Hematopoietic cell targeting conjugates and related methods

Abstract

Provided herein, inter alia, are conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) comprising a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); (b) at least one oligonucleotide operably linked 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; as well as methods of making the conjugates and pharmaceutical compositions comprising the conjugates. Further provided herein are methods of utilizing the conjugates, including, for example, methods of treating hemoglobinopathy (e.g., sickle cell disease (SCD) or thalassemia (e.g., alpha-thalassemia, beta-thalassemia, delta-thalassemia, or gamma-thalassemia)).

Description

Related applications

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

[0002] This application contains a sequence list that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on July 16, 2024, is named 62992_6WO01_SL.xml and has a size of 2,241,941 bytes. Technical Field

[0003] This disclosure particularly relates to conjugates comprising: a target agent (e.g., a hematopoietic cell (e.g., erythroid progenitor cell) target agent) comprising a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); and an oligonucleotide that regulates the expression and / or activity of a target gene expressed by the target cell. This disclosure further relates to pharmaceutical compositions comprising said conjugates; and methods of using said conjugates, including, for example, methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD)) or thalassemias (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia). Background Technology

[0004] Bone marrow is a soft, gel-like tissue that fills the cavities of bones. Adult bone marrow is red or yellow, depending on the dominance of hematopoietic (red) or fatty (yellow) tissue. Genetic perturbations (including, for example, gene mutations, gene overexpression, and defects in genes (and consequently their encoded products, such as proteins) in the subsets of cells within the bone marrow) are associated with a variety of genetic disorders, including, for example, inherited blood disorders (such as hemoglobinopathies and hereditary bone marrow failure syndromes).

[0005] In humans, all blood cells, including red blood cells, are formed in the red bone marrow, except for lymphocytes, which are produced in the bone marrow and reach their mature form in lymphoid organs. Normal red blood cells contain the protein hemoglobin, whose function is to transport oxygen (O2) from the lungs to peripheral tissues and to transport carbon dioxide (CO2) from tissues to the lungs. Hemoglobin is a heterotetramer composed of α-globin subunits and β-globin subunits, each of which is bound to a heme prosthetic group. Hemoglobin is synthesized from separate α-globin gene clusters and β-globin gene clusters, with different types of hemoglobin produced through different subunit combinations. Fetal hemoglobin is the major hemoglobin produced by the fetus. In healthy individuals, the transition from γ-globin gene expression to β-globin gene expression around birth is the basis for the conversion of fetal hemoglobin to adult hemoglobin production, resulting in adult hemoglobin being the major hemoglobin at 6 months of age. Hemoglobin conversion is not complete or irreversible; because adults retain a residual level (<1% of total hemoglobin) of fetal hemoglobin. The conversion from fetal to adult hemoglobin depends on the repression or silencing of upstream γ-globin genes via a repressor protein network. Hemoglobinopathies are a group of genetic disorders associated with abnormal hemoglobin production and / or abnormal structure, and are the most common inherited blood disorders. Summary of the Invention

[0006] This document provides, in particular, conjugates comprising a target agent (e.g., a hematopoietic cell (e.g., erythroid progenitor cell) target agent) comprising a protein (e.g., an antibody) that specifically binds to a TFR (e.g., hTFR (e.g., hTFR1)); and an oligonucleotide that regulates the expression and / or activity of a target gene expressed by a target cell; and methods for manufacturing the conjugates and pharmaceutical compositions comprising the conjugates. This document further provides methods utilizing the conjugates, including, for example, methods for treating hemoglobinopathies (e.g., sickle cell disease (SCD)) or thalassemias (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia (e.g., β-thalassemia)).

[0007] Accordingly, in one aspect, this document provides conjugates comprising: (a) a hematopoietic cell target comprising a protein (e.g., an antibody) that specifically binds to a transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); operably linked to (b) at least one oligonucleotide that regulates (e.g., inhibits) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by the hematopoietic cells.

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

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

[0011] In some embodiments, proteins (e.g., antibodies) that specifically bind to TFRs (e.g., hTFRs (e.g., hTFR1)) exhibit one or more of the following properties: (a) upon binding to a TFR (e.g., hTFRs (e.g., TFR1)) expressed on the surface of hematopoietic cells, the proteins (e.g., antibodies) or conjugates that specifically bind to said TFR (e.g., hTFRs (e.g., hTFR1)) do not induce the death of the target cells; (c) upon binding to a TFR (e.g., hTFRs (e.g., TFR1)) expressed on the surface of hematopoietic cells, the hematopoietic cells remain viable; (d) After being internalized into hematopoietic cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the death of the hematopoietic cells; and / or (e) after binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)).

[0012] In some embodiments, the protein that specifically binds to 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 substantially does not) block the binding of TF (e.g., hTF) to TFR (e.g., hTFR1). In some embodiments, the antibody comprises or is composed of the following: full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, single-domain antibody (sdAb) (e.g., VHH), sdAb-Fc (e.g., VHH-Fc), (sdAb)2 (e.g., (VHH)2), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody is an IgG (e.g., human IgG (hIgG)) antibody. In some embodiments, the antibody is an hIgG1, hIgG2, hIgG3, or hIgG4 antibody (e.g., an hIgG1 or hIgG4 antibody).

[0013] In some embodiments, the antibody comprises an immunoglobulin (Ig) (e.g., human Ig (hIg)) Fc region. In some embodiments, the antibody comprises or consists of the following: full-length antibody, Fab-Fc, scFv-Fc, (scFv)2-Fc, sdAb-Fc (e.g., VHH-Fc), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the Ig (e.g., hIg) 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., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is hIg. In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1 or hIgG4.

[0014] In some embodiments, the Ig (e.g., hIg) Fc region contains one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region that reduce or eliminate one or more of the following effector functions relative to the reference hIg Fc region: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or affinity for one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))). In some embodiments, the Ig (e.g., hIg) Fc region substantially does not mediate ADCC, substantially does not mediate CDC, and / or does not bind to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))).

[0015] In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234 and / or amino acid substitution at amino acid position L235 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234 and / or alanine at amino acid position L235 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234, alanine at amino acid position L235, and / or glycine, alanine, or serine at position P329 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234, serine at amino acid position L235, and / or glycine, alanine, or serine at position P329 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region contains alanine at amino acid position N297 according to the EU index number of Kabat.

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

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

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

[0019] In some embodiments, the protein that specifically binds to TFR (e.g., hTFR (e.g., hTFR1)) 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, oligonucleotides enhance the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins. In some embodiments, oligonucleotides inhibit the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins. In some embodiments, oligonucleotides regulate (e.g., enhance or inhibit) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins by binding to target nucleic acid molecules encoded by target genes, nucleic acids (e.g., mRNA), and / or proteins (e.g., binding to target mRNA molecules (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, oligonucleotides mediate one or more of the following: degradation of a target nucleic acid molecule (e.g., mRNA), inactivation of a target nucleic acid molecule (e.g., mRNA), modification of a target nucleic acid molecule (e.g., mRNA), alteration of splicing of a target nucleic acid molecule (e.g., mRNA), alteration (e.g., reduction) of the stability of a target nucleic acid molecule (e.g., mRNA), or blockade of translation of a target nucleic acid molecule (e.g., mRNA), or any combination thereof.

[0021] In some embodiments, the oligonucleotide comprises or consists of: antisense oligonucleotide (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA). In some embodiments, the oligonucleotide comprises or consists of: an antisense strand containing a region complementary to a target sequence (e.g., an mRNA sequence encoded by a target gene, a nucleic acid (e.g., mRNA), and / or a protein). In some embodiments, the oligonucleotide is single-stranded or double-stranded. In some embodiments, the oligonucleotide is DNA, RNA, or a hybrid RNA 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 antisense strand are part of a single nucleic acid molecule (e.g., where a hairpin loop is located between the sense strand and antisense strand of the single nucleic acid molecule). In some embodiments, the sense strand and antisense strand are separate nucleic acid molecules (i.e., connected only by the double-stranded region). In some embodiments, the length of the double-stranded region is approximately 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.

[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 in 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., a 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 nucleoside linker (e.g., at least one phosphate thioester nucleoside linker). In some embodiments, at least one modified nucleotide is a 2'-modified nucleotide (e.g., 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-MOE), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), 2'-ON-methylacetamido(2'-O-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt) (e.g., the 2'-modified nucleotide is 2'-O-methyl or 2'-fluoro(2'-F))).

[0023] In some embodiments, proteins, nucleic acids (e.g., mRNA), and / or proteins encoded by target genes are associated with one or more hemoglobinopathies. In some embodiments, inhibition or reduction of the expression and / or activity of proteins, nucleic acids (e.g., mRNA), and / or proteins encoded by target genes is associated with increased fetal hemoglobin levels, induction of fetal hemoglobin expression, and / or an increased fetal hemoglobin to adult hemoglobin ratio. In some embodiments, the expression and / or activity of proteins, nucleic acids (e.g., mRNA), and / or proteins encoded by target genes is associated with repression of fetal hemoglobin, decreased fetal hemoglobin levels, increased adult hemoglobin levels, and / or an increased adult hemoglobin to fetal hemoglobin ratio. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein are transcription factors. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein are highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

[0024] In some embodiments, the target genes are B-cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCL11A)), zinc finger and BTB domain 7A (ZBTB7A) (e.g., hZBTB7A), KLF transcription factor 1 (KLF1) (e.g., hKLF1), FA complementa A (FANCA) (e.g., human FANCA), dyskeratin pseudouridine synthase 1 (DKC1) (e.g., human DKC1), regulator of telomere elongation helicase 1 (RTEL1) (e.g., human RTEL1), and telomerase reverse transcriptase (TERT) (e.g., human TERT). ), telomerase RNA component (TERC) (e.g., human TERC), TERF1 interacting nuclear factor 2 (TINF2) (e.g., human TINF2), ribosomal protein S19 (RPS19) (e.g., human RPS19), ribosomal protein L11 (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), neutrophil-expressed elastase (ELA2) (e.g., human ELA2), HCLS1-associated protein X-1 (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.) For example, 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 α-motif domain-containing protein 9 (SAMD9) (e.g., human SAMD9), sterile α-motif domain-containing protein 9-like protein (SAMD9L) (e.g., human SAMD9L), or MDS1 and EVI1 complex locus (MECOM) (e.g., human MECOM).

[0025] In some embodiments, the target genes are B-cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCL11A)), zinc finger and BTB domain 7A (ZBTB7A) (e.g., hZBTB7A), and KLF transcription factor 1 (KLF1) (e.g., hKLF1).

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

[0027] In some embodiments, 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 is individually conjugated to a protein (e.g., an antibody) that specifically binds to TFR (e.g., as described herein).

[0028] In one aspect, this document provides conjugates comprising: (a) an erythroid precursor cell target comprising a protein (e.g., an antibody) that specifically binds to a transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); operably linked to (b) at least one oligonucleotide that regulates (e.g., inhibits) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by the erythroid precursor cells.

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

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

[0032] In some embodiments, a protein (e.g., an antibody) that specifically binds to a TFR (e.g., hTFR (e.g., hTFR1)) exhibits one or more of the following properties: (a) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., an antibody) or the conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of erythroid precursor cells does not induce the death of the target cells; (c) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the erythroid precursor cells remain viable; (d) Upon internalization into erythroid precursor cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the death of the erythroid precursor cells; and / or (e) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)).

[0033] In some embodiments, the protein that specifically binds to 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 substantially does not) block the binding of TF (e.g., hTF) to TFR (e.g., hTFR1). In some embodiments, the antibody comprises or is composed of the following: full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, single-domain antibody (sdAb) (e.g., VHH), sdAb-Fc (e.g., VHH-Fc), (sdAb)2 (e.g., (VHH)2), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody is an IgG (e.g., human IgG (hIgG)) antibody. In some embodiments, the antibody is an hIgG1, hIgG2, hIgG3, or hIgG4 antibody (e.g., an hIgG1 or hIgG4 antibody).

[0034] In some embodiments, the antibody comprises an immunoglobulin (Ig) (e.g., human Ig (hIg)) Fc region. In some embodiments, the antibody comprises or consists of the following: full-length antibody, Fab-Fc, scFv-Fc, (scFv)2-Fc, sdAb-Fc (e.g., VHH-Fc), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the Ig (e.g., hIg) 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., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is hIg. In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1 or hIgG4.

[0035] In some embodiments, the Ig (e.g., hIg) Fc region contains one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region that reduce or eliminate one or more of the following effector functions relative to the reference hIg Fc region: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or affinity for one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))). In some embodiments, the Ig (e.g., hIg) Fc region substantially does not mediate ADCC, substantially does not mediate CDC, and / or does not bind to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))).

[0036] In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234 and / or amino acid substitution at amino acid position L235 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234 and / or alanine at amino acid position L235 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234, alanine at amino acid position L235, and / or glycine, alanine, or serine at position P329 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234, serine at amino acid position L235, and / or glycine, alanine, or serine at position P329 according to the Kabat EU index number. In some embodiments, Ig is hIgG1, and the amino acid sequence of the Fc region contains alanine at amino acid position N297 according to the EU index number of Kabat.

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

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

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

[0040] In some embodiments, the protein that specifically binds to TFR (e.g., hTFR (e.g., hTFR1)) 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, oligonucleotides enhance the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins. In some embodiments, oligonucleotides inhibit the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins. In some embodiments, oligonucleotides regulate (e.g., enhance or inhibit) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins by binding to target nucleic acid molecules encoded by target genes, nucleic acids (e.g., mRNA), and / or proteins (e.g., binding to target mRNA molecules (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, oligonucleotides mediate one or more of the following: degradation of a target nucleic acid molecule (e.g., mRNA), inactivation of a target nucleic acid molecule (e.g., mRNA), modification of a target nucleic acid molecule (e.g., mRNA), alteration of splicing of a target nucleic acid molecule (e.g., mRNA), alteration (e.g., reduction) of the stability of a target nucleic acid molecule (e.g., mRNA), or blockade of translation of a target nucleic acid molecule (e.g., mRNA), or any combination thereof.

[0042] In some embodiments, the oligonucleotide comprises or consists of: antisense oligonucleotide (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA). In some embodiments, the oligonucleotide comprises or consists of: an antisense strand containing a region complementary to a target sequence (e.g., an mRNA sequence encoded by a target gene, a nucleic acid (e.g., mRNA), and / or a protein). In some embodiments, the oligonucleotide is single-stranded or double-stranded. In some embodiments, the oligonucleotide is DNA, RNA, or a hybrid RNA 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 antisense strand are part of a single nucleic acid molecule (e.g., where a hairpin loop is located between the sense strand and antisense strand of the single nucleic acid molecule). In some embodiments, the sense strand and antisense strand are separate nucleic acid molecules (i.e., connected only by the double-stranded region). In some embodiments, the length of the double-stranded region is approximately 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.

[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 in 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., a 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 nucleoside linker (e.g., at least one phosphate thioester nucleoside linker). In some embodiments, at least one modified nucleotide is a 2'-modified nucleotide (e.g., 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-MOE), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), 2'-ON-methylacetamido(2'-O-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt) (e.g., the 2'-modified nucleotide is 2'-O-methyl or 2'-fluoro(2'-F))).

[0044] In some embodiments, proteins, nucleic acids (e.g., mRNA), and / or proteins encoded by target genes are associated with one or more hemoglobinopathies. In some embodiments, inhibition or reduction of the expression and / or activity of proteins, nucleic acids (e.g., mRNA), and / or proteins encoded by target genes is associated with increased fetal hemoglobin levels, induction of fetal hemoglobin expression, and / or an increased fetal hemoglobin to adult hemoglobin ratio. In some embodiments, the expression and / or activity of proteins, nucleic acids (e.g., mRNA), and / or proteins encoded by target genes is associated with repression of fetal hemoglobin, decreased fetal hemoglobin levels, increased adult hemoglobin levels, and / or an increased adult hemoglobin to fetal hemoglobin ratio. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein are transcription factors.

[0045] In some embodiments, target genes and / or proteins are highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

[0046] In some embodiments, the target genes are B-cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCL11A)), zinc finger and BTB domain 7A (ZBTB7A) (e.g., hZBTB7A), KLF transcription factor 1 (KLF1) (e.g., hKLF1), FA complementa A (FANCA) (e.g., human FANCA), dyskeratin pseudouridine synthase 1 (DKC1) (e.g., human DKC1), regulator of telomere elongation helicase 1 (RTEL1) (e.g., human RTEL1), and telomerase reverse transcriptase (TERT) (e.g., human TERT). ), telomerase RNA component (TERC) (e.g., human TERC), TERF1 interacting nuclear factor 2 (TINF2) (e.g., human TINF2), ribosomal protein S19 (RPS19) (e.g., human RPS19), ribosomal protein L11 (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), neutrophil-expressed elastase (ELA2) (e.g., human ELA2), HCLS1-associated protein X-1 (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.) For example, 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 α-motif domain-containing protein 9 (SAMD9) (e.g., human SAMD9), sterile α-motif domain-containing protein 9-like protein (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., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

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

[0049] In some embodiments, 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 is individually conjugated to a protein (e.g., an antibody) that specifically binds to TFR (e.g., as described herein).

[0050] In one respect, this document provides cells that contain the conjugates described herein. In some embodiments, the cells are in vitro, ex vivo, or in vivo.

[0051] In one respect, this article provides pharmaceutical compositions comprising the conjugates described herein and pharmaceutically acceptable excipients.

[0052] In one respect, this article provides kits containing the conjugates or pharmaceutical compositions described herein.

[0053] In one aspect, this document provides a method for delivering a conjugate or pharmaceutical composition to cells, the method comprising introducing the conjugate or pharmaceutical composition described herein into cells, thereby delivering the conjugate or pharmaceutical composition to the cells. In some embodiments, the cells are in vitro, ex vivo, or in vivo. In some embodiments, the cells are a subject (e.g., a human subject).

[0054] In one aspect, this document provides a method for delivering a conjugate, cell, or pharmaceutical composition to a subject, the method comprising administering the conjugate, cell, or pharmaceutical composition described herein to the subject, thereby delivering the conjugate, cell, or pharmaceutical composition to the subject.

[0055] In one aspect, this document provides methods for regulating (e.g., inhibiting or enhancing) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by hematopoietic cells in cells, said methods comprising introducing conjugates or pharmaceutical compositions described herein to regulate (e.g., inhibit or enhance) the expression and / or activity of said target genes, said nucleic acids (e.g., mRNA), and / or said proteins. In some embodiments, the cells are in vitro, ex vivo, or in vivo. In some embodiments, the cells are a subject (e.g., a human subject).

[0056] In one aspect, this document provides methods for regulating (e.g., inhibiting or enhancing) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by erythroid precursor cells in cells, said methods comprising introducing into said cells a conjugate or a pharmaceutical composition described herein, thereby regulating (e.g., inhibiting or enhancing) said expression and / or activity of said target genes, said nucleic acids (e.g., mRNA), and / or said proteins. In some embodiments, the cells are in vitro, ex vivo, or in vivo. In some embodiments, the cells are a subject (e.g., a human subject).

[0057] In one aspect, this article provides a method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of target genes, nucleic acids (e.g., mRNA) and / or proteins expressed by hematopoietic cells in the cells of a subject, the method comprising administering to the subject a conjugate or a pharmaceutical composition described herein, thereby regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, the nucleic acid (e.g., mRNA) and / or the protein in the subject.

[0058] In one aspect, this document provides a method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by erythroid precursor cells in the cells of a subject, the method comprising administering to the subject a conjugate or pharmaceutical composition described herein, thereby regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, the nucleic acid (e.g., mRNA), and / or the protein in the subject.

[0059] In one aspect, this document provides methods for reducing and / or inhibiting the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by hematopoietic cells in cells, said methods comprising introducing into said cells a conjugate or a pharmaceutical composition described herein, thereby reducing or inhibiting said expression and / or activity of said target genes, said nucleic acids (e.g., mRNA), and / or said proteins. In some embodiments, the cells are in vitro, ex vivo, or in vivo. In some embodiments, the cells are a subject (e.g., a human subject).

[0060] In one aspect, this document provides methods for reducing and / or inhibiting the expression and / or activity of target genes, nucleic acids (e.g., mRNA), and / or proteins expressed by erythroid precursor cells in cells, said methods comprising introducing into said cells a conjugate or pharmaceutical composition described herein, thereby reducing or inhibiting said expression and / or activity of said target genes, said nucleic acids (e.g., mRNA), and / or said proteins. In some embodiments, the cells are in vitro, ex vivo, or in vivo. In some embodiments, the cells are a subject (e.g., a human subject). In some embodiments, the target is BCL11A (e.g., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

[0061] In one aspect, this article provides a method for reducing and / or inhibiting the expression and / or activity of target genes, nucleic acids (e.g., mRNA) and / or proteins expressed by hematopoietic cells in the cells of a subject, the method comprising administering to the subject the conjugates or pharmaceutical compositions described herein, thereby reducing or inhibiting the expression and / or activity of the target genes, nucleic acids (e.g., mRNA) and / or proteins in the subject.

[0062] In one aspect, this article provides a method for reducing and / or inhibiting the expression and / or activity of target genes, nucleic acids (e.g., mRNA) and / or proteins expressed by erythroid precursor cells in the cells of a subject, the method comprising administering to the subject the conjugates or pharmaceutical compositions described herein, thereby reducing or inhibiting the expression and / or activity of the target genes, nucleic acids (e.g., mRNA) and / or proteins in the subject.

[0063] In one aspect, this article provides a method for inducing the expression of fetal hemoglobin in a subject, the method comprising administering to the subject the conjugate or pharmaceutical composition described herein, thereby inducing the expression of fetal hemoglobin in the subject.

[0064] In one respect, this article provides a method for increasing the level of fetal hemoglobin in a subject, the method comprising administering to the subject the conjugate or pharmaceutical composition described herein, thereby increasing the level of fetal hemoglobin in the subject.

[0065] In one respect, this article provides a method for increasing the ratio of fetal hemoglobin to adult hemoglobin in a subject, the method comprising administering to the subject the conjugate or pharmaceutical composition described herein, thereby increasing the ratio of fetal hemoglobin to adult hemoglobin in the subject.

[0066] In one aspect, this document provides methods for treating, improving, or preventing hereditary blood disorders in subjects, the methods comprising administering to the subject the conjugates or pharmaceutical compositions described herein, thereby treating, improving, or preventing the hereditary blood disorder in the subject. In some embodiments, the hereditary blood disorder is a hemoglobinopathy or a hereditary bone marrow failure syndrome.

[0067] In one aspect, this document provides a method for treating, improving, or preventing hemoglobinopathies in a subject, the method comprising administering to the subject the conjugate or pharmaceutical composition described herein, thereby treating, improving, or preventing the hemoglobinopathies in the subject. In some embodiments, the subject is a human being.

[0068] In some embodiments, hemoglobinopathies are sickle cell disease, sickle cell phenotype, hemoglobin C disease, hemoglobin C phenotype, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia), a condition associated with hemoglobins having increased oxygen affinity, a condition associated with hemoglobins having decreased oxygen affinity, unstable hemoglobinopathies, methemoglobinemia, or any combination thereof.

[0069] In some embodiments, hemoglobinopathies are sickle cell diseases or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

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

[0071] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease or thalassemia (e.g., alpha-thalassemia, beta-thalassemia, delta-thalassemia, or gamma-thalassemia).

[0072] In one aspect, this article provides a method for treating, improving, or preventing hereditary bone marrow failure syndrome in a subject, the method comprising administering the conjugate or pharmaceutical composition described herein to the subject, thereby treating, improving, or preventing the hereditary bone marrow failure syndrome in the subject.

[0073] In some embodiments, hereditary bone marrow failure syndromes include amegacytic thrombocytopenic purpura (Amega), diamond blackfan anemia (DBA), congenital dyskeratosis (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman-Diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, and thrombocytopenic radial agenesis (TAR).

[0074] In one respect, this article provides the conjugates, cells, or pharmaceutical compositions described herein for use in treating diseases in subjects in need.

[0075] In one respect, this article provides the conjugates, cells, or pharmaceutical compositions described herein for use as medicines.

[0076] In one respect, this document provides the use of the conjugates, cells, or pharmaceutical compositions described herein for the manufacture of a medicament for the treatment of a disease in a subject in need. Attached Figure Description

[0077] Figure 1A-1D The line graph shows the binding of recombinantly expressed anti-TFR1 monoclonal antibody (mAb) (shown in Example 1) (or isotype control) to soluble TFR1 (using Octet BLI). Figure 1A The line graph shows the binding of the isotype control antibody to soluble TFR1 (showing a lack of binding). Figure 1B The line graph shows the binding of anti-TFR1 mAb2 to soluble TFR1. Figure 1CThe line graph shows the binding of anti-TFR1 mAb3 to soluble TFR1. Figure 1D The line graph shows the binding of anti-TFR1 Fab to soluble TFR1.

[0078] Figure 2A-2D The line graph shows the binding of anti-TFR1 mAb (shown in Example 1) to the surface of erythroid progenitor cells. Figure 2A The line graph shows the percentage of positive cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentration). Figure 2B The line graph shows the MFI of cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentration). Figure 2C The line graph shows the percentage of positive cells treated with anti-TFR1 Fab (at the indicated concentration). Figure 2D The line graph shows the MFI of cells treated with anti-TFR1 Fab (at the indicated concentration).

[0079] Figures 3A-3D The figure shows the internalization of anti-TFR1 mAb (shown in Example 1) (or isotype control) into erythroid progenitor cells. Figure 3A The figure shows the percentage of positive cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentrations). Figure 3B The figure shows the MFI of cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentrations). Figure 3C The figure shows the percentage of positive cells treated with anti-TFR1 Fab (at the indicated concentration). Figure 3D The figure shows the MFI of cells treated with anti-TFR1 Fab (at the indicated concentration).

[0080] Figure 4A The -B line graph shows the extent to which the mRNA of the indicated target gene is knocked down by the indicated siRNA in erythroid progenitor cells. Figure 4A The line graph shows HPRT1-2 siRNA-mediated knockdown of HPRT1 mRNA (at the indicated siRNA concentration). Figure 4B The line graph shows BCL11A mRNA knockdown mediated by BCL11A-11 siRNA or BCL11A-5 siRNA (at the indicated siRNA concentration).

[0081] Figures 5A-5B The bar chart shows the extent to which the indicated target protein levels are reduced in erythroid progenitor cells by the indicated siRNA. Figure 5AThe bar chart shows the reduction in BCL11A protein levels mediated by BCL11A-11 siRNA or BCA11A-5 siRNA (at the indicated siRNA concentration). Figure 5B The bar chart shows the HPRT1 protein level reduction mediated by HPRT1-2 siRNA (at the indicated siRNA concentration).

[0082] Figure 6A-6K The line graph shows the binding of each of the anti-TFR1 antibody-siRNA conjugates (AOC#1-11) to the recombinant TFR1 protein (using Octet BLI). Figure 6A The line graph shows the binding of AOC#1 with recombinant TFR1. Figure 6B The line graph shows the binding of AOC#2 with recombinant TFR1. Figure 6C The line graph shows the binding of AOC#3 with recombinant TFR1. Figure 6D The line graph shows the binding of AOC#4 with recombinant TFR1. Figure 6E The line graph shows the binding of AOC#5 with recombinant TFR1. Figure 6F The line graph shows the binding of AOC#6 with recombinant TFR1. Figure 6G The line graph shows the binding of AOC#7 with recombinant TFR1. Figure 6H The line graph shows the binding of AOC#8 with recombinant TFR1. Figure 6I The line graph shows the binding of AOC#9 with recombinant TFR1. Figure 6J The line graph shows the binding of AOC#10 with recombinant TFR1. Figure 6K The line graph shows the binding of AOC#11 with recombinant TFR1.

[0083] Figures 7A-7B The line graphs show the viability of erythroid precursor cells at indicated concentrations, 48 ​​and 72 hours after the first administration with the indicated AOC (AOC#1 or AOC#2). Figure 7A The line graph shows the viability of erythroid precursor cells 48 hours after the first administration with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. Figure 7B The line graph shows the viability of erythroid precursor cells 72 hours after the first administration of the indicated AOC at the indicated concentration.

[0084] Figures 7C-7D The line graph shows the knockdown of HPRT1 transcripts in erythroid progenitor cells at indicated concentrations, 48 ​​and 72 hours after the first administration of the indicated AOC (AOC#1 or AOC#2). Figure 7CThe line graph shows the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells at the indicated concentration 48 hours after the first administration of the indicated AOC (AOC#1 or AOC#2). Figure 7D The line graph shows the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells at the indicated concentration 72 hours after the first administration with the indicated AOC (AOC#1 or AOC#2).

[0085] Figures 8A-8B The line graphs show the viability of erythroid precursor cells at indicated concentrations, 48 ​​and 72 hours after the first administration with the indicated AOC (AOC#3 or AOC#4). Figure 8A The line graph shows the viability of erythroid precursor cells 48 hours after the first administration with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. Figure 8B The line graph shows the viability of erythroid precursor cells 72 hours after the first administration with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration.

[0086] Figure 8C-8D The line graph shows the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells at indicated concentrations, 48 ​​and 72 hours after the first administration with the indicated AOC (AOC#3 or AOC#4). Figure 8C The line graph shows the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells at the indicated concentration 48 hours after the first administration with the indicated AOC (AOC#3 or AOC#4). Figure 8D The line graph shows the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells at the indicated concentration 72 hours after the first administration with the indicated AOC (AOC#3 or AOC#4).

[0087] Figure 9 The line graph shows the knockdown of HPRT1 protein in erythroid progenitor cells 72 hours after the first administration of the indicated AOC (AOC#2 or AOC#4).

[0088] Figure 10A The line graph shows the viability of erythroid precursor cells 72 hours after the first administration of the indicated AOC (AOC#5) at the indicated concentration. Figure 10B The line graph shows the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells at the indicated concentration 72 hours after the first administration of the indicated AOC (AOC#5).

[0089] Figure 11A The line graph shows the viability of erythroid precursor cells 72 hours after the first administration with the indicated AOC (AOC#10 or AOC#11) at the indicated concentration. Figure 11B The line graph shows the knockdown of BCL11A mRNA transcripts in erythroid progenitor cells 72 hours after the first administration of the indicated AOC (AOC#10 or AOC#11) at the indicated concentration.

[0090] Figure 12A The line graph shows the viability of erythroid precursor cells 72 hours after the first administration of the indicated AOC (AOC#9) at the indicated concentration. Figure 12B The line graph shows the knockdown of BCL11A transcripts in erythroid progenitor cells at the indicated concentration 72 hours after the first administration of the indicated AOC (AOC#9).

[0091] Figure 13A The line graph shows the viability of erythroid precursor cells 72 hours after the first administration with the indicated AOC (AOC#7 or AOC#8) at the indicated concentration. Figure 13B The line graph shows the knockdown of BCL11A mRNA transcripts in erythroid progenitor cells 72 hours after the first administration of the indicated AOC (AOC#7 or AOC#8) at the indicated concentration. Detailed Implementation

[0092] The inventors have particularly discovered that molecular payloads (e.g., oligonucleotides) capable of regulating (e.g., inhibiting) the expression and / or activity of genes (which can regulate (e.g., repress) such as fetal hemoglobin production) can specifically target hematopoietic cells (e.g., erythroid precursor cells) by conjugation with a targeting agent (e.g., an anti-TFR antibody). Therefore, the conjugates described herein are particularly useful for treating hemoglobinopathies (including, for example, sickle cell disease and thalassemia). Thus, this disclosure provides conjugates; and their use, particularly in pharmaceutical compositions and methods of treating diseases (e.g., hemoglobinopathies). Table of contents 5.1 Definition 5.2 Conjugates 5.3 Hematopoietic cell targeting agents 5.3.1 TFR-targeting agents 5.3.1.1 TF Protein 5.3.1.1(i) Exemplary hTF variant protein 5.3.1.1(ii) Heterogeneous components 5.3.1.2 TFR-binding peptides and antibody-like scaffolds 5.3.1.2(i) Exemplary TFR-binding peptides and antibody-like scaffolds 5.3.1.2(ii) Heterogeneous Part 5.3.1.3 Anti-TFR (e.g., anti-TFR1) antibodies 5.3.1.3(i) Exemplary anti-TFR (e.g., anti-TFR1) antibody 5.3.1.4 Ig effector function 5.3.1.5 Promotion of heterodimerization 5.3.1.6 Ig constant region variation for site-specific conjugation 5.3.1.7 Exemplary variant Fc region 5.4 Methods for preparing proteins (e.g., targeting agents) 5.5 molecular effective load 5.5.1 Oligonucleotides 5.5.1.1 Total Length 5.5.1.2 Target Area 5.5.1.3 Antisense Oligonucleotides 5.5.1.3(i) Total Length 5.5.1.3(ii) Target Region 5.5.1.4 RNAi agents 5.5.1.4(i) Antisense Chain 5.5.1.4(i)(a) Total Length 5.5.1.4(i)(b) Target Region 5.5.1.4(ii) with a semantic chain 5.5.1.4(ii)(a) Antisense chain complementarity 5.5.1.4(ii)(b) Total Length 5.5.1.4(iii) dsRNA agents 5.5.1.4(iii)(a) Single and multiple nucleic acid molecules 5.5.1.4 (iii)(b) Length of the double-stranded region 5.5.1.4 (iii)(c) Nucleotide overhangs and blunt ends 5.5.1.4 (iii)(d) Exemplary structural combinations of sense chains and antisense chains 5.5.1.5 Modified oligonucleotides 5.5.1.5 (i) Properties of nucleotide modifications 5.5.1.5(i)(a) Modified nucleosides 5.5.1.5(i)(b) Nucleoside linkage modification 5.5.1.5(i)(c) Other exemplary nucleotide modifications 5.5.1.5(ii) Degree of modification of nucleotides 5.5.1.6 Exemplary Oligonucleotide Targets 5.5.1.6(i) Exemplary BCL11A-targeting oligonucleotide 5.5.1.6(ii) Exemplary ZBTB7A Targeted Oligonucleotide 5.5.1.6(iii) Exemplary KLF1-targeting oligonucleotides 5.5.1.6(iv) Other exemplary oligonucleotide targets 5.6 Methods for preparing oligonucleotide molecular payloads 5.7 connector 5.7.1 Cuttable Connector 5.7.2 Non-cuttable joints 5.7.3 Joint Assembly 5.8 Joining 5.9 Activity of molecular payloads (e.g., oligonucleotides) and their conjugates 5.10 cells 5.11 Pharmaceutical Composition 5.12 Usage Instructions 5.12.1 Delivery Method 5.12.2 Methods for regulating the expression of target genes, nucleic acids (e.g., mRNA), and / or proteins 5.12.3 Methods for reducing or inhibiting the expression of target genes, nucleic acids (e.g., mRNA), and / or proteins 5.12.4 Methods for regulating target mRNA splicing 5.12.5 Methods for inducing fetal hemoglobin expression 5.12.6 Methods for treating hereditary blood disorders 5.12.7 Methods for treating hemoglobinopathies 5.12.8 Methods for treating hereditary bone marrow failure syndrome 5.13 Reagent Kit 5.1 Definition

[0093] The section headings used herein are for typographical purposes only and should not be construed as limiting the scope of the topics discussed.

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

[0095] In this disclosure, the use of the singular includes the plural unless otherwise expressly stated. For example, as used in the specification and appended claims, the singular forms “a / an” and “the” include plural indicators unless the context clearly indicates otherwise. Furthermore, the use of the term “including” and other forms (e.g., include, includes, and included) is not restrictive.

[0096] It should be understood that, regardless of the use of the term "comprising" to describe various aspects herein, this document also provides other similar aspects described in terms of "composed of" and "essentially composed of".

[0097] Furthermore, as used herein, the term “and / or” should be considered as a specific disclosure of each of two designated features or components, together or apart from each other. Therefore, the term “and / or” as used herein, such as in phrases like “A and / or B”, is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover 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 stated herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, to include fractions thereof (such as one-tenth and one-hundredth of an integer).

[0099] The term "about" refers to a value or composition within an acceptable range of error for a particular value or composition, as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable range of error for that particular value or composition.

[0100] When this article describes proteins, it should be understood that this article also provides nucleic acid molecules (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) that encode proteins.

[0101] When this article describes proteins, nucleic acid molecules, carriers, and loading agents, it should be understood that this article also provides the isolation forms of proteins, nucleic acid molecules, carriers, and loading agents.

[0102] When this article describes proteins, nucleic acid molecules, etc., it should be understood that this article also provides recombinant forms of proteins, nucleic acid molecules, etc.

[0103] When this article describes a polypeptide or polypeptide group, it should be understood that this article also provides proteins that comprise polypeptides or polypeptide groups folded into their three-dimensional structures (i.e., tertiary or quaternary structures), and vice versa.

[0104] When this article describes proteins, it should be understood that this article also provides polypeptides that contain the same amino acid sequence either linearly or folded into their three-dimensional structure (i.e., tertiary or quaternary structure).

[0105] As used herein, the term "application" means the physical introduction of a pharmaceutical agent (e.g., the conjugates described herein), such as a therapeutic agent (or a precursor of a pharmaceutical agent that is metabolized or altered in the body to produce a pharmaceutical agent (e.g., a therapeutic agent)) into a subject using any of the various methods and delivery systems known to those skilled in the art. Application may also be performed, for example, once, multiple times, and / or over one or more extended periods of time. Application includes self-application.

[0106] The terms “pharmaceutical” and “part” are used interchangeably herein and are generally used to describe any macromolecule or micromolecule (and any combination thereof). Exemplary pharmaceuticals include, but are not limited to, proteins, peptides, nucleic acid molecules (e.g., DNA, RNA), small molecules, carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG), conjugates (e.g., those described herein), and any combination thereof. A pharmaceutical may contain more than one individual pharmaceutical (where said individual pharmaceuticals are the same or different). For example, a pharmaceutical may contain antibodies (e.g., targeting agents described herein) and oligonucleotides (e.g., oligonucleotides described herein). Pharmaceuticals as defined herein include, for example, conjugates described herein.

[0107] As used herein, the term “affinity” refers to the strength of binding between one protein (e.g., an antibody) and another protein (e.g., an antigen). Protein affinity 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 unbound antibody, and [ligand] is the molar concentration of unbound antigen. The affinity constant Ka is defined as 1 / Kd. Standard methods for measuring affinity are known to those skilled in the art. Exemplary methods for measuring affinity include surface plasmon resonance (SPR) (e.g., BIAcore®-based assays), a commonly used method known in the art (see, for example, 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 entire contents of each of which are incorporated herein by reference for all purposes).

[0108] As used herein, the term "antibody" is used in the broadest sense to include various immunoglobulin (Ig) structures (e.g., human Ig (hIg), mouse Ig (mIg)), including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity (i.e., antigen-binding fragments or variants). Therefore, the term antibody includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules containing antibody CDR, VH region, and / or VL region; and antibody-like scaffolds (e.g., fibronectin). Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camel-derived antibodies, intracellular antibodies, variable domains (VNAR fragments) of novel antigen receptor β-lactamases, affinity molecules, biantibodies, triantibodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies (e.g., VHH, (VHH)2), single-chain antibodies, single-chain Fv (scFv; (scFv)2), Fab fragments (e.g., Fab, single-chain Fab (scFab), F(a) b')2 fragments, disulfide-linked Fv (sdFv), Fc fusions (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc), and any of the above-described antigen-binding fragments, as well as conjugates or fusion proteins containing any of the above-described fragments. Antibodies can be Ig isotypes (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG of Ig). 2aor IgG 2b In some embodiments, the antibodies described herein are IgG antibodies or a class (e.g., human IgG1 or IgG4) or subclass. In some embodiments, the antibodies described herein are mIgG antibodies or a class (e.g., mIgG1 or mIgG2a) or subclass. In some embodiments, the antibody is a human, humanized, or chimeric IgG1 or IgG4 monoclonal antibody. In some embodiments, the term antibody refers to a population of monoclonal or polyclonal antibodies. The antibodies described herein can be produced by any standard method known in the art, for example, recombinant production in host cells, see, for example, §5.4; or synthetic production.

[0109] As used herein, the term “antibody-like scaffold” refers to an antigen-binding domain based on a non-Ig structure. Various antibody-like scaffolds are known in the art. For example, the 10th type III domain of fibronectin (e.g., AdNectins®) and engineered ankylosing repeats (e.g., DARPins®) have been used as alternative scaffolds for antigen-binding domains; see, for example, Gebauer and Skera, Engineered protein scaffolds as next-generation antibody therapies. 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 are incorporated herein by reference for all purposes.Exemplary antibody-like scaffolds include, but are not limited to, lipid transport proteins (see, for example, US 7250297) (e.g., Antiticalin®), the z-domain of protein A-derived molecules such as protein a (see, for example, US 5831012) (e.g., Affibody®), the domain of membrane receptors stabilized by disulfide bonds and Ca2+ (see, for example, US 7803907) (e.g., Avimer / Maxibody®), serum transferrin (see, for example, US 2004023334) (e.g., Transbody®); designed ankyrin repeat proteins (see, for example, US 7417130) (e.g., DARPin®), fibronectin (see, for example, US 6818418) (e.g., AdNectin®), C-type lectin domains (see, for example, US 2004132094) (e.g., Tetranectin®); human γ-lens proteins or ubiquitin (see, for example, US 7250297) (see, for example, US 7250297) (e.g., Antiticalin®); and other proteins such as protein a (see, for example, US 7250297). 7838629 (e.g., Affilin®); Kunitz-type domains of human protease inhibitors (see, for example, US 2004209243), C-type lectins (see, for example, US 2004132094) (e.g., Tetranectins®), cysteine ​​knots or knotting agents (see, for example, US 7186524) (e.g., Microbodies®), nucleic acid aptamers (see, for example, US 5475096), thioredoxin A scaffolds (see, for example, US 6004746) (peptide aptamers), and the 10th type III domain of fibronectin (see, for example, US 6818418) (e.g., AdNectins®) and cysteine-dense peptides (see, for example, WO 2023023031). Other exemplary antibody-like scaffolds are known in the art and are described, for example, in: Storz U. Intellectual property protection: strategies for antibody inventions. MAbs. [Monoclonal Antibodies] 2011; 3(3):310-317. doi:10.4161 / mabs.3.3.15530. For all purposes, the entire contents of each of the foregoing references are incorporated herein by reference. Antibody-like scaffolds include, for example, naturally occurring antigen conjugates, variants of naturally occurring conjugates (e.g., functional variants), fragments of naturally occurring antigen conjugates (e.g., functional fragments), and synthetic antigen conjugates (i.e., non-naturally occurring antigen conjugates).

[0110] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to an immune mechanism that causes immune effector cells (e.g., NK cells) to lyse antibody-coated target cells (or Fc-containing proteins) (e.g., the IgFc-containing fusion proteins described herein). As used herein, the terms “reduced ADCC”, etc., refer to a reduction in the number of target cells lysed by the ADCC mechanism defined above, given a given concentration of antibody (or IgFc-containing proteins) (e.g., the Fc-containing fusion proteins described herein) in the culture medium surrounding the target cells within a given time, and / or an increase in the concentration of antibody (or Fc-containing proteins) (e.g., the Fc-containing fusion proteins described herein) in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells within a given time, via the ADCC mechanism defined above. The reduction in ADCC is relative to ADCC mediated by the same antibody (or protein containing an Fc region) (e.g., the Fc-containing fusion protein described herein), which is produced from the same type of host cells using the same standard methods of production, purification, formulation, and storage (known to those skilled in the art), but without engineering modifications (e.g., not containing one or more amino acid variations, such as amino acid substitutions, that mediate ADCC reduction). For example, the reduction in ADCC mediated by an antibody (or protein containing an Fc region) (e.g., the Fc-containing fusion protein described herein) that contains an amino acid substitution that reduces ADCC is relative to ADCC mediated by the same antibody (or protein containing an Fc region) (e.g., the Fc-containing fusion protein described herein) without the amino acid substitution in its Fc region.

[0111] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a standard single-stranded oligonucleotide known in the art, which, for example, is capable of regulating the expression of a target gene (or protein) by hybridization with a target nucleic acid (e.g., mRNA encoded by a target gene and encoding a target protein), particularly with a sequential sequence on the target nucleic acid. Antisense oligonucleotides include DNA, RNA, and hybrid DNA / RNA oligonucleotides.

[0112] As used herein, the term "antisense strand" in relation to oligonucleotides described herein (e.g., RNAi agents (e.g., siRNA agents), antisense oligonucleotides) refers to an oligonucleotide containing a complementary region that is at least partially (e.g., substantially, completely) complementary to a target nucleic acid sequence (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., siRNA), the sense strand will typically pair with the sense strand (as described herein).

[0113] As used herein, the term "BCL11 transcription factor A" or "BCL11A" refers to a C2H2 type zinc finger transcription factor that plays a role, particularly in the repression of fetal hemoglobin and the conversion from fetal hemoglobin to adult hemoglobin. The amino acid sequence of the reference human BCL11A (hBCL11A) protein is shown in SEQ ID NO: 291 (NCBI reference number: NP_075044.2).

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

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

[0116] As used herein, the term "blunt end" refers to a double-stranded oligonucleotide that does not contain any unpaired nucleotides (i.e., no one or more nucleotide overhangs) at the ends (e.g., 3′ end, 5′ end) of the molecule. Double-stranded oligonucleotides may have blunt ends, for example, at the 3′ end, 5′ end, or both the 3′ end and 5′ end of the molecule.

[0117] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable region of a heavy-chain or light-chain polypeptide. These specific regions have been described in: Kabat et al., J. Biol. Chem., 252, 6609-6616 (1977); and Kabat et al., Sequences of protein of immunological interest. (1991), the full contents of each of which are incorporated herein by reference for all purposes. Unless otherwise stated, the term “CDR” is the CDR defined by: Kabat et al., J. Biol. Chem., 252, 6609-6616 (1977); and Kabat et al., Sequences of protein of immunological interest. (1991). Those skilled in the art will be able to determine CDRs as defined by other schemes (e.g., Chothia, IMGT) using common methods known in the art.

[0118] The terms “CH1” and “CH1 region” are used interchangeably herein and refer to the first constant region of the immunoglobulin heavy chain. An exemplary reference to the hIgG1 CH1 region is shown in SEQ ID NO: 171; and an exemplary reference to the hIgG4 CH1 region is shown in SEQ ID NO: 184.

[0119] The terms “CH2” and “CH2 region” are used interchangeably herein and refer to the second constant region of the immunoglobulin heavy chain. An exemplary reference to the hIgG1 CH2 region is shown in SEQ ID NO: 173; and an exemplary reference to the hIgG4 CH2 region is shown in SEQ ID NO: 186.

[0120] The terms “CH3” and “CH3 region” are used interchangeably herein and refer to the third constant region of the immunoglobulin heavy chain. An exemplary reference to the hIgG1 CH3 region is shown in SEQ ID NO: 174; and an exemplary reference to the hIgG4 CH3 region is shown in SEQ ID NO: 187.

[0121] As used herein, the term "complementarity" relating to a first nucleotide sequence (e.g., sense strand or target mRNA) associated with a second nucleotide sequence (e.g., an antisense strand or antisense oligonucleotide) refers to the ability of a nucleic acid molecule containing the first nucleotide sequence to hybridize with a nucleic acid molecule containing the second nucleotide sequence and form a double-stranded region (via base pair hydrogen bonds) under suitable in vivo or in vitro conditions (e.g., under certain standard conditions, under physiological conditions in mammals (e.g., humans)). Those skilled in the art will be able to select the set of conditions most suitable for hybridization testing. Complementary sequences include, for example, 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 native and modified nucleotides, as well as nucleotide mimics, to at least the extent required to satisfy the hybridization requirements described above. Therefore, the determination of complementarity (as described herein) is independent of nucleotide chemical modifications (e.g., as described herein). For example, both (C) and 5-methylcytosine (mC) are complementary to (G).

[0122] As used herein, the term "conjugation" refers to the operative linking (e.g., chemical conjugation) of at least a first agent (e.g., an oligonucleotide (e.g., the oligonucleotide described herein)) to a second agent (e.g., a protein (e.g., a target agent (e.g., the target agent described herein (e.g., a hematopoietic cell target agent (e.g., the erythroid progenitor cell target agent described herein (e.g., the anti-TFR (e.g., the anti-hTFR (e.g., the anti-hTFR1 antibody described herein))))). The first agent may be directly or indirectly linked to the second agent (e.g., via a linker (e.g., as described herein)). Methods for operatively linking two agents (e.g., chemical conjugation methods) are technically important. The same applies to commercially available conjugation reagents and kits, which are well-known in the field, and detailed instructions for use are readily available from commercial suppliers. Operable links (e.g., chemical conjugations) include both covalent and non-covalent conjugations. In some embodiments, an operable link (e.g., a chemical conjugation) comprises a covalent link of a first agent (e.g., an oligonucleotide (e.g., the oligonucleotide described herein)) to a second agent (e.g., a protein (e.g., a target agent (e.g., the target agents described herein (e.g., hematopoietic cell target agents (e.g., the erythroid progenitor cell target agents described herein (e.g., the anti-TFR (e.g., anti-hTFR (e.g., anti-hTFR1) antibody described herein (e.g., the anti-TFR antibody described herein (e.g., anti-hTFR1 antibody (e.g., the anti-TFR antibody (e.g., anti-hTFR1 antibody (e.g., the anti-TFR antibody (e.g., the ...TFR1 antibody (e.g., the anti-TFR antibody (e.g., the anti-TFR1 antibody (e.g., the anti-TFR antibody (e.g., the anti-TFR1 antibody (e.g.

[0123] The terms "constant region" and "constant domain" are used interchangeably in this document. They refer to the carboxyl-terminal portion of the light and / or heavy chains of a full-length antibody that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Ig Fc receptors (e.g., Fcγ receptors). The constant region of an Ig molecule typically has a more conserved amino acid sequence than the variable domain of Ig.

[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, disease, abnormality, pathology, condition, symptom, or syndrome in which physiological function is impaired, regardless of its etiological nature. The term disease includes infections (e.g., viral, bacterial, fungal, protozoan infections).

[0125] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to a macromolecule comprising multiple deoxyribonucleotides polymerized by phosphodiester bonds. A deoxyribonucleotide is a nucleotide 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 antiparallel and at least partially (e.g., substantially, completely) complementary nucleic acid sequences forming the double-stranded region. For example, in some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand.

[0127] When used to refer to the Ig Fc region or proteins containing the Ig Fc region (e.g., full-length antibodies), the term "effective function" refers to those biological activities attributable to the Ig Fc region of a typical full-length antibody, and therefore varies with antibody isotypes. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa)) 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 erythrocyte). Therefore, erythroid precursor cells include, but are not limited to, megakaryocyte-erythroid progenitor cells, proerythrocytes, early erythroblasts, intermediate erythroblasts, late erythroblasts, and reticulocytes. In some embodiments, erythroid precursor cells include megakaryocyte-erythroid progenitor cells, proerythrocytes, early erythroblasts, intermediate erythroblasts, and late erythroblasts. In some embodiments, erythroid precursor cells include proerythrocytes, early erythroblasts, intermediate erythroblasts, and late erythroblasts.

[0129] As used herein, the term "erythroid progenitor cell target" refers to an agent that specifically binds to antigens expressed on erythroid progenitor cells (or a subset thereof). For example, antigens expressed on or in erythroid progenitor cells may be membrane proteins, such as integrated membrane proteins or peripheral membrane proteins. Typically, an erythroid progenitor cell target specifically binds to antigens on erythroid progenitor cells, which promote the internalization of the erythroid progenitor cell target (and any associated molecular payload) into the erythroid progenitor cells. In some embodiments, the erythroid progenitor cell target specifically binds to internalized cell surface receptors on erythroid progenitor cells and can be internalized into erythroid progenitor cells via receptor-mediated internalization. In some embodiments, the erythroid progenitor cell target is a protein (e.g., an antibody), peptide, nucleic acid (e.g., an aptamer), or small molecule. In some embodiments, the erythroid progenitor cell target is linked to a molecular payload.

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

[0131] As used herein, the term "Fab" refers to an antigen-binding domain comprising a Fab heavy chain that includes a VH region and a CH1 region from the N-terminus to the C-terminus; and a light chain that includes a VL region and a CL region from the N-terminus to the C-terminus; and wherein the Fab heavy chain and the light chain are associated to form an antigen-binding domain.

[0132] As used in this article, the term "Fab-Fc" refers to an antibody containing a Fab that is operatively linked to an Fc region.

[0133] As used herein, the term "Fc region" refers to the C-terminal region of an Ig (e.g., human Ig) heavy chain that includes at least a CH2 region operatively connected to a CH3 region from the N-terminus to the C-terminus. In some embodiments, the Fc region includes an Ig hinge region or at least a portion of an Ig hinge region operatively 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., including one or more amino acid modifications), see, for example, §§ 5.3.2.1, 5.3.2.2, 5.3.2.3, 5.3.2.4. Other examples of proteins with engineered Fc regions can be found in Saunders 2019 (KOSaunders, “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 are incorporated herein by reference for all purposes).

[0134] As used herein, the terms “first” and “second” regarding Fc regions, etc., are used for convenience in distinguishing when more than one of each type of part exists. Unless explicitly stated otherwise, the use of these terms is not intended to assign a particular order or orientation to a protein. For example, antibodies described herein (e.g., in the case of full-length antibodies) may contain, for example, two Fc regions associated via one or more covalent bonds (e.g., disulfide bonds).

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

[0136] As used herein, the term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody: (i) a first Ig light chain comprising a light chain variable region (VL) and a light chain constant region (CL) from the N-terminus to the C-terminus; (ii) a first Ig heavy chain comprising a heavy chain variable region (VH), a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; (iii) a second Ig heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; and (iv) a second Ig light chain comprising a VL region and a VH region from the N-terminus to the C-terminus; wherein the first light chain and the first heavy chain are associated to form a first antigen-binding domain; wherein the second light chain and the second heavy chain are associated to form a second antigen-binding domain; and wherein the first heavy chain and the second heavy chain are associated to form a dimer. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences; and the two light chains comprise substantially identical amino acid sequences. In some embodiments, the two heavy chains contain substantially the same amino acid sequence, except for one or more amino acid modifications that promote proper heavy chain heterodimerization (e.g., as described herein); and the two light chains contain substantially the same amino acid sequence. If the antibody chains differ due to post-translational modifications (e.g., C-terminal cleavage of lysine residues, alternative glycosylation patterns, etc.), they may be substantially the same but not identical.

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

[0138] As used herein, the term "functional variant" in relation to a polypeptide or protein refers to a protein that contains at least one, but no more than 20%, 15%, 12%, 10%, or 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 specific function of the reference protein. Not all functions of the reference protein (e.g., wild-type) need to be retained by the functional variant of the protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild-type protein.

[0139] As used herein, the term "functional fragment" in relation to proteins refers to a segment of a reference protein that retains at least one specific function. Not all functions of a reference polypeptide or protein need to be retained by a functional fragment of the protein. In some cases, 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 "fusion" and its grammatical equivalents refer to the operative linking of at least one polypeptide derived from a first polypeptide to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are distinct. The term fusion encompasses both direct linking of at least two polypeptides via peptide bonds and indirect linking via linkers (e.g., peptide linkers).

[0141] As used herein, the term "fusion protein" and its grammatical equivalents refer to a protein comprising at least one polypeptide derived from a first polypeptide and another polypeptide derived from a second polypeptide, wherein the first polypeptide and the second polypeptide are not naturally found to be operatively linked together. The at least two polypeptides in a fusion protein may be operatively linked directly via peptide bonds or operatively linked indirectly via a linker (e.g., a peptide linker). Thus, for example, the term fusion protein encompasses embodiments in which polypeptide A is operatively linked directly to polypeptide B via peptide bonds (polypeptide A-polypeptide B), and embodiments in which polypeptide A is operatively linked to polypeptide B via 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" refers to the portion of a peptide or protein that, when conjugated to or otherwise operatively linked (e.g., fused) in vitro to a subject (e.g., a human subject) and thus increases the half-life of the subject peptide or protein (e.g., small molecules, peptides, polynucleotides, carbohydrates, lipids, synthetic polymers (e.g., PEG polymers), etc.). Pharmacokinetic properties of peptides or proteins can be evaluated using in vitro models known in the art.

[0143] As used herein, the term "half-life extended peptide" refers to a peptide that, when operatively linked to another peptide (the subject peptide), increases the half-life of the subject peptide in vitro upon administration to a subject (e.g., a human subject). Pharmacokinetic properties of peptides or proteins can be evaluated using in vitro models known in the art.

[0144] As used herein, the term "heavy chain" refers to a portion of an immunoglobulin (e.g., human Ig) that typically comprises a heavy chain variable region (VH), a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. The heavy chain constant regions (i.e., the CH1, hinge, CH2, and CH3 regions) can be any distinct isotype, such as human alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the constant domains. These produce human antibodies of the hIgA, hIgD, IgE, hIgG, and hIgM classes, including subclasses of hIgG, such as hIgG1, hIgG2, hIgG3, and hIgG4. As used herein, when referring to human antibodies, the term “heavy chain” can refer to any different type based on the amino acid sequence of the constant domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which respectively produce human IgA, IgD, IgE, IgG, and IgM antibodies, including subclasses of human IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0145] As used herein, the term "hematopoietic cell" refers to any blood cell. Therefore, the term hematopoietic cell includes, but is not limited to, hematopoietic pluripotent stem cells (HSPCs), common myeloid progenitor cells, megakaryocytes, erythroid progenitor cells, erythroid progenitor cells, proerythrocytes, early erythroblasts, intermediate erythroblasts, late erythroblasts, reticulocytes, megakaryocytes, platelets, granulocytes, monocytes, promonocytes, monocytes, macrophages, myelocytes, 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 target" refers to an agent that specifically binds to an antigen expressed on hematopoietic cells (or a subset thereof). For example, the antigen expressed on or in hematopoietic cells may be a membrane protein, such as an integrated membrane protein or a peripheral membrane protein. Typically, a hematopoietic cell target specifically binds to an antigen on a hematopoietic cell, which promotes the internalization of the hematopoietic cell target (and any associated molecular payload) into the hematopoietic cell. In some embodiments, the hematopoietic cell target specifically binds to an internalized cell surface receptor on a hematopoietic cell and can be internalized into the hematopoietic cell via receptor-mediated internalization. In some embodiments, the hematopoietic cell target is a protein (e.g., an antibody), a peptide, a nucleic acid (e.g., an aptamer), or a small molecule. In some embodiments, the hematopoietic cell target is linked to a molecular payload.

[0147] As used herein, when referring to a second element to describe a first element, the term "heterogeneous" means that the first and second elements do not exist in nature in the arrangement described. For example, a nucleic acid molecule containing a "heterogeneous portion" means a nucleic acid molecule with a portion (e.g., a carbohydrate, a small molecule, a polypeptide, a polynucleotide, a lipid, a synthetic polymer (e.g., a polymer of PEG)) that is not naturally linked to the nucleic acid molecule.

[0148] The terms “hinge” or “hinge region” are used interchangeably herein and refer to the hinge region of the immunoglobulin heavy chain. An exemplary reference to the hIgG1 hinge region is shown in SEQ ID NO: 172; and an exemplary reference to the hIgG4 hinge region is shown in SEQ ID NO: 185.

[0149] As used herein, the term “isolated” for agents (e.g., proteins, nucleic acid molecules, etc.) refers to agents (e.g., proteins, nucleic acid molecules, etc.) that are substantially free of other cellular components associated with them in their natural state.

[0150] As used herein, the term "KLF transcription factor 1" or "KLF1" refers to a zinc finger transcription factor that plays a role, particularly in the repression of fetal hemoglobin and the conversion from fetal hemoglobin to adult hemoglobin. The amino acid sequence of the reference human KLF1 (hKLF1) protein is shown in SEQ ID NO: 297 (NCBI reference number: NP_006554.1).

[0151] As used herein, the terms “modified nucleotide,” “nucleotide modification,” or the term “modification,” etc., relating to nucleotide or nucleic acid sequences, are used to refer to nucleotides that contain chemical modifications (e.g., modified sugar moieties, modified nucleobases, and / or modified internucleotide linkages, or any combination thereof). Exemplary modifications are provided herein, see, for example, §5.5.1.5. In some embodiments of this disclosure, deoxynucleotides (which are considered to be naturally occurring nucleotide forms) are considered to constitute modified nucleotides if present within RNA oligonucleotides.

[0152] As used herein, the term "molecular payload" refers to a pharmaceutical agent that modulates biological outcomes. In some embodiments, the molecular payload is operatively linked to a target agent (e.g., a target agent described herein, such as an anti-TFR antibody). In some embodiments, the molecular payload is a small molecule, protein, peptide, or oligonucleotide. In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide described herein, see, for example, § 5.5.1). In some embodiments, the molecular payload is used to regulate (e.g., inhibit) the transcription of DNA molecules, regulate (e.g., inhibit) the translation of RNA (e.g., mRNA) molecules, regulate (e.g., inhibit) the expression of proteins, or regulate (e.g., inhibit) the activity of proteins. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to a target nucleic acid molecule (e.g., an RNA molecule encoded by a target gene, such as an mRNA molecule encoded by a target gene).

[0153] As used herein, the term “non-complementary nucleotide mismatch” refers to a nucleotide in a complementary region (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 polymers of DNA or RNA. Nucleic acid molecules can be single-stranded or double-stranded; contain natural, non-natural, or modified nucleotides; and contain natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, rather than phosphodiesters found between nucleotides in unmodified nucleic acid molecules. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules obtained by any means available in the art, including but not limited to recombinant means, such as cloning nucleic acid molecules from recombinant libraries or cell genomes using common cloning techniques and polymerase chain reactions, as well as synthetic means. Those skilled in the art will understand that, unless otherwise stated, the nucleic acid sequences presented in this application will enumerate thymidine (T) in a representative DNA sequence, but where the sequence represents RNA (e.g., mRNA), thymidine (T) will be replaced with uracil (U). Thus, any RNA polynucleotide encoded by DNA identified by a specific sequence identification number may also contain a corresponding RNA (e.g., mRNA) sequence encoded by that DNA, wherein each thymidine (T) sequence of that DNA is replaced by uracil (U).

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

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

[0157] As used herein, the term "partial complementarity" means that in a hybridization pair of a first nucleic acid molecule and a second nucleic acid molecule, at least 70% but not all of the bases in the sequential sequence of the first nucleic acid molecule will hybridize with the same number of bases in the sequential sequence of the second nucleic acid molecule. The sequential sequence may comprise all or part of the first or second nucleic acid molecule.

[0158] The determination of the “percentage of identity” between two sequences (e.g., proteins (amino acid sequences) or oligonucleotides (nucleic acid sequences)) can be accomplished using mathematical algorithms. The determination of identity (as described herein) is independent of nucleotide chemical modifications (e.g., as described herein). For example, for the purpose of determining identity, (mC) is identical to (C). A specific non-limiting example of a mathematical algorithm for comparing 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 incorporated herein by reference in its entirety. Such algorithms are incorporated in the NBLAST and XBLAST procedures of Altschul SF et al., (1990) J Mol Biol [Journal of Molecular Biology] 215:403 (which is incorporated herein by reference in its entirety). BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, for example, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST procedure parameter set, for example, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, Gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res [Nucleic Acid Research] 25: 3389-3402 (which is incorporated herein by reference in its entirety). Alternatively, PSIBLAST can be used to perform iterative searches that detect long-distance relationships between molecules (ibid.). When using BLAST, GappedBLAST, and PSI Blast procedures, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17 (which is incorporated herein by reference in its entirety). This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, regardless of the presence of vacancy. When calculating the percentage of identity, only perfect matches are typically counted.

[0159] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to animals (e.g., human subjects) and comprising a therapeutic agent (e.g., the conjugate described herein) and a pharmaceutically acceptable carrier or diluent. "Pharmaceutically acceptable carrier or diluent" means a substance intended for contact with tissues of humans and / or non-human animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and commensurate with a reasonable therapeutic benefit / risk ratio.

[0160] As used herein, the term “multiple” means two or more (e.g., three or more, four or more, five or more, six or more, seven or more, nine or more, or ten or more).

[0161] As used herein, the terms “protein,” “polypeptide,” and “peptide” refer to a polymer of at least two (e.g., at least five) amino acids linked by peptide bonds. The term “polypeptide” does not indicate a specific length of amino acid polymer chain. Shorter amino acid polymers (e.g., about 2–50 amino acids) are generally referred to as peptides in the art, and longer amino acid polymers (e.g., about 50 amino acids) are referred to as polypeptides. However, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein. In some embodiments, proteins are folded into their three-dimensional structure. When considering polypeptides (e.g., having a linear (i.e., primary) structure) 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, for example, naturally occurring proteins, variants of naturally occurring proteins (e.g., functional variants), fragments of naturally occurring proteins (e.g., functional fragments), and synthetic proteins (i.e., non-naturally occurring proteins).

[0162] As used herein, the term "complementary region" refers to a portion of a first nucleic acid molecule that contains 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 “polynucleotide” are used interchangeably herein and refer to a macromolecule comprising multiple ribonucleotides polymerized by phosphodiester bonds. A ribonucleotide is a nucleotide in which the sugar is ribose. RNA may contain modified nucleotides; and contains native, non-native, or altered internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, rather than the phosphodiester linkages found between nucleotides in unmodified nucleic acid molecules.

[0164] As used herein, the term "RNAi agent" refers to an agent containing one or more RNA molecules that can mediate the targeted cleavage of RNA molecules (e.g., mRNA molecules) via the RNA-induced silencing complex (RISC) pathway. Therefore, RNAi agents can, for example, regulate (e.g., inhibit) the expression of target genes or proteins in cells (e.g., cells within a subject, such as mammalian subjects). RNAi agents include, for example, siRNA, miRNA, and shRNA.

[0165] The term "scFv" or "single-chain variable fragment" refers to an antibody comprising a VH region operatively linked to a VL region via a peptide linker, wherein the VH and VL regions are associated to specifically bind an antigen (e.g., to form an antigen-binding domain). In some embodiments, the scFv comprises a VH region, a peptide linker, and a VL region from its N-terminus to its C-terminus.

[0166] As used herein, the term "(scFv)2" refers to an antibody comprising a first scFv and a second scFv operatively linked (e.g., via a peptide linker). The first and second scFvs may specifically bind to the same or different antigens. In some embodiments, the first and second scFvs are operatively linked via a peptide linker.

[0167] As used herein, the term "scFv-Fc" refers to an antibody comprising an scFv operatively linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the scFv is operatively linked only to the first Fc domain of a first-first-second Fc domain pair. In some embodiments, a first scFv is operatively linked to the first Fc domain, and a second scFv is operatively linked to the second Fc domain of a first-first-second Fc domain pair.

[0168] As used herein, the term "(scFv)2-Fc" refers to (scFv)2 operatively linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, (scFv)2 is operatively linked only to the first Fc domain of a first-first-second Fc domain pair. In some embodiments, a first (scFv)2 is operatively linked to the first Fc domain, and a second (scFv)2 is operatively linked to the second Fc domain of a first-first-second Fc domain pair.

[0169] As used herein, the term "sense strand" refers to a portion of an RNA molecule (e.g., a portion of an RNAi agent (e.g., described herein), or a portion of a dsRNA agent (e.g., described herein)) that contains a region that is at least partially (e.g., substantially, completely) complementary to a region of the antisense strand (as defined herein). The sense strand is generally referred to as such because its sequence is oriented in the same direction relative to the target RNA (e.g., the mRNA sequence).

[0170] As used herein, the term "single-domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. sdAbs are capable of specifically binding to a particular antigen. VHHs (as defined herein) are examples of sdAbs.

[0171] As used herein, the term "specific binding" refers to a preferential interaction between a first protein (e.g., an antibody) and a second protein (e.g., an antigen) relative to other amino acid sequences, i.e., a significantly higher binding affinity. In this text, when a first protein is referred to as "specifically binding" a second protein, it should be understood that the first protein specifically binds to the epitope of the second protein. The term "epitaph" refers to the portion of the second protein that is specifically recognized by the first protein. Specific binding includes molecules that cross-react with the same epitope in different species. For example, an antibody that specifically binds to human TFR may cross-react with TFR in another species (e.g., cynomolgus monkeys, mice, etc.) and is still considered to specifically bind to human TFR in this text. A protein can specifically bind to more than one different protein. Specific binding can be measured, for example, by measuring binding affinity (e.g., using standard methods known in the art and described herein (e.g., surface plasmon resonance (SPR) (e.g., BIAcore®-based determination)), which is a common method known in the art (see, for example, Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173 and 5,468,614, the entire contents of each of which are incorporated herein by reference 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 (e.g., a 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, such as a non-human primate (e.g., monkey, ape), an ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), a carnivore (e.g., dog, cat), a rodent (e.g., rat, mouse), or a rabbit (e.g., rabbit). In some embodiments, the subject is a bird, such as a member of the bird groups Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathea (e.g., ostrich, emu), Columbiformes (e.g., pigeon, wild pigeon), or Psittaciformes (e.g., parrot).

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

[0174] As used herein, the term "target nucleic acid sequence" refers to a continuous 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 mRNA, which is the 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 the oligonucleotides (e.g., antisense oligonucleotides, RNAi agents, etc.) described herein.

[0175] As used herein, the term "therapeutic agent" means a pharmaceutical agent (e.g., the conjugate described herein) that, when administered in a therapeutically effective amount, is capable of achieving a desired therapeutic outcome (e.g., treating a disease as defined herein) in a subject or outside the body.

[0176] As used herein, the term "therapeuticly effective amount" of a therapeutic agent means any amount of a therapeutic agent that, when used alone or in combination with another therapeutic agent, improves the condition of a disease, such as protecting a subject from the onset of a disease (or infection); improves the symptoms of a disease or infection, such as reducing the severity, frequency, or duration of symptoms, increasing the asymptomatic period of a disease or infection; prevents or reduces damage or disability caused by a disease or infection; or promotes the resolution of a disease (or infection). The ability of a therapeutic agent to improve the condition of a disease can be assessed using a variety of methods known to a skilled technician, such as in human subjects during clinical trials, in animal model systems where efficacy in predictable humans is known, or by measuring the activity of the agent in an in vitro assay.

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

[0178] As used herein, the term "transferrin" or "TF" refers to the plasma glycoprotein transferrin, which plays a role, particularly 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 the reference human TF (hTF) protein is shown in SEQ ID NO: 3 (UniProt accession number P02787).

[0179] As used herein, the term "transferrin receptor" or "TFR" refers to a transmembrane homodimeric glycoprotein that plays a role, particularly in the cellular uptake of iron from the plasma glycoprotein transferrin. The term TFR, where applicable, includes several isotypes and homologs. For example, human TFR (hTFR) includes homologs hTFR1 and hTFR2. The amino acid sequence of the reference hTFR1 protein is shown in SEQ ID NO: 1 (UniProt accession number P02786|). TFR1 is also commonly referred to in the art as CD71. The terms TFR1 and CD71 are used interchangeably herein. The amino acid sequence of the reference hTFR2 protein is shown in SEQ ID NO: 2 (UniProt accession number Q9UP52).

[0180] As used herein, the terms "treat," "treating," "treatment," etc., refer to the reduction or improvement of a disease and / or one or more symptoms associated with it, or the attainment of a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a disease does not necessarily require the complete elimination of the disease or one or more symptoms associated with it. In some embodiments, the effect is therapeutic, i.e., but not limited to, the effect partially or completely reduces, weakens, eliminates, alleviates, relieves, reduces the intensity of the disease and / or adverse symptoms caused by the disease, or cures the disease and / or adverse symptoms caused by the disease. In some embodiments, the effect is preventative, i.e., the effect protects against or prevents the occurrence or recurrence of the disease. For this purpose, the methods disclosed herein include administering a therapeutically effective amount, for example, the conjugates described herein (or carriers, pharmaceutical compositions, etc., containing them).

[0181] As used herein, the term "variation" or "variant" or similar usage regarding nucleotide or nucleic acid sequences refers to a nucleic acid molecule that contains at least one substitution, addition, deletion, or inversion of one or more nucleotides compared to a reference nucleic acid molecule. Similarly, as used herein, the term "variation" or "variant" or similar usage regarding peptides or proteins refers to a peptide or protein that contains at least one substitution, addition, deletion, or inversion of an amino acid residue compared to a reference peptide or protein.

[0182] "Modulations that promote heterodimerization of the first and second Fc regions" (or similar terminology) are post-translational modifications of the peptide backbone or Fc regions that reduce or prevent the formation of homodimers by associating the Fc-containing peptides with the same peptides. As used herein, association-promoting modifications specifically include individual modifications to each of the two Fc regions (i.e., the first and second Fc regions) to which association is desired, wherein the modifications are complementary to each other to promote association between the two Fc regions. For example, association-promoting modifications may alter the structure or charge of one or both Fc regions to enable them to associate favorably, either spatially or electrostatically. Thus, heterodimerization occurs between peptides containing the first Fc region and peptides containing the second Fc region, which may be different in the sense of their fusion with other components (e.g., antigen-binding domains) of each Fc region. In some embodiments, association-promoting modifications include amino acid mutations, particularly amino acid substitutions, in the Fc regions. In particular embodiments, association-promoting modifications include individual amino acid mutations, particularly one or more amino acid substitutions, in each of the first and second Fc regions. See, for example, § 5.3.2.2.

[0183] As used herein, the term "variable region" refers to a portion of an antibody, typically a portion of the light or heavy chain, usually about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain, and about 90 to 115 amino acids from the mature light chain. These vary considerably in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while highly conserved regions within the variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is assumed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human frame region (FR). In certain embodiments, the variable region is a primate (e.g., a non-human primate) variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a primate (e.g., a non-human primate) frame region (FR).

[0184] The terms "VL" and "VL region" are used interchangeably to refer to the variable region of the immunoglobulin light chain. The VL region can be incorporated into antibodies (e.g., scFv, Fab, full-length antibodies). For example, scFv contains a VL region operatively linked to the VH region via a peptide linker.

[0185] The terms "VH" and "VH region" are used interchangeably to refer to the variable region of the immunoglobulin heavy chain. The VH region can be incorporated into antibodies (e.g., scFv, Fab, full-length antibodies). For example, scFv contains a VH region operatively linked to the VL region via a peptide linker.

[0186] The term "VHH" used in this article refers to a class of single-domain antibodies (sdAbs) that possess a single single-chain variable antibody domain (VH). These antibodies can be found, produced, or synthesized in camelid mammals (such as camels and llamas) that naturally lack light chains.

[0187] As used herein, the term "(VHH)2" refers to an antibody comprising a first VHH and a second VHH operatively linked (e.g., via a peptide linker). The first and second VHHs may specifically bind to the same or different antigens. In some embodiments, the first and second VHHs are operatively linked via a peptide linker.

[0188] As used herein, the term "VHH-Fc" refers to an antibody comprising a VHH operatively linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the VHH is operatively linked only to the first Fc domain of a first and second Fc domain pair. In some embodiments, a first VHH is operatively linked to the first Fc domain, and a second VHH is operatively linked to the second Fc domain of a first Fc and a second Fc pair.

[0189] As used herein, the term "(VHH)2-Fc" refers to (VHH)2 operatively linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, (VHH)2 is operatively linked only to the first Fc domain of a first-first-second Fc domain pair. In some embodiments, a first (VHH)2 is operatively linked to the first Fc domain, and a second (VHH)2 is operatively linked to the second Fc domain of a first-second-Fc pair.

[0190] As used herein, the term “ZBTB7A” or “zinc-finger and BTB domain 7A” specifically refers to a transcription factor that plays a role in the repression of fetal hemoglobin and the conversion from fetal hemoglobin to adult hemoglobin. The amino acid sequence of the reference human ZBTB7A (hZBTB7A) protein is shown in SEQ ID NO: 294 (NCBI reference number: NP_056982.1). 5.2 Conjugates

[0191] This article provides, in particular, conjugates (e.g., antibody-oligonucleotide conjugates) that can be used to regulate (e.g., inhibit, reduce, enhance) the expression and / or activity of target genes or proteins (e.g., within cells (e.g., erythroid precursor cells), such as within the cells of a subject (e.g., mammalian subjects, such as human subjects)) (e.g., by binding to target nucleic acid molecules (e.g., mRNA molecules)).

[0192] The conjugates described herein comprise a target agent (e.g., a hematopoietic cell target 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) the expression or activity of a target gene or protein (e.g., a target gene or protein expressed by hematopoietic cells (e.g., erythroid progenitor cells))).

[0193] In some embodiments, the conjugate is internalized into the hematopoietic cells after binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells.

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

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

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

[0198] In some embodiments, a protein (e.g., an antibody) that specifically binds to a TFR (e.g., hTFR (e.g., hTFR1)) exhibits one or more of the following properties: (a) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., an antibody) or the conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of erythroid precursor cells does not induce the death of the target cells; (c) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the erythroid precursor cells remain viable; (d) Upon internalization into erythroid precursor cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the death of the erythroid precursor cells; and / or (e) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the degradation of the 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., hTFR1)) expressed on the surface of a target cell (e.g., erythroid precursor cells), the conjugate is internalized into the target cell; (b) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of a target cell (e.g., erythroid precursor cells), the conjugate does not induce the death of the target cell; (c) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of a target cell (e.g., erythroid precursor cells), the target cell remains viable; (d) upon internalization into the target cell (e.g., erythroid precursor cells), the conjugate does not induce the death of the target cell; and / or (e) Upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1))) expressed on the surface of a target cell (e.g., erythroid precursor cells), the conjugate does not induce the degradation of the target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1))). 5.3 Hematopoietic cell targeting agents

[0200] As described above, the conjugates described herein comprise a targeting agent (e.g., for targeting a molecular payload (e.g., an oligonucleotide described herein) to one or more specific cells (e.g., within a subject)). In some embodiments, the targeting agent is a hematopoietic cell (e.g., 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 hematopoietic cells (e.g., erythroid precursor cells). In some embodiments, hematopoietic cells (e.g., erythroid precursor cells) are present in (e.g., the subject's) bone marrow. In some embodiments, the targeting agent is capable of targeting a molecular payload to one or more cells 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)) by specifically binding to an antigen expressed on the surface of the erythroid precursor cells (e.g., TFR (e.g., hTFR (e.g., hTFR1)).

[0201] It should be understood that various types of targeting agents (e.g., hematopoietic cell (e.g., erythroid progenitor cell) targeting agents) can be used according to this disclosure. For example, a targeting agent (e.g., a hematopoietic cell (e.g., erythroid progenitor cell) targeting agent) may comprise (or consist of): small molecules, oligonucleotides (e.g., DNA, RNA, RNA / DNA hybrids) (e.g., aptamers), proteins (e.g., antibodies, peptides), lipids (e.g., microvesicles), or carbohydrates (e.g., polysaccharides). 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 progenitor cell) targeting agents) are further described in detail herein; however, it should be understood that the exemplary targeting agents (e.g., hematopoietic cell (e.g., erythroid progenitor cell) targeting agents provided herein are not intended to be limiting.

[0202] In some embodiments, a hematopoietic cell (e.g., erythroid progenitor cell) target specifically binds to antigens (e.g., TFR (e.g., hTFR (e.g., hTFR1))) expressed on the surface of hematopoietic cells (or one or more subsets thereof) (e.g., erythroid progenitor cells) (e.g., within the bone marrow). In some embodiments, a hematopoietic cell (e.g., erythroid progenitor cell) target specifically binds to antigens (e.g., TFR (e.g., hTFR (e.g., hTFR1))) expressed on the surface of erythroid progenitor cells. In some embodiments, a hematopoietic cell (e.g., erythroid progenitor cell) target specifically binds to antigens (e.g., TFR (e.g., hTFR (e.g., hTFR1))) expressed on the surface of erythroid progenitor cells within the bone marrow.

[0203] By interacting with one or more molecules (e.g., proteins) expressed on the surface of target hematopoietic cells (e.g., erythroid progenitor cells), tissue (e.g., bone marrow) localization and selective or preferred uptake into hematopoietic cells (e.g., erythroid progenitor cells) can be achieved. In some embodiments, molecules (e.g., proteins) that serve as substrates for hematopoietic cell (e.g., erythroid progenitor cell) uptake transport proteins can be used to deliver molecular payloads (e.g., oligonucleotides as described herein) into hematopoietic cells (e.g., erythroid progenitor cells). Binding to molecules (e.g., proteins) expressed on the surface of hematopoietic cells (e.g., erythroid progenitor cells), followed by endocytosis, can allow macromolecules (such as antibodies) to enter hematopoietic cells (e.g., erythroid progenitor cells). For example, as described in detail below, molecular payloads (e.g., oligonucleotides described herein) conjugated with transferrin (or a functional fragment or functional variant thereof) or anti-TFR (e.g., hTFR (e.g., hTFR1)) antibodies can be taken up by hematopoietic cells (e.g., erythroid progenitor cells) via binding to TFR (e.g., hTFR (e.g., hTFR1)) and can then be endocytosed, for example, via endocytosis (e.g., clathrin-mediated endocytosis).

[0204] The use of hematopoietic cell (e.g., erythroid progenitor cells) targets can be used to concentrate molecular payloads (e.g., oligonucleotides described herein) in hematopoietic cells (e.g., erythroid progenitor cells (e.g., within the bone marrow (e.g., within the subject)) while reducing toxicity associated with effects in other cells or tissues. In some embodiments, the hematopoietic cell (e.g., erythroid progenitor cells) target concentrates the bound molecular payload (e.g., oligonucleotides described herein) in hematopoietic cells (e.g., erythroid progenitor cells (e.g., within the bone marrow (e.g., within the subject)) compared to another tissue or cell type within the subject. In some embodiments, the hematopoietic cell (e.g., erythroid progenitor cells) target agent concentrates the molecular payload (e.g., oligonucleotides described herein) bound to hematopoietic cells (e.g., erythroid progenitor cells) in an amount 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 that in non-hematopoietic cells (e.g., non-erythroid progenitor cells). In some embodiments, when the molecular payload (e.g., oligonucleotides described herein) is delivered to a subject in conjunction with the hematopoietic cell (e.g., erythroid progenitor cells) target agent, the toxicity of the molecular payload (e.g., oligonucleotides described herein) in the 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%.

[0205] In some embodiments, the hematopoietic cell (e.g., erythroid progenitor) target agent exhibits one or more of the following properties: (a) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of a target cell (e.g., erythroid progenitor) and the target agent is internalized into the target cell; (b) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of a target cell (e.g., erythroid progenitor) and the target agent does not induce the death of the target cell; (c) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of a target cell (e.g., erythroid progenitor) and the target cell remains viable; (d) After being internalized into target cells (e.g., erythroid progenitor cells), the hematopoietic cell (e.g., erythroid progenitor cell) target agent does not induce the death of said target cells; and / or (e) after binding to target molecules (e.g., TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of target cells (e.g., erythroid progenitor cells), the hematopoietic cell (e.g., erythroid progenitor cell) target agent does not induce the degradation of said target molecules (e.g., TFR (e.g., hTFR (e.g., hTFR1))). 5.3.1 TFR-targeting agents

[0206] In some embodiments, the target agent (e.g., a hematopoietic cell (e.g., erythroid progenitor cell) target agent) specifically binds to transferrin receptor (TFR) (e.g., hTFR (e.g., hTFR1)). In some embodiments, the target agent (e.g., a hematopoietic cell (e.g., erythroid progenitor cell) target agent) specifically binds to hTFR. In some embodiments, the target agent (e.g., a hematopoietic cell (e.g., erythroid progenitor cell) target agent) specifically binds to hTFR1.

[0207] TFR1 is a transmembrane homodimeric glycoprotein that plays a role, particularly in cellular iron uptake from the plasma glycoprotein transferrin (TF). Iron uptake from transferrin involves the binding of TF to TFRs (e.g., TFR1), the internalization of TF within endocytic vesicles via receptor-mediated endocytosis, and the release of iron induced by a decrease in endosome pH. TFR1 is expressed by, for example, placental syncytiotrophoblasts, myocytes, basal keratinocytes, hepatocytes, endocrine pancreas, spermatocytes, and erythroid precursor cells. While TFR expression is known to be highly expressed in erythroid precursor cells, it is not expressed in mature erythrocytes. TFR2 is a known homolog of TFR1, but TFR1 is considered the major protein responsible for iron uptake due to its higher affinity and expression pattern. See, for example, Derek K. Marsee et al., CD71 (Transferrin Receptor): An Effective Marker for Erythroid Precursors in Bone Marrow Biopsy Specimens, American Journal of Clinical Pathology, Vol. 134, No. 3, September 2010, pp. 429-435, https: / / doi.org / 10.1309 / AJCPCRK3MOAOJ6AT; C. Sieff et al., Changes in CellSurface Antigen Expression During Hemopoietic Differentiation, Blood, Vol. 60(3), 1982, pp. 703-713. https: / / doi.org / 10.1182 / blood.V60.3.703.703; for all purposes, the full content of each of these articles is incorporated herein by reference.

[0208] The amino acid sequence of the reference hTFR1 protein is shown in SEQ ID NO: 1. The amino acid sequence of the reference hTFR2 protein is shown in SEQ ID NO: 2. The amino acid sequence of the reference hTF protein is shown in SEQ ID NO: 3. See Table 1 in this document. Table 1. Amino acid sequences of reference hTFR1, hTFR2 and hTF proteins.

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

[0210] In some embodiments, the target (e.g., a hematopoietic cell (e.g., an erythroid progenitor cell) target) specifically binds to one or more of hTFR1 and mouse TFR1, rat TFR1, and non-human primate TFR1 (e.g., cynomolgus monkey TFR1).

[0211] In some embodiments, TFR (e.g., hTFR (e.g., hTFR1)) targets enhance the distribution and / or uptake (e.g., into cells, such as into the subject's cells, such as cells expressing TFR (e.g., hTFR (e.g., hTFR1)) (e.g., erythroid precursor cells (e.g., in bone marrow)) of molecular payloads (e.g., oligonucleotides described herein) (e.g., compared to oligonucleotides lacking a targeting moiety). In some embodiments, TFR (e.g., hTFR (e.g., hTFR1)) targets alter (e.g., prolong) the lifespan (e.g., in vivo) of molecular payloads (e.g., oligonucleotides described herein) (e.g., RNAi agents (e.g., siRNA), ASO, etc.) (e.g., compared to oligonucleotides lacking a targeting moiety). In some embodiments, TFR (e.g., hTFR (e.g., hTFR1)) targets provide enhanced affinity for selected targets (e.g., selected cell types, compartments (e.g., cell types, tissues, organs, or body regions)) (e.g., compared to oligonucleotides lacking the target moiety) (e.g., erythroid precursor cells (e.g., in bone marrow)).

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

[0213] In some embodiments, the TFR target is a protein. In some embodiments, the target is transferrin. In some embodiments, the target is an antibody. In some embodiments, the target comprises an antibody-like scaffold (e.g., a cysteine-dense peptide, see, for example, WO 2023023031, the entire contents of which are incorporated herein by reference for all purposes). 5.3.1.1 TF Protein

[0214] In some embodiments, the TFR targeter comprises a TF (e.g., hTF) (or a functional fragment or variant thereof). In some embodiments, the TFR targeter comprises a TFR1 binding domain of a 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 that 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 shown in SEQ ID NO: 3.

[0215] Variant hTF proteins are known in the art, see, for example, WO 2009019314 A1, WO 2008152140 A2, EP 2216341 A1, WO 2009149393 A2, US 8158579 B2, the entire contents of each of which are incorporated herein by reference for all purposes. In some embodiments, variant hTF proteins exhibit increased stability and / or a longer plasma half-life (e.g., relative to a reference hTF protein that does not contain one or more variants). In some embodiments, variant hTF proteins comprise substitutions of non-cysteine ​​amino acid residues with cysteine ​​(e.g., at positions V1, P2, D3, K4 according to SEQ ID NO: 3). T5, H14, Q20, S21, D24, K27, S28, V29, P31, S32, D33, A43, E89, D104, G106, G114, L1 22. G123, P145, S155, D163, T165, D166, P168, P175, G176, G178, C179, S180, T181, L182, Q184, F187, S189, D197, G198, E212, A215, N216, A218, D221, D229, G257, N26 8. D277, K278, K280, E281, S287, P288, H289, K291, S298, P307, L326, T330, P335, T 336, 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, K5 45, 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 (e.g., as described in WO 2009019314 A1). In some embodiments, the variant hTF protein includes the addition of cysteine ​​residues (e.g., as described in WO 2009019314 A1). (i) Exemplary hTF variant protein

[0216] Table 2 provides the amino acid sequences of exemplary hTF variants. Table 2. Amino acid sequences of exemplary hTF variants.

[0217] In some embodiments, the TFR targeter comprises a TF variant (e.g., an hTF variant) (or a functional fragment or variant thereof). In some embodiments, the TF variant (or a functional fragment or variant thereof) comprises or consists of the following: an amino acid sequence that 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 the TF variants shown in Table 2, wherein the amino acid sequence of the TF variant contains 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, the TF variant (or a functional fragment or variant thereof) comprises or consists of the following: an amino acid sequence that 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 the TF variant shown in any one of SEQ ID NO: 4-12, wherein the amino acid sequence of the TF variant contains at least one amino acid variation compared to the amino acid sequence of a reference TF protein (e.g., SEQ ID NO: 3). (ii) Heterogeneous components

[0218] In some embodiments, the TF (e.g., an hTF variant) is operatively linked to a heterologous portion (e.g., an Fc region (e.g., the Fc region described herein (see, for example, § 5.3.2))). In some embodiments, the heterologous portion is a half-life extension portion. Exemplary half-life extension portions include, but are not limited to, immunoglobulins (e.g., human Ig (hIg)), fragments of Ig (e.g., hIg), Ig (e.g., hIg) constant regions, fragments of Ig (e.g., hIg) constant regions, Ig (e.g., hIg) Fc regions, human serum albumin (HSA), HSA-binding proteins or peptides, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life-extended peptides include, but are not limited to, Ig, fragments of Ig, one or more Ig heavy chain constant regions, fragments of Ig constant regions, Ig Fc regions, hIg, fragments of hIg, one or more hIg heavy chain constant regions, fragments of hIg constant regions, hIg Fc regions, human serum albumin (HSA), and HSA-binding proteins or peptides. The pharmacokinetic properties of immunomodulatory proteins fused to or conjugated to the half-life-extended moiety or the peptides described herein can be evaluated using standard in vivo methods known in the art.

[0219] In some embodiments, the heterologous portion is a heterologous polypeptide. In some embodiments, the heterologous polypeptide includes one or more Ig heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, the Ig is IgG. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4.

[0220] In some embodiments, the heteropeptide comprises or consists of: an IgG CH2 region and an IgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partially IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partially IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partially IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the heteropeptide comprises or is composed of the following: an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0221] In some embodiments, the heteropeptide comprises or is composed of: an Ig Fc region. In some embodiments, the Ig Fc region comprises or is composed 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 is composed of: a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or is composed 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 is composed of: an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or is composed of: at least a portion of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises or is composed of: an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises or is composed 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 the following: IgG4 hinge region, IgG4 CH2 region and IgG4 CH3 region.

[0222] In some embodiments, the heteropeptide comprises one or more hIg heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1, IgG2, IgG3, or IgG4. In some embodiments, hIgG is IgG1 or IgG4. In some embodiments, hIgG is hIgG1. In some embodiments, hIgG is hIgG4.

[0223] In some embodiments, the heteropeptide comprises or consists of the following: an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partial hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partial hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partial hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the heteropeptide comprises or is composed of the following: hIgG4 hinge region, hIgG4CH2 region and hIgG4 CH3 region.

[0224] In some embodiments, the heteropeptide comprises or is composed of: an hIg Fc region. In some embodiments, the hIg Fc region comprises or is composed of: at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: at least a portion of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: at least a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or consists of at least a portion of the hIgG4 hinge region, the hIgG4 CH2 region, and the hIgG4 CH3 region. 5.3.1.2 TFR-binding peptides and antibody-like scaffolds

[0225] In some embodiments, the TFR targeter comprises a peptide that specifically binds to a TFR (e.g., hTFR). TFR (e.g., hTFR (e.g., hTFR1)) binding peptides that can be used in the conjugates described herein are known in the art. See, for example, US6743893 and US 8399653, the entire contents of each of which are incorporated herein by reference for all purposes.

[0226] In some embodiments, the TFR target comprises an antibody-like scaffold. Anti-TFR antibody-like scaffolds that can be used in the conjugates described herein are known in the art. See, for example, WO 2023023031 (which describes an anti-TFR cysteine-dense peptide) and WO 2021076546 (which describes a TFR-binding fibronectin type III domain), the entire contents of each of which are incorporated herein by reference for all purposes. (i) Exemplary TFR-binding peptides and antibody-like scaffolds

[0227] Table 3 provides the amino acid sequences of exemplary TFR-specific antibody-like scaffolds. Table 3. Amino acid sequences of exemplary TFR-specific peptides and antibody-like scaffolds.

[0228] In some embodiments, the TFR target comprises a TFR-specific peptide or an antibody-like scaffold (or a functional fragment or variant thereof). In some embodiments, the TFR target comprises a TFR-specific peptide (or a functional fragment or variant thereof). In some embodiments, the TFR-specific peptide (or a functional fragment or variant thereof) comprises or consists of an amino acid sequence that 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 the TFR-specific peptide (or a functional fragment or variant thereof) shown in Table 3. In some embodiments, the TFR-specific peptide (or a functional fragment or variant thereof) comprises or consists of an amino acid sequence that 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 shown in any one of SEQ ID NO: 13-41. In some embodiments, the TFR target comprises an antibody-like scaffold (or a functional fragment or variant thereof). In some embodiments, the antibody-like scaffold (or a functional fragment or variant thereof) comprises or consists of an amino acid sequence that 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 the antibody-like scaffold (or a functional fragment or variant thereof) shown in Table 3. In some embodiments, the antibody-like scaffold (or a functional fragment or variant thereof) comprises or consists of an amino acid sequence that 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 shown in any one of SEQ ID NO:42-61. (ii) Heterogeneous components

[0229] In some embodiments, a TFR-binding peptide or antibody-like scaffold is operatively linked to a heterologous portion (e.g., an Fc region (e.g., the Fc region described herein (see, for example, § 5.3.2))). In some embodiments, the heterologous portion is a half-life extension portion. Exemplary half-life extension portions include, but are not limited to, immunoglobulins (e.g., human Ig (hIg)), fragments of Ig (e.g., hIg), Ig (e.g., hIg) constant regions, fragments of Ig (e.g., hIg) constant regions, Ig (e.g., hIg) Fc regions, human transferrin, human serum albumin (HSA), HSA-binding proteins or peptides, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life-extended peptides include, but are not limited to, Ig, fragments of Ig, one or more Ig heavy chain constant regions, fragments of Ig constant regions, Ig Fc regions, hIg, fragments of hIg, one or more hIg heavy chain constant regions, fragments of hIg constant regions, hIg Fc regions, human serum albumin (HSA), and HSA-binding proteins or peptides. The pharmacokinetic properties of immunomodulatory proteins fused to or conjugated to the half-life-extended moiety or the peptides described herein can be evaluated using standard in vivo methods known in the art.

[0230] In some embodiments, the heterologous portion is a heterologous polypeptide. In some embodiments, the heterologous polypeptide includes one or more Ig heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, the Ig is IgG. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4.

[0231] In some embodiments, the heteropeptide comprises or consists of: an IgG CH2 region and an IgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partially IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partially IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partially IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the heteropeptide comprises or is composed of the following: an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0232] In some embodiments, the heteropeptide comprises or is composed of: an Ig Fc region. In some embodiments, the Ig Fc region comprises or is composed 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 is composed of: a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or is composed 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 is composed of: an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or is composed of: at least a portion of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises or is composed of: an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises or is composed 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 the following: IgG4 hinge region, IgG4 CH2 region and IgG4 CH3 region.

[0233] In some embodiments, the heteropeptide comprises one or more hIg heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1, IgG2, IgG3, or IgG4. In some embodiments, hIgG is IgG1 or IgG4. In some embodiments, hIgG is hIgG1. In some embodiments, hIgG is hIgG4.

[0234] In some embodiments, the heteropeptide comprises or consists of the following: an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partial hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heteropeptide comprises or consists of: an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partial hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heteropeptide comprises or consists of: an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the heteropeptide comprises or consists of: a partial hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the heteropeptide comprises or is composed of the following: hIgG4 hinge region, hIgG4CH2 region and hIgG4 CH3 region.

[0235] In some embodiments, the heteropeptide comprises or is composed of: an hIg Fc region. In some embodiments, the hIg Fc region comprises or is composed of: at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: at least a portion of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: at least a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or is composed of: an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or consists of at least a portion of the hIgG4 hinge region, the hIgG4 CH2 region, and the hIgG4 CH3 region. 5.3.1.3 Anti-TFR (e.g., anti-TFR1) antibodies

[0236] In some embodiments, the TFR target is an anti-TFR antibody (e.g., an anti-hTFR antibody) (e.g., an anti-hTFR1 antibody). In some embodiments, the antibody comprises or consists of the following: full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, single-domain antibody (sdAb) (e.g., VHH), sdAb-Fc (e.g., VHH-Fc), (sdAb)2 (e.g., (VHH)2), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody comprises or consists of the following: full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, sdAb-Fc (e.g., 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: Fab. In some embodiments, the antibody comprises or consists of: F(ab')2. In some embodiments, the antibody comprises or consists of: Fab-Fc. In some embodiments, the antibody comprises or consists of: scFv-Fc. In some embodiments, the antibody comprises or consists of: (scFv)2-Fc. In some embodiments, the antibody comprises or consists of: sdAb-Fc (e.g., VHH-Fc). In some embodiments, the antibody comprises or consists of: o(sdAb)2-Fc (e.g., (VHH)2-Fc).

[0237] In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is an IgG1 or IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is an hIgG1, hIgG2, hIgG3, or hIgG4 antibody. In some embodiments, the antibody is an hIgG1 or hIgG4 antibody. In some embodiments, the antibody is an hIgG1 antibody. In some embodiments, the antibody is an hIgG4 antibody. (i) Exemplary anti-TFR (e.g., anti-TFR1) antibody

[0238] In some embodiments, the target (e.g., a hematopoietic cell (e.g., erythroid precursor cell) target) comprises an anti-TFR antibody. Anti-hTFR1 antibodies that can be used in the conjugates described herein are known in the art.

[0239] Exemplary anti-TFR1 antibodies known in the art that can be used in the conjugates described herein include, but are not limited to, for example, OKT9 (see, for example, US 4364934); M11, M23, M27, B84 (see, for example, WO 2015098989 and US9994641); 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4 (see, for example, WO 2016081643 and US 9708406); 8D3 (see, for example, US 2010 / 077498 and Lee et al., “Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”). [Targeting rat anti-8D3 mouse transferrin receptor monoclonal antibody across mouse blood-brain barrier]” 2000, J Pharmacol. Exp. Ther. [Journal of Pharmacology and Experimental Therapeutics], 292:1048-1052); OX26 (see, for example, Haobam, B. et al. 2014. Rab17-mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion. Cellular microbiology. 16: 1806-21); DF1513 (see, for example, Ortiz-Zapater E et al. Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A).[Transportation of human transferrin receptor in plant cells: the role of tyrosine phosphorylation inhibitor A23 and brefidobacterium A] Plant J 48:757-70 (2006); the following commercially available clones (e.g., Novus Biologicals) are: 1A1B2, 661G1, MEM-189, JF0956, 29806, 1A1B2, TFRC / 1818, 1E6, 66Ig10, TFRC / 1059, Q1 / 71, 23D10, 13E4, TFRC / 1149, ER-MP21, YTA74.4, BU54, 2B6, RI7 217; BA120g (see, for example, US20110311544 A1 and US 7572895); B3 / 25 and T58 / 30 (see, for example, Trowbridge, IS et al., “Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumor cells”, Nature, 1981, Vol. 294, pp. 171-173; the following commercially available clones (e.g., BioXcell) are R17 217.1.3, 5E9C11, OKT9 (BE0023 clone), BK19.9, B3 / 25, T56 / 14, and T58 / 1 (see, for example, Gatter, KC et al., “Transferrin receptors in human tissues: their distribution and possible clinical relevance”, J Clin Pathol, May 1983). 36(5):539-45); 5E9C11, R17 217.1.3 (available from BioXel), BE0175 (available from BioXel); the full content of each of these articles is incorporated herein by reference for all purposes.

[0240] Exemplary anti-TFR (e.g., hTFR (e.g., hTFR1)) antibodies that can be used in the conjugates described herein are described in, for example, WO 2023283531; WO 2021154477 A1; WO 2020132584 A1; WO 2021154476 A1; WO2021150382 A1; WO 2023023031 A2; WO 2021146256 A1; WO 2021142275 A1; US20220017635 A1; WO 2016207240 A1; US ​​11267896 B2; US 20220143206 A1, US 11028179B2; US 11286305 B2; WO 2023087017 A1; WO 2023086864 A1; WO 2023044398 A1; WO2023039611 A2; WO 2023034409 A1; WO 2023283623 A1; WO 2023283624 A2; WO2023283619 A2; WO 2023283620 A1; WO 2023283615 A1; WO 2023283613 A1; WO2023283614 A2; US 11672872 B2; US 11648318 B2; WO 2022271549 A1; WO 2022201122A1; WO 2022174114 A1;WO 2022026152 A2; WO 2022020107 A1; WO 2022020106 A1; WO2022020105 A1; WO 2022020108 A; WO 2022020109 A1; WO 2021205358 A1; US20230174646A1; US ​​20210299266 A1; WO 2021195469 A1; US ​​11446387 B2; US 20220409735 A1; US20210301290 A1; US ​​20210369762 A1; US 11525137 B2; US 11555190 B2; US 11111308B2; US 10550188 B2; US 10508151 B2; US 20160208008 A1; US ​​20150291697 A1; US20130171061 A1; US ​​9562230 B2; US 7976841 B2; US 4364934; WO 2015098989; US9994641; WO 2016081643;US 9708406; US 2010077498; US 20110311544; US 7572895; WO2019075417; US 20060286030 A1; US ​​20190240346 A1; US ​​20130216476 A1; WO2023283531; US ​​20130177579 A1; US ​​9598496 B2; US 20130045206 A1; US ​​20060039908A1; US ​​6015555 A; US 6008326 A; US 5648469 A; EP 79696B1;WO 2023034409 A1; US4364934; US 8409573; US 9708406; US 9611323; WO 2015098989; Schneider C. et al., “Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9”, 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”, J Pharmacol. Exp. Ther. 2000, 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. Rab17-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, IS et al. "Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumor." cells. "Anti-transferrin receptor monoclonal antibodies and toxin-antibody conjugates affect the growth of human tumor cells" Nature, 1981, Vol. 294, pp. 171-173; Gatter, KC et al. "Transferrin receptors in human tissues: their distribution and possible clinical relevance" J Clin Pathol. May 1983; 36(5):539-4; For all purposes, the full content of each of these articles is incorporated herein by reference.

[0241] Table 4 provides the amino acid sequences of exemplary anti-hTFR antibodies that can be used in the conjugates described herein. The CDRs of the anti-hTFR antibodies in Table 4 are represented according to Kabat. Those skilled in the art will be able to determine the CDRs as defined by other schemes (e.g., Chothia, IMGT) using common methods known in the art. Table 4. Amino acid sequences of exemplary anti-hTFR antibodies.

[0242] Table 26 provides the amino acid sequences of other exemplary anti-hTFR1 antibodies that can be used in the conjugates described herein. The CDRs of the anti-hTFR antibodies in Table 26 are represented according to Kabat. Those skilled in the art will be able to determine the CDRs as defined by other schemes (e.g., Chothia, IMGT) using common methods known in the art. Table 26. Amino acid sequences of exemplary anti-TFR1 antibodies.

[0243] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) antibodies comprise anti-TFR (e.g., hTFR, e.g., hTFR1) antibodies named and / or incorporated herein by reference.

[0244] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VH, which includes: VH CDR1, VH CDR2 and VH CDR3.

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

[0249] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VL, which includes: VL CDR1, VL CDR2 and VL CDR3.

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

[0254] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VH, which includes VH CDR1, VH CDR2 and VH CDR3; and VL, which includes VL CDR1, VL CDR2 and VL CDR3.

[0255] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the following: the amino acid sequence of VH CDR1 of an anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of an anti-TFR antibody named and / or incorporated herein by reference, comprising 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 following: the amino acid sequence of VH CDR2 of an anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of an anti-TFR antibody named and / or incorporated herein by reference, comprising 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 following: the amino acid sequence of VH CDR2 of an anti-TFR antibody named and / or incorporated herein by reference. The amino acid sequence of CDR3, or the amino acid sequence of VH anti-TFR antibody comprising or consisting of the following, or which is incorporated herein by reference: 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 following, or the amino acid sequence of VL anti-TFR antibody comprising or consisting of the following, or the amino acid sequence of VL anti-TFR antibody comprising or consisting of the following, or which is incorporated herein by reference: 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 following, or the amino acid sequence of VL anti-TFR antibody comprising or consisting of the following, or which is incorporated herein by reference: The amino acid sequence of CDR2: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 contains or consists of the following: the amino acid sequence of VL CDR3 of the anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of VL CDR3 of the anti-TFR antibody named and / or incorporated herein by reference, containing or consisting of the following: 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 following: the amino acid sequence of an anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of an anti-TFR antibody named and / or incorporated herein by reference, comprising 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 following: the amino acid sequence of an anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of an anti-TFR antibody named and / or incorporated herein by reference, comprising 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 following: the amino acid sequence of an anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of an anti-TFR antibody named and / or incorporated herein by reference, comprising 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); The amino acid sequence of CDR3: 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 following: the amino acid sequence CDR1 of the anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence CDR1 of the anti-TFR antibody named and / or incorporated herein by reference, comprising or consisting of the following: 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 following: the amino acid sequence of the anti-TFR antibody named and / or incorporated herein by reference, or the amino acid sequence of the anti-TFR antibody VLCDR2 named and / or incorporated herein by reference, comprising or consisting of the following: 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 following: the amino acid sequence of the anti-TFR antibody named and / or incorporated herein by reference, comprising or consisting of the following: The amino acid sequence of CDR3, or the amino acid sequence of VLCDR3 containing an anti-TFR antibody that is named by reference and / or incorporated herein by reference, consisting of 1, 2 or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

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

[0258] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFR1) antibody comprises the anti-TFR (e.g., hTFR, e.g., hTFR1) antibody provided in Table 4.

[0259] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VH, which includes: VH CDR1, VH CDR2 and VH CDR3.

[0260] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the following: the amino acid sequence of VH CDR1 of VH shown in Table 4, or the amino acid sequence of VH CDR1 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR2 of VH shown in Table 4, or the amino acid sequence of VH CDR2 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR3 of VH shown in Table 4, or the amino acid sequence of VH CDR3 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR1 of VH shown in Table 4, or the amino acid sequence of VH CDR1 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR2 of VH shown in Table 4, or the amino acid sequence of VH CDR2 of VH shown in Table 4 comprising or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VHCDR3 comprises or consists of the following: the amino acid sequence of VH CDR3 of VH shown in Table 4, or the amino acid sequence of VH CDR3 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown 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 following: the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown 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 following: the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown 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 CDR1 comprises or consists of the following: the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown 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 following: the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown 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 following: the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0264] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VL, which includes: VL CDR1, VL CDR2 and VL CDR3.

[0265] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the following: the amino acid sequence of VL CDR1 of VL shown in Table 4, or the amino acid sequence of VL CDR1 of VL shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR2 of VL shown in Table 4, or the amino acid sequence of VL CDR2 of VL shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR3 of VL shown in Table 4, or the amino acid sequence of VL CDR3 of VL shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR1 of VL shown in Table 4, or the amino acid sequence of VL CDR1 of VL shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR2 of VL shown in Table 4, or the amino acid sequence of VL CDR2 of VL shown in Table 4 comprising or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VLCDR3 comprises or consists of the following: the amino acid sequence of VL CDR3 of VL shown in Table 4, or the amino acid sequence of VL CDR3 of VL shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VLCDR1 shown in Table 4, or the amino acid sequence of VL CDR1 shown 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 following: the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown 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 following: the amino acid sequence of VL CDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown 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 following: the amino acid sequence of VLCDR1 shown in Table 4, or the amino acid sequence of VL CDR1 shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown in Table 4 comprising or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0269] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VH, which includes VH CDR1, VH CDR2 and VH CDR3; and VL, which includes VL CDR1, VL CDR2 and VL CDR3.

[0270] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the following: the amino acid sequence of VH CDR1 of VH shown in Table 4, or the amino acid sequence of VH CDR1 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR2 of VH shown in Table 4, or the amino acid sequence of VH CDR2 of VH shown in Table 4 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR3 of VH shown in Table 4, or the amino acid sequence of VH CDR3 of VH shown in Table 4 comprising or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); VL The amino acid sequence of CDR1 contains or consists of the following: the amino acid sequence of VLCDR1 of VL shown in Table 4, or the amino acid sequence of VL CDR1 of VL shown in Table 4 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 contains or consists of the following: the amino acid sequence of VL CDR2 of VL shown in Table 4, or the amino acid sequence of VL CDR2 of VL shown in Table 4 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 contains or consists of the following: the amino acid sequence of VL CDR3 of VL shown in Table 4, or the amino acid sequence of VL CDR3 of VL shown in Table 4 containing or consisting of the following: 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 following: the amino acid sequence of VHCDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown 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 following: the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown 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 following: the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown 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 following: the amino acid sequence of VL CDR1 shown in Table 4. The amino acid sequence of CDR1, or the amino acid sequence of VL CDR1 shown in Table 4 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 containing or consisting of the following: the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown in Table 4 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 containing or consisting of the following: the amino acid sequence of VLCDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown in Table 4 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0272] In some embodiments, the amino acid sequence of VH comprises or consists of an amino acid sequence that 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 VH shown in Table 4. In some embodiments, the amino acid sequence of VL comprises or consists of an amino acid sequence that 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 VL shown in Table 4. In some embodiments, the amino acid sequence of VH comprises or consists of the following: an amino acid sequence that 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 VH shown in Table 4; and the amino acid sequence of VL comprises or consists of the following: an amino acid sequence that 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 VL shown in Table 4.

[0273] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) antibodies comprise the anti-TFR (e.g., hTFR, e.g., hTFR1) antibodies provided in Table 26.

[0274] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VH, which includes: VH CDR1, VH CDR2 and VH CDR3.

[0275] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the following: the amino acid sequence of VH CDR1 of VH shown in Table 26, or the amino acid sequence of VH CDR1 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR2 of VH shown in Table 26, or the amino acid sequence of VH CDR2 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR3 of VH shown in Table 26, or the amino acid sequence of VH CDR3 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR1 of VH shown in Table 26, or the amino acid sequence of VH CDR1 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR2 of VH shown in Table 26, or the amino acid sequence of VH CDR2 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR3 of VH shown in Table 26, or the amino acid sequence of VH CDR3 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown 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 following: the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown 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 following: the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown 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 following: the amino acid sequence of VHCDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown 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 following: the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VHCDR2 shown 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 following: the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0279] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VL, which includes: VL CDR1, VL CDR2 and VL CDR3.

[0280] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the following: the amino acid sequence of VL CDR1 of VL shown in Table 26, or the amino acid sequence of VL CDR1 of VL shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR2 of VL shown in Table 26, or the amino acid sequence of VL CDR2 of VL shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR3 of VL shown in Table 26, or the amino acid sequence of VL CDR3 of VL shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR1 of VL shown in Table 26, or the amino acid sequence of VL CDR1 of VL shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR2 of VL shown in Table 26, or the amino acid sequence of VL CDR2 of VL shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VL CDR3 of VL shown in Table 26, or the amino acid sequence of VL CDR3 of VL shown in Table 26 comprising or consisting of the following: 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 VLCDR1 shown in Table 26, or the amino acid sequence of VL CDR1 shown 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 VL CDR2 shown in Table 26, or the amino acid sequence of VL CDR2 shown 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 VL CDR3 shown in Table 26, or the amino acid sequence of VL CDR3 shown 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 VLCDR1 shown in Table 26, or the amino acid sequence of VL CDR1 shown 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 VL CDR2 shown in Table 26, or the amino acid sequence of VLCDR2 shown 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 VL CDR3 shown in Table 26, or the amino acid sequence of VL CDR3 shown in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0284] In some embodiments, anti-TFR (e.g., hTFR, e.g., hTFR1) includes VH, which includes VH CDR1, VH CDR2 and VH CDR3; and VL, which includes VL CDR1, VL CDR2 and VL CDR3.

[0285] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the following: the amino acid sequence of VH CDR1 of VH shown in Table 26, or the amino acid sequence of VH CDR1 of VH shown in Table 26 comprising or consisting of the following: 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 following: the amino acid sequence of VH CDR2 of VH shown in Table 26, or the amino acid sequence of VH CDR2 of VH shown in Table 26 comprising or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VHCDR3 comprises or consists of the following: the amino acid sequence of VH CDR3 of VH shown in Table 26, or the amino acid sequence of VH CDR3 of VH shown in Table 26 comprising or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); VL The amino acid sequence of CDR1 contains or consists of the following: the amino acid sequence of VL CDR1 of VL shown in Table 26, or the amino acid sequence of VL CDR1 of VL shown in Table 26 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 contains or consists of the following: the amino acid sequence of VL CDR2 of VL shown in Table 26, or the amino acid sequence of VL CDR2 of VL shown in Table 26 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VLCDR3 contains or consists of the following: the amino acid sequence of VL CDR3 of VL shown in Table 26, or the amino acid sequence of VL CDR3 of VL shown in Table 26 containing or consisting of the following: 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 following: the amino acid sequence of VHCDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown 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 following: the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VHCDR2 shown 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 following: the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown 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 following: the amino acid sequence of VL CDR1 shown in Table 26. The amino acid sequence of CDR1, or the amino acid sequence of VL CDR1 shown in Table 26 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 containing or consisting of the following: the amino acid sequence of VL CDR2 shown in Table 26, or the amino acid sequence of VL CDR2 shown in Table 26 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 containing or consisting of the following: the amino acid sequence of VL CDR3 shown in Table 26, or the amino acid sequence of VL CDR3 shown in Table 26 containing or consisting of the following: 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[0287] In some embodiments, the amino acid sequence of VH comprises or consists of an amino acid sequence that 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 VH shown in Table 26. In some embodiments, the amino acid sequence of VL comprises or consists of an amino acid sequence that 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 VL shown in Table 26. In some embodiments, the amino acid sequence of VH comprises or consists of the following: an amino acid sequence that 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 VH shown in Table 26; and the amino acid sequence of VL comprises or consists of the following: an amino acid sequence that 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 VL shown in Table 26. 5.3.2 Ig constant region

[0288] In some embodiments, the antibody (or heteropeptide (e.g., operatively linked to a protein that specifically binds to TFR (e.g., hTF) (see, for example, § 5.3.1.3))) comprises an IgG CH2 region and an IgG CH3 region. In some embodiments, the antibody (or heteropeptide) comprises a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises a portion of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0289] In some embodiments, the antibody (or heteropeptide) 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 IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 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 the following: IgG4 hinge region, IgG4 CH2 region and IgG4 CH3 region.

[0290] In some embodiments, the antibody (or heteropeptide) comprises a first Ig Fc region and a second Ig Fc region. In some embodiments, the first Ig Fc region and / or the 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 Ig Fc region and / or the second Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Ig Fc region and / or the 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 Ig Fc region and / or the 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 Ig Fc region and / or the second Ig Fc region comprises or consists of at least a portion of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the first Ig Fc region and / or the second Ig Fc region comprises or consists of the following: an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the first Ig Fc region and / or the second Ig Fc region comprises or consists of the following: at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the first Ig Fc region and / or the second Ig Fc region comprises or consists of the following: an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0291] In some embodiments, the antibody (or heteropeptide) comprises one or more hIg heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1, IgG2, IgG3, or IgG4. In some embodiments, hIgG is IgG1 or IgG4. In some embodiments, hIgG is hIgG1. In some embodiments, hIgG is hIgG4.

[0292] In some embodiments, the antibody (or heteropeptide) comprises an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the antibody (or heteropeptide) comprises a portion of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises a portion of an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the antibody (or heteropeptide) comprises an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0293] In some embodiments, the antibody (or heteropeptide) comprises an hIg Fc region. In some embodiments, the hIg 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 hIg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or consists of at least a portion of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region comprises or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region comprises or consists of at least a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or consists of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or consists of at least a portion of the hIgG4 hinge region, the hIgG4 CH2 region, and the hIgG4 CH3 region. In some embodiments, the hIg Fc region comprises or consists of the hIgG4 hinge region, the hIgG4 CH2 region, and the hIgG4 CH3 region.

[0294] In some embodiments, the antibody (or heteropeptide) comprises a first hIg Fc region and a second hIg Fc region. In some embodiments, the first hIg Fc region and / or the second hIg 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 hIg Fc region and / or the second hIg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first hIg Fc region and / or the second hIg Fc region comprises or consists of at least a portion of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the first hIg Fc region and / or the second hIg Fc region comprises or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the first hIg Fc region and / or the second hIg Fc region comprises or consists of at least a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the first hIg Fc region and / or the second hIg Fc region comprises or consists of the following: an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the first hIg Fc region and / or the second hIg Fc region comprises or consists of the following: at least a portion of an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the first hIg Fc region and / or the second hIg Fc region comprises or consists of the following: an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0295] In some embodiments, the antibody (or heterologous polypeptide) contains one or more Ig (e.g., hIg) light chain constant regions (e.g., hIg light chain κ constant region (κCL) or hIg light chain λ constant region (λCL)).

[0296] Table 5 provides exemplary references to the amino acid sequences of the heavy and light chain constant regions of hIgG1 and hIgG4, which may be incorporated into one or more of the embodiments described herein (e.g., anti-TFR (e.g., hTFR (e.g., hTFR1)) antibodies and heterologous peptides). Table 5. Amino acid sequences of exemplary hIg heavy and light chain constant regions and their components.

[0297] In some embodiments, the antibody (or heterologous peptide) comprises one or more hIg constant regions, wherein the amino acid sequence of the one or more hIg constant regions comprises or consists of the following: an amino acid sequence that 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 the peptides shown in Table 5. In some embodiments, the amino acid sequence of the one or more hIg constant regions comprises or consists of the following: the amino acid sequence shown in Table 5.

[0298] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequences shown in Table 5, and further comprises one 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown in Table 5, which comprises or consists 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown in Table 5, which comprises or consists 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown in Table 5, which comprise or consist of approximately 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown in Table 5, and further comprises one 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown 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 one or more hIg constant regions comprises or consists of the amino acid sequences shown 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 one or more hIg constant regions comprises or consists of the following: an amino acid sequence that 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: 171-198. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the following: an amino acid sequence of any one of SEQ ID NO: 171-198.

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

[0302] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NO: 171-198, and further comprises one 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 one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NO: 171-198, which comprises or consists 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 one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NO: 171-198, which 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 one or more hIg constant regions comprises or consists of the following: an amino acid sequence of any one of SEQ ID NO: 171-198, comprising or consisting of the following: 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 heteropeptide) comprises an Fc region (see, for example, § 5.3.2). In some embodiments, the Fc region of the antibody (or heteropeptide) described herein exhibits a reduction in one or more Fc effector functions relative to a reference (e.g., wild-type) Fc region. Exemplary Fc effector functions include, but are not limited to, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and binding affinity to one or more human Fc receptors, such as Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa)).

[0304] Fc effector functions can be assessed using standard in vitro and / or in vitro assays known in the art, including any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fcγ receptor) binding affinity and C1q binding affinity.

[0305] For example, ADCC activity can be assessed using standard (radioactive and non-radioactive) methods known in the art (see, for example, WO 2006 / 082515, WO 2012 / 130831, the entire contents of each of which are incorporated herein by reference for all purposes). For example, ADCC activity can be assessed using pentavalent chromium ( 51 The Cr content was evaluated using a determination. In short, the Cr content was... 51Cr is preloaded into target cells, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicating NK cell lysis of target cells). A similar non-radioactive assay can also be used, employing a similar method, but with target cells preloaded with fluorescent dyes such as calcein-AM, CFSE, BCECF, or lanthanide fluorophores (europium). See, for example, Parekh, Bhavin S et al., “Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay.” mAbs [Monoclonal Antibodies] Vol. 4, 3(2012): 310-8. Doi:10.4161 / mabs.19873, the full contents of which are incorporated herein by reference for all purposes. Exemplary commercially available non-radioactive assays include, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA; and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Other non-limiting examples of in vitro assays that can be used to evaluate the ADCC activity of the fusion proteins described herein include those described in the following: US 5500362; US 5821337; Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 83 (1986) 7059-7063; Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 82 (1985) 1499-1502; and Bruggemann, M., et al., J. Exp. Med. [Journal of Experimental Medicine] 166 (1987) 1351-1361, the full contents of each of which are incorporated herein by reference. Alternatively or additionally, the ADCC activity of the fusion proteins described herein may be assessed in vivo, for example, in animal models (e.g., Clynes, et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 95 (1998) 652-656 (the full contents of which are incorporated herein by reference for all purposes)).

[0306] C1q binding assays can be used to assess the ability of the antibodies (or heteropeptides) described herein to bind to C1q (or bind with a lower affinity than the reference fusion protein) and thus lack (or have reduced) CDC activity. The binding of the antibodies (or heteropeptides) described herein to C1q can be determined by a variety of in vitro assays known in the art (e.g., biochemical or immunological-based assays), including, for example, equilibration methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), as well as 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 can utilize labeling on one or more components being examined and / or employ a variety of detection methods, including but not limited to colorimetric, fluorescent, luminescent, or isotopic labeling. Detailed descriptions of affinity and kinetics can be found, for example, Paul, WE, ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), the full contents of which are incorporated herein by reference. See, for example, WO 2006 / 029879 and WO 2005 / 100402, which describe C1q and C3c binding ELISAs, the full contents of each of which are incorporated herein by reference for all purposes. Other CDC activity assays include those described below: for example, Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, MS, et al., Blood 101 (2003) 1045-1052; and Cragg, MS, and Glennie, MJ., Blood 103 (2004) 2738-2743), the full contents of each of which are incorporated herein by reference for all purposes.

[0307] ADCP activity can be measured by in vitro or in vivo methods known in the art, as well as commercially available assays (see, for example, 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 full contents of each of which are incorporated herein by reference for all purposes). For example, a primary cell-based ADCP assay can be used, in which fresh human peripheral blood mononuclear cells (PBMCs) are isolated using a standard procedure, the PBMCs are isolated and differentiated into macrophages in a culture. A fluorescent label of the macrophages is added to the culture containing the fluorescently labeled target cells. Phagocytic events can be analyzed using FACS screening and / or microscopy. A modified report version of the above assay can also be used, employing engineered cell lines stably expressing FcγRIIa (CD32a) as effector cell lines (e.g., engineered T cell lines, such as THP-1), which eliminates the requirement for primary cells. Exemplary ADCP analyses are described, for example, in Ackerman, ME et al., A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J. Immunol. Methods 366, 8-19 (2011); and Mcandrew, EG et al., Determining the phagocytic activity of clinical antibody samples. J. Vis. Exp. 3588 (2011). Doi:10.3791 / 3588; the full contents of each of these articles are incorporated herein by reference.

[0308] The binding of the antibody (or heteropeptide) described herein to the Fc receptor can be determined using a variety of in vitro assays (e.g., biochemical or immunological-based assays) known in the art for measuring Fc-Fc receptor interactions (i.e., specific binding of the Fc region to the 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), as well as 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 can utilize labeling on one or more components being examined and / or employ a variety of detection methods, including but not limited to colorimetric, fluorescent, luminescent, or isotopic labeling. Detailed descriptions of affinity and kinetics can be found, for example, Paul, WE, ed., Fundamental Immunology, 4th edition, Lippincott-Raven, Philadelphia (1999), the full contents of each of which are incorporated herein by reference for all purposes.

[0309] In some embodiments, the Fc region of the antibody (or heterologous peptide) described herein is altered (e.g., includes one or more amino acid variations (e.g., one or more amino acid substitutions, deletions, additions, etc.)) relative to the amino acid sequence of a reference Fc region (e.g., a wild-type Fc region, such as in Table 5 herein (e.g., SEQ ID NO: 178, 180, 191, or 195)) (referred to herein as the “altered Fc region”). In some embodiments, one or more amino acid variations (e.g., one or more amino acid substitutions, deletions, additions, etc.) reduce or eliminate one or more Fc effector functions relative to a reference Fc that does not contain variations (e.g., one or more variations (e.g., one or more amino acid substitutions, deletions, additions, etc.)).

[0310] In some embodiments, antibodies (or heteropeptides) containing altered Fc regions do not exhibit detectable or reduced ADCC compared to reference antibodies (or heteropeptides) that do not contain Fc region variations (e.g., one or more amino acid variations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, antibodies (or heteropeptides) containing altered Fc regions do not exhibit detectable or reduced CDC compared to reference antibodies (or heteropeptides) that do not contain Fc region variations (e.g., one or more amino acid variations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, antibodies (or heteropeptides) containing modified Fc regions do not exhibit detectable or reduced ADCP compared to reference antibodies (or heteropeptides) that do not contain Fc region variations (e.g., one or more variations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, antibodies (or heteropeptides) containing altered Fc regions exhibit reduced or undetectable specific binding affinity for one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa)) compared to reference antibodies (or heteropeptides) not containing Fc region variations (e.g., one or more variations (e.g., one or more amino acid substitutions, deletions or additions)). In some embodiments, antibodies (or heteropeptides) containing altered Fc regions exhibit reduced or undetectable specific binding affinity for FcγRI, FcγIIa and / or FcγIIIa compared to antibodies (or heteropeptides) not containing Fc region variations (e.g., one or more variations (e.g., one or more amino acid substitutions, deletions or additions)). In some embodiments, antibodies (or heteropeptides) containing altered Fc regions exhibit reduced or undetectable specific binding affinity for FcγRI compared to reference antibodies (or heteropeptides) that do not contain Fc variants (e.g., one or more variants (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, antibodies (or heteropeptides) containing altered Fc regions exhibit reduced or undetectable specific binding affinity for FcγIIa compared to reference antibodies (or heteropeptides) that do not contain Fc region variants (e.g., one or more variants (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, antibodies (or heteropeptides) containing altered Fc regions exhibit reduced or undetectable specific binding affinity for FcγIIIa compared to antibodies (or heteropeptides) that do not contain Fc region variants (e.g., one or more variants (e.g., one or more amino acid substitutions, deletions, or additions)).In some embodiments, antibodies (or heteropeptides) containing altered Fc regions exhibit reduced or undetectable specific binding affinity for C1q compared to reference antibodies (or heteropeptides) that do not contain Fc region variations (e.g., one or more variations (e.g., one or more amino acid substitutions, deletions, or additions)).

[0311] In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable ADCC. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable CDC. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable ADCP. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable specific binding affinity to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))). In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable specific binding affinity to FcγRI, FcγIIa and / or FcγRI. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable specific binding affinity to FcγRI. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable specific binding affinity for FcγIIa. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable specific binding affinity for FcγIIIa. In some embodiments, antibodies (or heteropeptides) containing the Fc region do not exhibit detectable specific binding affinity for C1q.

[0312] Amino acid substitutions that reduce or eliminate the function of one or more Fc effectors are known in the art. See, for example, Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, v10 (June 7, 2019) DOI=10.3389 / fimmu.2019.01296, the entire contents of which are incorporated herein by reference for all purposes, see, more particularly, for example, Saunders’ Table 4.

[0313] In some embodiments, the modified Fc region comprises an hIgG1 Fc region containing one or more amino acid variations (e.g., one or more amino acid substitutions). In some embodiments, the hIgG1 Fc region contains amino acid substitutions at amino acid positions L234, L235, and / or P329 according to the Kabat EU number. In some embodiments, the hIgG1 Fc region contains the following amino acid substitutions L234A and / or L235A according to the Kabat EU number. In some embodiments, the hIgG1 Fc region contains the following amino acid substitutions L234A, L235A, and P329G according to the Kabat EU number. In some embodiments, the hIgG1 Fc region contains the following amino acid substitutions L234A, L235A, and P329A according to the Kabat EU number.

[0314] In some embodiments, the modified Fc region comprises an hIg4 Fc region containing one or more amino acid variations (e.g., one or more amino acid substitutions). In some embodiments, the hIgG4 Fc region contains amino acid substitutions at amino acid positions S228, F234, and / or L235 according to the Kabat EU number. In some embodiments, the hIgG4 Fc region contains the following amino acid substitutions S228P, F234A, and / or L235A according to the Kabat EU number. In some embodiments, the hIgG4 Fc region contains the following amino acid substitutions S228P, F234A, and / or L235E according to the Kabat EU number. In some embodiments, the hIgG4 Fc region contains the following amino acid substitutions S228P and / or L235E according to the Kabat EU number.

[0315] Table 6 provides exemplary modified Fc region amino acid sequences known in the art that exhibit reduced function of one or more effectors. Table 6. Exemplary modified amino acid sequences of the Fc region.

[0316] In some embodiments, the variant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of the polypeptides shown in Table 6.

[0317] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of the following: the amino acid sequence of the polypeptide shown in Table 6, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid variations (e.g., amino acid substitution, deletion, or addition). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of the following: the amino acid sequence of the polypeptide shown in Table 6, and further comprises or consists of the following: at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitution, addition, deletion, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of the following: the amino acid sequence of the polypeptide shown in Table 6, and further comprises or consists of the following: about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitution, addition, deletion, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide includes an hIg Fc region comprising or consisting of the following: the amino acid sequence of the polypeptide shown in Table 6, and further comprising or consisting of the following: no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid variations (e.g., substitution, addition, deletion, etc.).

[0318] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of the amino acid sequences of the polypeptides shown in Table 6, and further comprises one 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of the amino acid sequences of the polypeptides shown 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of the amino acid sequences of the polypeptides shown 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 hIg Fc fusion protein or polypeptide includes an hIg Fc region comprising or consisting of the following: the amino acid sequence of the polypeptide shown in Table 6, and further comprising or consisting of the following: 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 hIg Fc fusion protein or polypeptide includes an hIg Fc region, which comprises or consists of an amino acid sequence that is 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 NO: 199-224.

[0320] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following: SEQ ID NO: 199-224, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid variations (e.g., amino acid substitution, deletion, or addition). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following: SEQ ID NO: 199-224, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitution, addition, deletion, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following: SEQ ID NO: 199-224, and further comprises or comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitution, addition, deletion, etc.). In some embodiments, the amino acid sequence of the variant hIgFc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following: SEQ ID NO: 199-224, and further comprises or comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitution, addition, deletion, etc.).

[0321] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following amino acid sequences in SEQ ID NO: 199-224, and further comprises one 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following amino acid sequences in SEQ ID NO: 199-224, and further comprises 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing any one of the following amino acid sequences in SEQ ID NO: 199-224, and further comprises 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 hIgFc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of any one of the following: SEQ ID NO: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 hIgG1 Fc region comprises alanine at position L234 and / or position L235 according to the Kabat EU number. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and position L235 according to the Kabat EU number. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine, glycine, or serine at position P329 according to the Kabat EU number.

[0323] In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 according to the Kabat EU number; alanine at position L235; and alanine, glycine, or serine at position P329. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 according to the Kabat EU number; alanine at position L235; and alanine at position P329. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 according to the Kabat EU number; alanine at position L235; and glycine at position P329. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 according to the Kabat EU number; alanine at position L235; and serine at position P329.

[0324] In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and / or alanine, glycine, or serine at position P329 according to the Kabat EU number; and comprises an amino acid sequence that 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 the polypeptides shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and / or alanine at position L235 according to the Kabat EU number; and comprises an amino acid sequence that 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 the polypeptides shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and position L235 according to the Kabat EU number; and comprises an amino acid sequence that 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 the polypeptides shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and alanine, glycine, or serine at position P329 according to the Kabat EU number; and comprises an amino acid sequence that 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 the polypeptides shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and alanine at position P329 according to the EU number of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of the polypeptides shown in Table 6.In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and glycine at position P329 according to the Kabat EU number; and comprises an amino acid sequence that 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 the peptides shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and serine at position P329 according to the Kabat EU number; and comprises an amino acid sequence that 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 the peptides shown in Table 6.

[0325] In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and / or alanine, glycine, or serine at position P329 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and / or alanine at position L235 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and position L235 according to the EU numbering of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and alanine, glycine, or serine at position P329 according to the EU numbering of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and alanine at position P329 according to the EU number of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224.In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and glycine at position P329 according to the EU numbering of the Kabat; and comprises an amino acid sequence that 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: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and serine at position P329 according to the EU numbering of the Kabat; and comprises an amino acid sequence that 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: 199-224.

[0326] In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and / or alanine, glycine, or serine at position P329 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and / or alanine at position L235 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234 and position L235 according to the EU numbering of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and alanine, glycine, or serine at position P329 according to the EU numbering of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and alanine at position P329 according to the EU number of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224.In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and glycine at position P329 according to the EU numbering of the Kabat; and comprises an amino acid sequence that 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: 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region comprises alanine at position L234, alanine at position L235, and serine at position P329 according to the EU numbering of the Kabat; and comprises an amino acid sequence that 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: 199-224.

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

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

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

[0330] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises phenylalanine at position L234, alanine at position L235, and / or proline at position S228 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises phenylalanine at position L234 and / or alanine at position L235 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises phenylalanine at position L234, alanine at position L235, and proline at position S228 according to the EU number of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224.

[0331] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises phenylalanine at position L234, alanine at position L235, and / or proline at position S228 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises phenylalanine at position L234 and / or alanine at position L235 according to the EU number of the Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to any one of SEQ ID NO: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises phenylalanine at position L234, alanine at position L235, and proline at position S228 according to the EU number of Kabat; and comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 199-224. 5.3.2.2 Promotion of heterodimerization

[0332] As described herein, in some embodiments, the antibody (or heterodipeptide) comprises a first Fc region and a second Fc region (see, for example, § 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 facilitate heterodimerization. The IgG-derived heterodimeric form can be produced by methods known in the art, such as by forced heavy chain heterodimerization. Forced heavy chain heterodimerization can be achieved using methods known in the art, such as mortars or chain exchange engineered domains (SEED), see, for example, 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 referred to as “Ji-Hee 2016”), the entire contents of which are incorporated herein by reference for all purposes.

[0333] In some embodiments, the interface between the first and second Ig Fc regions is modified, for example, by introducing amino acid substitutions to increase heterodimerization, for example, relative to an unmodified interface, such as a naturally occurring interface. For example, dimerization of the first and second Ig Fc regions can be enhanced by providing the Ig Fc interface of the first and second Fc regions with one or more of the following: paired protrusion-cavities (“mortars”), electrostatic interactions, or chain exchange, such that, for example, the heteropolymer is present in a larger proportion than the homopolymer form relative to the unmodified interface.

[0334] Knobs-into-holes amino acid pairing modifications are known in the art and described, for example, in US 5731116; US7476724; Ji-Hee 2016; and Ridgway, J. “'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization” et al. Prot. Engineering 9(7): 617-621 (1996), the entire contents of each of which are incorporated herein by reference. Generally, koning involves 1) introducing one or more amino acid substitutions into 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 region under conditions that promote heterodimerization. "Pepper" is typically generated by replacing a small amino acid in the parental Ig Fc region (e.g., T366Y or T366W) with a larger amino acid; "mortar" is generated by replacing a larger residue in the parental Ig Fc region (e.g., Y407T, T366S, L11368A, or Y407V) with a smaller amino acid. Exemplary pestle-mortar mutations include S354C and T366W in the "pepper" Ig Fc region and Y349C, T366S, L368A, and Y407V in the "mortar" Ig Fc region. Table 7 provides other exemplary pestle-mortar mutations in wells that can be incorporated into any one or more embodiments, as well as additional exemplary optional stable Ig Fc cysteine ​​mutations. Table 7. Exemplary mortars and stable cysteine ​​modifications.

[0335] Table 8 provides exemplary amino acid sequences of the Fc region known in the art for promoting heterodimerization. Table 8. Exemplary amino acid sequences of modified heterodimer Fc region pairs.

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

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

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

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

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

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

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

[0343] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366 according to the EU number of 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: 225-232; and the amino acid sequence of the second Ig Fc region comprises the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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: 241-248.

[0344] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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: 233-240; and the amino acid sequence of the second Ig Fc region comprises the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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: 249-256.

[0345] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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: 227; and the amino acid sequence of the second Ig Fc region comprises the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 according to the EU number of 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 the S amino acid at position T366 according to the EU number of Kabat, the A amino acid at position L368, and the V amino acid at position Y407, 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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: 250.

[0355] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 the W amino acid at position T366 and the C amino acid at position S354 according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, and the C amino acid at position Y349 according to the EU number of 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 variation for site-specific conjugation

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

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

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

[0364] As described herein, in some embodiments, the antibody (or heterologous portion) comprises a first Ig Fc region and a second Ig Fc region (see, for example, § 5.3.2). In some embodiments, the first Ig Fc region and the second Ig Fc region each comprise a plurality of amino acid variations described herein, such as one or more amino acid variations that reduce or eliminate one or more Ig Fc effector functions (e.g., ADCC, ADCP, CDC, and binding affinity to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa)))) (see, for example, § 5.3.2.1); and one or more amino acid modifications that promote heterodimerization of the first and second Fc regions (see, for example, § 5.3.2.2).

[0365] In some embodiments, the first and second Fc regions each contain one or more amino acid variations that reduce or eliminate one or more Fc effector functions (e.g., ADCC, ADCP, CDC, and binding affinity to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa)))) (see, for example, § 5.3.2.1); and one or more amino acid variations that promote heterodimerization of the first and second Fc regions (see, for example, § 5.3.2.2).

[0366] Table 9 provides the amino acid sequences of the Fc region of exemplary variants. Table 9. Amino acid sequences of the Fc region of exemplary variants.

[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, according to the EU number of 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: 257-264; 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, 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, according to the EU number of Kabat, and is identical to the amino acid sequence of any one of SEQ ID NO: 257-264. The amino acid sequences of any one of 265-272 are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0368] In some embodiments, the amino acid sequence of the first Fc region comprises tryptophan amino acid at position T366, cysteine ​​amino acid residue at position S354, leucine amino acid residue at position L234, leucine amino acid residue at position L235, and alanine amino acid residue at position P329, according to the EU number of 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: 273-280; and the second Ig The amino acid sequence of the Fc region contains the serine amino acid at position T366, the alanine amino acid at position L368, the valine amino acid at position Y407, the cysteine ​​amino acid at position Y349, the cysteine ​​amino acid residue at position Y349, the leucine amino acid residue at position L234, the leucine amino acid residue at position L235, and the alanine amino acid residue at position P329, 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: 281-288.

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

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

[0371] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366, the C amino acid residue at position S354, the L amino acid residue at position L234, the L amino acid residue at position L235, and the A amino acid residue at position P329, according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the V amino acid at position Y407, the C amino acid residue at position Y349, the L amino acid residue at position L234, the L amino acid residue at position L235, and the A amino acid residue at position P329, according to the EU number of 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 is identical to the amino acid sequence of any one of SEQ ID NO: 259. The amino acid sequences of any one of 267 are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0372] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366, the C amino acid residue at position S354, the L amino acid residue at position L234, the L amino acid residue at position L235, and the A amino acid residue at position P329, according to the EU number of 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 the S amino acid at position T366, the C amino acid residue at position Y349, the L amino acid residue at position L234, the L amino acid residue at position L235, the A amino acid residue at position P329, the A amino acid at position L368, and the V amino acid at position Y407, according to the EU number of 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; The amino acid sequences of any one of the 268 are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

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

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

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

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

[0377] In some embodiments, the amino acid sequence of the first Fc region comprises the W amino acid at position T366, the C amino acid residue at position S354, the L amino acid residue at position L234, the L amino acid residue at position L235, and the G amino acid residue at position P329, according to the EU number of 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 the S amino acid at position T366, the A amino acid at position L368, the C amino acid residue at position Y349, the ...

Claims

1. A conjugate comprising: (a) A hematopoietic cell targeting agent comprising a protein (e.g., an antibody) that specifically binds to a transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); operably linked to (b) At least one oligonucleotide that regulates (e.g., inhibits) the expression and / or activity of target genes, nucleic acids (e.g., mRNA) and / or proteins expressed by the hematopoietic cells.

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

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

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

5. The conjugate of any one of claims 1-4, wherein the protein (e.g., antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) exhibits one or more of the following properties: (a) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the protein (e.g., antibody) or the conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the hematopoietic cells; (b) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the protein (e.g., antibody) or the conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce death of the target cells; (c) (d) After binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the hematopoietic cells remain viable; and (e) after internalization into hematopoietic cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the death of the hematopoietic cells; and / or (e) after binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)).

6. A conjugate comprising: (a) An erythroid progenitor cell target comprising a protein (e.g., an antibody) that specifically binds to a transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); operably linked to (b) At least one oligonucleotide that regulates (e.g., inhibits) the expression and / or activity of target genes, nucleic acids (e.g., mRNA) and / or proteins expressed by the erythroid precursor cells.

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

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

10. The conjugate of any one of claims 6-10, wherein the protein (e.g., antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) exhibits one or more of the following properties: (a) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., antibody) or the conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the erythroid precursor cells; (b) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., antibody) or the conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce death of the target cells; (c) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the erythroid precursor cells remain viable; (d) Upon internalization into erythroid precursor cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the death of the erythroid precursor cells; and / or (e) upon binding to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)).

11. The conjugate as claimed in any of the preceding claims, wherein the protein that specifically binds to 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 is composed of the following: full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, single-domain antibody (sdAb) (e.g., VHH), sdAb-Fc (e.g., VHH-Fc), (sdAb)2 (e.g., (VHH)2), or (sdAb)2-Fc (e.g., (VHH)2-Fc).

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

14. The conjugate according to any one of claims 11-13, wherein the antibody is an hIgG1, hIgG2, hIgG3 or hIgG4 antibody (e.g., hIgG1 or hIgG4 antibody).

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

16. The conjugate of any one of claims 11-15, wherein the antibody comprises or is composed of the following: Full-length antibodies, Fab-Fc, scFv-Fc, (scFv)2-Fc, sdAb-Fc (e.g., VHH-Fc) or (sdAb)2-Fc (e.g., (VHH)2-Fc).

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

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

19. The conjugate according to any one of claims 15-18, wherein the Ig is hIg.

20. The conjugate of claim 19, wherein the hIg is human IgG (hIgG).

21. The conjugate of claim 20, wherein the hIgG is hIgG1 or hIgG4.

22. The conjugate of any one of claims 15-21, wherein the Ig (e.g., hIg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region, the one or more amino acid substitutions reducing or eliminating one or more of the following effector functions relative to the reference hIg Fc region: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or affinity for one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))).

23. The conjugate of any one of claims 15-22, wherein the Ig (e.g., hIg) Fc region substantially does not mediate ADCC, substantially does not mediate CDC, and / or does not bind to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb (e.g., FcγRI, FcγIIa and / or FcγIIIa))).

24. The conjugate according to any one of claims 15-23, wherein the Ig is hIgG1, 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 according to the EU index number of Kabat.

25. The conjugate according to any one of claims 15-24, wherein the Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234 and / or alanine at amino acid position L235 according to the EU index number of Kabat.

26. The conjugate according to any one of claims 15-25, wherein the Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234, alanine at amino acid position L235, and / or glycine, alanine, or serine at position P329 according to the EU index number of Kabat.

27. The conjugate according to any one of claims 15-26, wherein the Ig is hIgG1, and the amino acid sequence of the Fc region comprises alanine at amino acid position L234, serine at amino acid position L235, and / or glycine, alanine, or serine at position P329 according to the EU index number of Kabat.

28. The conjugate of any one of claims 15-27, wherein the Ig is hIgG1, and the amino acid sequence of the Fc region contains alanine at amino acid position N297 according to the EU index number of Kabat.

29. The conjugate according to 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 according to the EU index number of Kabat.

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

31. The conjugate according to any one of claims 15-23 or 29-30, wherein the Ig is hIgG4, and the amino acid sequence of the Fc region contains alanine at amino acid position N297 according to the EU index number 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 associated via at least one covalent (e.g., disulfide) bond.

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

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

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

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

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

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

39. The conjugate according to any one of claims 11-38, wherein the amino acid sequence of the second Fc region comprises tryptophan at amino acid position T366 according to the EU index number 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 position S354 according to the EU index number of Kabat.

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

42. The conjugate of any one of claims 11-41, wherein the antibody does not (or substantially does not) block the binding of TF (e.g., hTF) to the TFR (e.g., hTFR1).

43. The conjugate as claimed in any of the preceding claims, wherein the protein that specifically binds to TFR (e.g., hTFR (e.g., hTFR1)) 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 as claimed in any of the preceding claims, wherein the oligonucleotide enhances the expression and / or activity of the target gene, the nucleic acid (e.g., mRNA), and / or the protein.

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

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

48. The conjugate as claimed in any 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 as claimed in any of the preceding claims, wherein the oligonucleotide mediates one or more of the following: degradation of the target nucleic acid molecule (e.g., mRNA), inactivation of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration of splicing of the target nucleic acid molecule (e.g., mRNA), alteration of stability (e.g., reduction) of the target nucleic acid molecule (e.g., mRNA), or blockade of translation of the target nucleic acid molecule (e.g., mRNA), or any combination thereof.

50. The conjugate as claimed in any of the preceding claims, wherein the oligonucleotide comprises or is composed of the following: Antisense oligonucleotides (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

51. The conjugate as claimed in any of the preceding claims, wherein the oligonucleotide comprises or is composed of the following: An antisense strand, wherein the antisense strand contains a region complementary to a target sequence (e.g., an mRNA sequence encoded by the target gene, a nucleic acid (e.g., mRNA), and / or a protein).

52. The conjugate as claimed in any of the preceding claims, wherein the oligonucleotide is single-stranded or double-stranded.

53. The conjugate as claimed in any of the preceding claims, wherein the oligonucleotide is DNA, RNA, or a hybrid of RNA and RNA.

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

55. The conjugate as claimed in any of the preceding claims, wherein the sense strand and the antisense strand are part of a single nucleic acid molecule (e.g., wherein a hairpin loop is located between the sense strand and the antisense strand of the single nucleic acid molecule).

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

57. The conjugate as claimed in any of the preceding claims, wherein the length of the double-stranded region is 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.

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

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

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

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

63. The conjugate as claimed in any of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleoside linker (e.g., at least one phosphate thioside linker).

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

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

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

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

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

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

70. The conjugate as claimed in any of the preceding claims, wherein the target gene is B-cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCL11A)), zinc finger and BTB domain 7A (ZBTB7A) (e.g., hZBTB7A), KLF transcription factor 1 (KLF1) (e.g., hKLF1), FA complement group A (FANCA) (e.g., human FANCA), dyskeratin pseudouridine synthase 1 (DKC1) (e.g., human DKC1), regulator of telomere elongation helicase 1 (RTEL1) (e.g., human RTEL1), telomerase reverse transcriptase (TE... RT (e.g., human TERT), telomerase RNA component (TERC) (e.g., human TERC), TERF1 interacting nuclear factor 2 (TINF2) (e.g., human TINF2), ribosomal protein S19 (RPS19) (e.g., human RPS19), ribosomal protein L11 (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), neutrophil-expressed elastase (ELA2) (e.g., human ELA2), HCLS1-associated protein X-1 (HAX1) (e.g., human HAX1), glucose-6-phosphatase catalytic subunit 3 (G6PC3) (e.g., human G6PC3), growth factor-independent 1 transcriptional repressor (GFI) 1) (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 α-motif domain-containing protein 9 (SAMD9) (e.g., human SAMD9), sterile α-motif domain-containing protein 9-like protein (SAMD9L) (e.g., human SAMD9L), or MDS1 and EVI1 complex locus (MECOM) (e.g., human MECOM).

71. The conjugate as claimed in any of the preceding claims, wherein the target gene is BCL11A (e.g., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

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

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

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

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

76. The conjugate as claimed in any of the preceding claims, wherein the conjugate is cuttable or incuttable.

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

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

79. A cell comprising the conjugate as described in 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 as described in any one of claims 1-78, and a pharmaceutically acceptable excipient.

82. A kit comprising a conjugate as described in any one of claims 1-78 or a pharmaceutical composition as described in any one of claims 81.

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

84. The method of claim 83, wherein the cells are 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 a conjugate as claimed in any one of claims 1-78, a cell as claimed in any one of claims 79-80, or a pharmaceutical composition as claimed in claim 81, thereby delivering the conjugate, the cell, or the pharmaceutical composition to the subject.

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

88. A method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by erythroid precursor cells in cells, the method comprising introducing into the cells a conjugate as described in any one of claims 1-78 or a pharmaceutical composition as described in claim 81, thereby regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, the nucleic acid (e.g., mRNA), and / or the protein.

89. The method of any one of claims 87-88, wherein the cells are 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 for regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA), and / or protein expressed by hematopoietic cells in the cells of a subject, the method comprising administering to the subject a conjugate as described in any one of claims 1-78 or a pharmaceutical composition as described in claim 81, thereby regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, the nucleic acid (e.g., mRNA), and / or the protein in the subject.

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

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

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

95. The method of any one of claims 93-94, wherein the cells are 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 BCL11A (e.g., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

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

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

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

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

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

103. A method for treating, improving, or preventing a hereditary blood disorder in a subject, the method comprising administering to the subject a conjugate as described in any one of claims 1-78 or a pharmaceutical composition as described in claim 81, thereby treating, improving, or preventing the hereditary blood disorder in the subject.

104. The method of claim 103, wherein the hereditary blood disorder is hemoglobinopathies or hereditary bone marrow failure syndromes.

105. A method of treating, improving, or preventing hemoglobinopathies in a subject, the method comprising administering to the subject a conjugate as described in any one of claims 1-78 or a pharmaceutical composition as described in claim 81, thereby treating, improving, or preventing the hemoglobinopathies in the subject.

106. The method of claim 105, wherein the hemoglobinopathy is sickle cell disease, sickle cell phenotype, hemoglobin C disease, hemoglobin C phenotype, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia), a condition associated with hemoglobins having increased oxygen affinity, a condition associated with hemoglobins having decreased oxygen affinity, unstable hemoglobinopathies, methemoglobinemia, or any combination thereof.

107. The method of claim 105 or 106, wherein the hemoglobinopathy is sickle cell disease or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-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 phenotype, hemoglobin C disease, hemoglobin C phenotype, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia), a condition associated with hemoglobin having increased oxygen affinity, a condition associated with hemoglobin having decreased oxygen affinity, unstable hemoglobinopathies, 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 thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

111. A method for treating, improving, or preventing hereditary bone marrow failure syndrome in a subject, the method comprising administering to the subject a conjugate as described in any one of claims 1-78 or a pharmaceutical composition as described in claim 81, thereby treating, improving, or preventing the hereditary bone marrow failure syndrome in the subject.

112. The method of claim 111, wherein the hereditary bone marrow failure syndrome is amegacytic thrombocytopenic purpura (Amega), congenital aplastic anemia (DBA), congenital dyskeratosis (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwarzman-Diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Doppowitz syndrome, Kosterman syndrome, refractory cytopenia, thrombocytopenic radius loss (TAR), SAMD9 / SAMD9L disorder, or MECOM-related 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 treating a disease in a subject in need.

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

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.

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