.

63. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified internucleoside linkage (e.g., at least one phosphorothioate intemucleoside linkage).

64. The conjugate of any one of the preceding claims, wherein the at least one modified nucleotide is a 2’ modified nucleotide (e.g., a 2'-fluoro (2'-F), 2'-O-methyl (2'-0-Me), 2'-O- methoxyethyl (2'-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), ethylene -bridged nucleic acid (ENA), and (S)- constrained ethyl-bridged nucleic acid (cEt) (e.g., a 2' modified nucleotide is 2'- O-methyl or 2'-fluoro (2'-F))).

65. The conjugate of any one of the preceding claims, wherein the protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with one or more hemoglobinopathy .

66. The conjugate of any one of the preceding claims, wherein inhibition of or a reduction in expression and / or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with an increase in the level of fetal hemoglobin, the induction of expression of fetal hemoglobin, and / or an increase in the ratio of fetal hemoglobin to adult hemoglobin.

67. The conjugate of any one of the preceding claims, wherein expression and / or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and / or protein is associated with a repression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the level of adult hemoglobin, and / or an increase in ratio of adult hemoglobin to fetal hemoglobin.

68. The conjugate of any one of the preceding claims, wherein the target gene, nucleic acid (e.g., mRNA), and / or protein is a transcription factor.

69. The conjugate of any one of the preceding claims, wherein the target gene, nucleic acid (e.g., mRNA), and / or protein is highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

70. The conjugate of any one of the preceding claims, wherein the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCLUA)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pseudouridine Synthase 1 (DKC1) (e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11), Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed(ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1 ) (e.g., human HAX1), Glucosc-6-Phosphatasc Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPE Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Eike (SAMD9L) (e.g., human SAMD9L), or MDS1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

71. The conjugate of any one of the preceding claims, wherein the target gene is BCL11A (e.g., hBCLUA), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

72. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is non-covalently conjugated to (b) the at least one oligonucleotide.

73. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is covalently conjugated to (b) the at least one oligonucleotide.

74. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is directly conjugated to (b) the at least one oligonucleotide.

75. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is indirectly conjugated to (b) the at least one oligonucleotide through (c) a linker.

76. The conjugate of any one of the preceding claims, wherein the linker is cleavable or non- cleavable.

77. The conjugate of any one of the preceding claims, wherein the wherein (b) comprises at least 2, 3, 4, 5, 6, or more oligonucleotides.

78. The conjugate of any one of the preceding claims, wherein each of the at least 2, 3, 4, 5, 6, or more oligonucleotides are individually conjugated to (a) the protein (e.g., antibody) that specifically binds TFR (e.g., as described herein).

79. A cell comprising the conjugate of any one of claims 1-78.

80. The cell of claim 79, wherein the cell is in vitro, ex vivo, or in vivo.

81. A pharmaceutical composition comprising the conjugate of any one of claims 1-78 and a pharmaceutically acceptable excipient.

82. A kit comprising the conjugate of any one of claims 1 -78 or the pharmaceutical composition of any one of claims 81.

83. A method of delivering a conjugate or pharmaceutical composition to a cell, the method comprising introducing into a cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby deliver the conjugate or pharmaceutical composition into the cell.

84. The method of claim 83, wherein the cell is in vitro, ex vivo, or in vivo.

85. The method of claim 83 or 84, wherein the cell is a subject (e.g., a human subject).

86. A method of delivering a conjugate, cell, or pharmaceutical composition to a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81, to thereby deliver the conjugate, cell, or pharmaceutical composition to the subject.

87. A method of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein.

88. A method of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein.

89. The method of any one of claims 87-88, wherein the cell is in vitro, ex vivo, or in vivo.

90. The method of any one of claims 87-89, wherein the cell is a subject (e.g., a human subject).

91. A method of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

92. A method of modulating (e.g., inhibiting or enhancing) expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

93. A method of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein.

94. A method of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein.

95. The method of any one of claims 93-94, wherein the cell is in vitro, ex vivo, or in vivo.

96. The method of any one of claims 93-95, wherein the cell is a subject (e.g., a human subject).

97. The method of any one of claims 93-96, wherein the target is BCL11 A (e.g., hBCLl 1 A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

98. A method of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

99. A method of reducing and / or inhibiting expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1- 78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and / or activity of the target gene, nucleic acid (e.g., mRNA), and / or protein in the subject.

100. A method of inducing expression of fetal hemoglobin in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby induce expression of fetal hemoglobin the subject.

101. A method of increasing the level of fetal hemoglobin in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby increase the level of fetal hemoglobin the subject.

102. A method of increasing the ratio of fetal hemoglobin to adult hemoglobin in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby increase the ratio of fetal hemoglobin to adult hemoglobin in the subject.

103. A method of treating, ameliorating, or preventing an inherited blood disorder in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby treat, ameliorate, or prevent the inherited blood disorder in the subject.

104. The method of claim 103, wherein the inherited blood disorder is a hemoglobinopathy or an inherited bone marrow failure syndrome.

105. A method of treating, ameliorating, or preventing a hemoglobinopathy in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby treat, ameliorate, or prevent the hemoglobinopathy in the subject.

106. The method of claim 105, wherein the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

107. The method of claim 105 or 106, wherein the hemoglobinopathy is sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y-thalassemia).

108. The method of any one of claims 105-107, wherein the subject is a human.

109. The method of any one of claims 105-108, wherein the subject is suspected of having or has been diagnosed with sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobinC trait, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalasscmia, P-thalasscmia, 8-thalasscmia, or ' / -thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

110. The method of any one of claims 105-109, wherein the subject is suspected of having or has been diagnosed with sickle cell disease or a thalassemia (e.g., a- thalassemia, P-thalassemia, 6-thalassemia, or ' / -thalassemia).

111. A method of treating, ameliorating, or preventing an inherited bone marrow failure syndrome in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby treat, ameliorate, or prevent the inherited bone marrow failure syndrome in the subject.

112. The method of claim 111, wherein the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, thrombocytopenia absent radii (TAR) a SAMD9 / SAMD9E disorder, or a MECOM-associated syndrome.

113. The conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81 for use in the treatment of a disease in a subject in need thereof.

114. The conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81 for use as a medicament.

115. Use of the conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81 for the manufacture of a medicament for the treatment of a disease in a subject in need thereof.

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