Hematopoietic cell targeting conjugates and related methods

Abstract

Provided herein, in particular, are a conjugate comprising a protein (e.g., an antibody) that specifically binds to a transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)), wherein the conjugate comprises (b) a conjugate operably linked to at least one oligonucleotide that modulates the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed by the target cell, as well as a method for producing the conjugate and a pharmaceutical composition comprising the conjugate. Furthermore, provided herein are methods of using the conjugate, including, for example, a method for treating hemoglobin disorders (e.g., sickle cell disease (SCD)) or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

Description

Technical Field

[0001] Related Applications

[0001] This application claims priority to U.S. Patent Application No. 63 / 514956, filed on July 21, 2023, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing

[0002] This application includes a sequence listing submitted electronically in XML format, the entire content of which is incorporated herein by reference. The XML file was created on July 16, 2024, named 62992_6WO01_SL.xml, and has a size of 2,241,941 bytes.

[0003] 1. Field

[0003] The present disclosure relates, inter alia, to 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 a 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 a target cell. The present disclosure further relates to pharmaceutical compositions containing the conjugates and methods of using them (e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia or γ-thalassemia))).

Background Art

[0004] 2. Background

[0004] Bone marrow is a soft gelatinous tissue that fills the cavities of bones. Adult bone marrow is either red or yellow depending on the predominance of hematopoietic (red) or adipose (yellow) tissue. Genetic perturbations, including, for example, gene mutations, overexpression of genes, and deficiencies of genes (and thus their encoded products (e.g., proteins)) in subsets of cells within the bone marrow are associated with various genetic diseases, including, for example, hereditary blood disorders (e.g., hemoglobinopathies and hereditary bone marrow failure syndromes).

[0005]

[0005] In humans, red bone marrow forms all blood cells, including red blood cells, but lymphocytes are an exception. They are produced in the bone marrow and mature into lymphocytes in lymphoid organs. Normal red blood cells contain a protein called hemoglobin, which is responsible for transporting oxygen (O2) from the lungs to peripheral tissues and carbon dioxide (CO2) from the tissues to the lungs. Hemoglobin is a heterotetramer composed of an α-like globin subunit and a β-like globin subunit, each bound to a heme prosthetic group. Hemoglobin is synthesized separately from the α-like globin gene cluster and the β-like globin gene cluster, and different types of hemoglobin are produced depending on the combination of subunits. Fetal hemoglobin is the main hemoglobin produced by the fetus. In healthy individuals, a shift in gene expression from gamma globin to beta globin around birth is the basis for the switch in production from fetal hemoglobin to adult hemoglobin, and by about 6 months of age, adult hemoglobin becomes the dominant hemoglobin. The switch in hemoglobin is neither complete nor irreversible. Adults retain the ability to produce small amounts of fetal hemoglobin (less than 1% of total hemoglobin). The switch from fetal to adult hemoglobin depends on the suppression or silencing of the upstream gamma globin gene via a network of repressor proteins. Hemoglobin disorders are a group of genetic disorders associated with abnormalities in hemoglobin production and / or structure, and are the most common genetic blood disorders. [Overview of the Initiative]

[0006]

[0006] Provided herein, in particular, are conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) containing a protein (e.g., an antibody) that specifically binds to TFRs (e.g., hTFR (e.g., hTFR1)), and an oligonucleotide that modulates the expression and / or activity of a target gene expressed by the target cell, as well as methods for producing the conjugate and pharmaceutical compositions comprising the conjugate. Furthermore, provided herein are methods for using the conjugate, including, for example, methods for treating hemoglobin disorders (e.g., sickle cell disease (SCD)) or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia (e.g., β-thalassemia)).

[0007]

[0007] Accordingly, in some embodiments, what is provided herein is 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)), and (b) a hematopoietic cell targeting agent operably linked to at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, nucleic acid (e.g., mRNA) and / or protein expressed by a hematopoietic cell.

[0008]

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

[0009]

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

[0010]

[0010] In some embodiments, the conjugate exhibits one or more of the following properties: (a) when it binds 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) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the conjugate does not induce target cell death; (c) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the hematopoietic cells remain alive; (d) when it is internalized into the hematopoietic cells, the conjugate does not induce hematopoietic cell death; and / or (e) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of hematopoietic cells, the conjugate does not induce degradation of the TFR (e.g., hTFR (e.g., TFR1)).

[0011]

[0011] 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) When it binds to a TFR (e.g., hTFR (e.g., hTFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) or conjugate that specifically binds to that TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the hematopoietic cell; (b) When it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) or conjugate that specifically binds to that TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the hematopoietic cell. (c) When binding to TFRs (e.g., hTFRs (e.g., TFR1)) expressed on the surface of hematopoietic cells, the hematopoietic cells remain alive; (d) When internalized into hematopoietic cells, proteins (e.g., antibodies) or conjugates that specifically bind to such TFRs (e.g., hTFRs (e.g., hTFR1)) do not induce hematopoietic cell death; and / or (e) when binding to TFRs (e.g., hTFRs (e.g., TFR1)) expressed on the surface of hematopoietic cells, proteins (e.g., antibodies) or conjugates that specifically bind to such TFRs (e.g., hTFRs (e.g., hTFR1)) do not induce degradation of the TFRs (e.g., hTFRs (e.g., TFR1)).

[0012]

[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 includes or consists of a full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, a single-domain antibody (sdAb) (e.g., 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]

[0013] In some embodiments, the antibody includes an immunoglobulin (Ig) (e.g., human Ig (hIg)) Fc region. In some embodiments, the antibody includes or consists of a 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 includes at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hIg) Fc region includes the hinge region, a CH2 region, and a CH3 region. In some embodiments, Ig is hIg. In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1 or hIgG4.

[0014]

[0014] In some embodiments, the Fc region of Ig (e.g., hIg) comprises one or more amino acid substitutions compared to the Fc region of reference Ig (e.g., hIg), such that the Fc region reduces or eliminates one or more of the following effector functions compared to the Fc region of reference hIg: 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 Fc region of Ig (e.g., hIg) is substantially non-ADCC-mediated, substantially non-CDC-mediated, 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]

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

[0016]

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

[0017]

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

[0018]

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

[0019]

[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 includes transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

[0020]

[0020] In some embodiments, oligonucleotides enhance the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or a target protein. In some embodiments, oligonucleotides inhibit the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or a target protein. In some embodiments, oligonucleotides regulate the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or a target protein by binding to a target nucleic acid molecule (e.g., a target mRNA molecule (e.g., a part of a target mRNA molecule)) encoded by the target gene, target nucleic acid (e.g., mRNA) and / or a target protein. In some embodiments, the target nucleic acid molecule is a target mRNA molecule (e.g., a part of a target mRNA molecule). In some embodiments, the oligonucleotide mediates one or more of the following: degradation of a target nucleic acid molecule (e.g., mRNA), dysfunction 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 stability of a target nucleic acid molecule (e.g., mRNA), or blockage of translation of a target nucleic acid molecule (e.g., mRNA), or any combination thereof.

[0021]

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

[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 constituting the oligonucleotide are modified. In some embodiments, substantially all (or all) of the nucleotides constituting 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 nucleic acid base. In some embodiments, the oligonucleotide comprises at least one modified internucleoside bond (e.g., at least one phosphorothioate internucleoside bond). 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'-M0E), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMA0E), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), 2'-ON-methylacetamide(2'-O-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)-restricted ethyl-bridged nucleic acid (cEt) (e.g., the 2'-modified nucleotide is 2'-O-methyl or 2'-fluoro(2'-F))).

[0023]

[0023] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and / or protein encoded by the target gene is associated with one or more hemoglobin disorders. In some embodiments, inhibition or reduction of the expression and / or activity of the protein encoded by the target gene, target nucleic acid (e.g., mRNA), and / or target protein 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. In some embodiments, the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA), and / or target protein is associated with suppression 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. In some embodiments, the target gene, target nucleic acid (e.g., mRNA), and / or target protein is a transcription factor. In some embodiments, target genes, target nucleic acids (e.g., mRNA), and / or target proteins are highly expressed in erythroid precursor cells (compared to other non-erythroid precursor cell types).

[0024]

[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-containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF transcription factor 1 (KLF1) (e.g., hKLF1), FA complement group A (FANCA) (e.g., human FANCA), diskelin pseudouridine synthase 1 (DKC1) (e.g., human DKC1), telomere elongation regulating 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 ribosomal 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-derived elastase (ELA2) (e.g., human ELA2), HCLS1-related protein X-1 (HAX1) (e.g., E.g., human HAX1), glucose-6-phosphatase catalytic subunit 3 (G6PC3) (e.g., human G6PC3), growth factor-independent transcriptional repressor 1 (GFI1) (e.g., human GFI1), WASP actin nucleation promoter (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), SAM (Sterile Alpha Motif) domain-containing protein 9 (SAMD9) (e.g., human SAMD9).These are either SAM domain-containing 9-like loci (SAMD9L) (e.g., human SAMD9L) or MDS1-EVI1 complex locus (MECOM) (e.g., human MECOM).

[0025]

[0025] In some embodiments, the target gene is B-cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCL11A)), zinc finger and BTB domain-containing 7A (ZBTB7A) (e.g., hBTB7A), or KLF transcription factor 1 (KLF) (e.g., hKLF1).

[0026]

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

[0027]

[0027] In some embodiments, (b) comprises at least two, three, four, five, six or more oligonucleotides. In some embodiments, at least two, three, four, five, six or more oligonucleotides are individually conjugated to a protein (e.g., an antibody as described herein) that specifically binds to (a) a TFR (e.g., as described herein).

[0028]

[0028] In one embodiment, the herein provides a conjugate comprising: (a) an erythroid precursor 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)), and (b) an erythroid precursor cell targeting agent operably ligated to at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed by erythroid precursor cells.

[0029]

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

[0030]

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

[0031]

[0031] In some embodiments, the conjugate exhibits one or more of the following properties: (a) When it binds to TFRs (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the conjugate is internalized into the erythroid precursor cells; (b) When it binds to TFRs (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the conjugate does not induce target cell death; (c) the erythroid precursor cells (d) When bound to TFRs expressed on the surface (e.g., hTFR (e.g., TFR1)), the erythroid precursor cells remain alive; (f) even when internalized into the erythroid precursor cells, the conjugate does not induce the death of the erythroid precursor cells; and / or (e) when bound to TFRs expressed on the surface of erythroid precursor cells (e.g., hTFR (e.g., TFR1)), the conjugate does not induce the degradation of the TFRs (e.g., hTFR (e.g., TFR1)).

[0032]

[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) When it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) or conjugate that specifically binds to a TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the erythroid precursor cell; (b) When it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) or conjugate that specifically binds to a TFR (e.g., hTFR (e.g., hTFR1)) does not induce target cell death. (c) When bound to TFRs (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the erythroid precursor cells remain alive; (d) When internalized into erythroid precursor cells, proteins (e.g., antibodies) or conjugates that specifically bind to TFRs (e.g., hTFR (e.g., hTFR1)) do not induce the death of erythroid precursor cells; and / or (e) when bound to TFRs (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, proteins (e.g., antibodies) or conjugates that specifically bind to TFRs (e.g., hTFR (e.g., hTFR1)) do not induce the degradation of TFRs (e.g., hTFR (e.g., TFR1)).

[0033]

[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 includes or consists of a full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, a single-domain antibody (sdAb) (e.g., 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]

[0034] In some embodiments, the antibody includes an immunoglobulin (Ig) (e.g., human Ig (hIg)) Fc region. In some embodiments, the antibody includes or consists of a 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 includes at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hIg) Fc region includes the hinge region, a CH2 region, and a CH3 region. In some embodiments, Ig is hIg. In some embodiments, hIg is human IgG (hIgG). In some embodiments, hIgG is hIgG1 or hIgG4.

[0035]

[0035] In some embodiments, the Fc region of Ig (e.g., hIg) comprises one or more amino acid substitutions compared to the Fc region of reference Ig (e.g., hIg), such that the Fc region reduces or eliminates one or more of the following effector functions compared to the Fc region of reference hIg: 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) FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)). In some embodiments, the Fc region of Ig (e.g., hIg) is substantially non-ADCC-mediated, substantially non-CDC-mediated, 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]

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

[0037]

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

[0038]

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

[0039]

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

[0040]

[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 includes transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

[0041]

[0041] In some embodiments, oligonucleotides enhance the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein. In some embodiments, oligonucleotides inhibit the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein. In some embodiments, oligonucleotides regulate the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein by binding to a target nucleic acid molecule (e.g., a target mRNA molecule (e.g., a part of a target mRNA molecule)) encoded by the target gene, target nucleic acid (e.g., mRNA) and / or target protein. In some embodiments, the target nucleic acid molecule is a target mRNA molecule (e.g., a part of a target mRNA molecule). In some embodiments, the oligonucleotide mediates one or more of the following: degradation of a target nucleic acid molecule (e.g., mRNA), dysfunction 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 stability of a target nucleic acid molecule (e.g., mRNA), or blockage of translation of a target nucleic acid molecule (e.g., mRNA), or any combination thereof.

[0042]

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

[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 constituting the oligonucleotide are modified. In some embodiments, substantially all (or all) of the creotides constituting 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 nucleic acid base. In some embodiments, the oligonucleotide comprises at least one modified internucleoside bond (e.g., at least one phosphorothioate internucleoside bond). 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'-M0E), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMA0E), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), 2'-ON-methylacetamide(2'-O-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)-restricted ethyl-bridged nucleic acid (cEt) (e.g., the 2'-modified nucleotide is 2'-O-methyl or 2'-fluoro(2'-F))).

[0044]

[0044] In some embodiments, the protein encoded by the target gene, target nucleic acid (e.g., mRNA) and / or target protein is associated with one or more hemoglobin disorders. In some embodiments, inhibition or reduction of the expression and / or activity of the protein encoded by the target gene, target nucleic acid (e.g., mRNA) and / or target protein 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. In some embodiments, the expression and / or activity of the protein encoded by the target gene, target nucleic acid (e.g., mRNA) and / or target protein is associated with suppression 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. In some embodiments, the target gene, target nucleic acid (e.g., mRNA) and / or target protein is a transcription factor.

[0045]

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

[0046]

[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-containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF transcription factor 1 (KLF1) (e.g., hKLF1), FA complement group A (FANCA) (e.g., human FANCA), diskelin pseudouridine synthase 1 (DKC1) (e.g., human DKC1), telomere elongation helicase 1 (RTEL1) (e.g., human RTEL1), telomerase inversion Ribosomal enzyme (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 ribosomal 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-derived elastase (ELA2) (e.g., human ELA2), HCLS1-related protein X-1 (HAX1) (e.g., (e.g., human HAX1), glucose-6-phosphatase catalytic subunit 3 (G6PC3) (e.g., human G6PC3), growth factor-independent transcription repressor 1 (GFI1) (e.g., human GFI1), WASP actin nucleation promoter (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), SAM (Sterile AlphaThese are Motif) domain-containing 9 (SAMD9) (e.g., human SAMD9), SAM domain-containing 9-like (SAMD9L) (e.g., human SAMD9L), or MDS1-EVI1 complex locus (MECOM) (e.g., human MECOM).

[0047]

[0047] In some embodiments, the target gene is BCL11A (e.g., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

[0048]

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

[0049]

[0049] In some embodiments, (b) comprises at least two, three, four, five, six or more oligonucleotides. In some embodiments, each of at least two, three, four, five, six or more oligonucleotides is individually conjugated to (a) a protein that specifically binds to TFR (e.g., an antibody as described herein).

[0050]

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

[0051]

[0051] In one embodiment, what is provided herein is a pharmaceutical composition comprising a conjugate described herein and a pharmaceutically acceptable excipient.

[0052]

[0052] In one embodiment, what is provided herein is a kit comprising a conjugate or a pharmaceutical composition described herein.

[0053]

[0053] In some embodiments, the Specified herein provides a method for delivering a conjugate or pharmaceutical composition to a cell, the method comprising delivering the conjugate or pharmaceutical composition described herein into the cell by introducing the conjugate or pharmaceutical composition described herein into the cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0054]

[0054] In one embodiment, the Specified herein provides a method for delivering a conjugate, cells, or pharmaceutical composition to a subject, the method comprising administering the conjugate, cells, or pharmaceutical composition described herein to the subject.

[0055]

[0055] In some embodiments, the foregoing provides a method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed in hematopoietic cells within a cell, the method comprising regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein by introducing a conjugate or a pharmaceutical composition described herein. 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]

[0056] In some embodiments, the foregoing provides a method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed in intracellular erythroid precursor cells, the method comprising regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein by introducing a conjugate or a pharmaceutical composition described herein. 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]

[0057] In one embodiment, the Specified provides a method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed by hematopoietic cells within a target cell, the method comprising regulating (e.g., inhibiting or enhancing) the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein in the target by administering the subject a conjugate or a pharmaceutical composition described herein.

[0058]

[0058] In one embodiment, the foregoing provides a method for modulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed in erythroid precursor cells within a target cell, the method comprising administering the subject a conjugate or pharmaceutical composition described herein to the subject to modulate (e.g., inhibit or enhance) the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein in the subject.

[0059]

[0059] In some embodiments, the foregoing provides a method for reducing and / or inhibiting the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed in hematopoietic cells within a cell, the method comprising reducing or inhibiting the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein by introducing the conjugate or pharmaceutical composition described herein into 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).

[0060]

[0060] In some embodiments, the foregoing provides a method for reducing and / or inhibiting the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed in intracellular erythroid precursor cells, the method comprising reducing or inhibiting the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein by introducing the conjugate or pharmaceutical composition described herein into 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). In some embodiments, the target is BCL11A (e.g., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

[0061]

[0061] In one embodiment, the foregoing provides a method for reducing and / or inhibiting the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed by hematopoietic cells in a subject cell, the method comprising reducing or inhibiting the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein in the subject by administering the conjugate or pharmaceutical composition described herein to the subject.

[0062]

[0062] In one embodiment, the foregoing provides a method for reducing and / or inhibiting the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA) and / or target protein expressed by erythroid precursor cells in a subject cell, the method comprising reducing or inhibiting the expression and / or activity of the target gene, target nucleic acid (e.g., mRNA) and / or target protein in the subject by administering the subject a conjugate or pharmaceutical composition described herein.

[0063]

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

[0064]

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

[0065]

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

[0066]

[0066] In some embodiments, the Specified herein provides a method for treating, improving or preventing a hereditary blood disorder in a subject, the method comprising administering to the subject a conjugate or pharmaceutical composition described herein to treat, improve or prevent a hereditary blood disorder in the subject. In some embodiments, the hereditary blood disorder is a hemoglobin disorder or a hereditary bone marrow failure syndrome.

[0067]

[0067] In some embodiments, the Specified herein provides a method for treating, improving or preventing a hemoglobin disorder in a subject, the method comprising administering to the subject a conjugate or pharmaceutical composition described herein to treat, improve or prevent a hemoglobin disorder in the subject. In some embodiments, the subject is a human.

[0068]

[0068] In some embodiments, hemoglobin disorders 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), conditions associated with hemoglobin with increased oxygen affinity, conditions associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

[0069]

[0069] In some embodiments, the hemoglobin disorder is sickle cell disease or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

[0070]

[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 hemoglobin disease, methemoglobinemia, or any combination thereof.

[0071]

[0071] In some embodiments, the subject is suspected of having sickle cell disease or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia) or has been diagnosed with sickle cell disease or thalassemia.

[0072]

[0072] In one embodiment, the Specified herein provides a method for treating, improving or preventing hereditary bone marrow failure syndrome in a subject, the method comprising administering to the subject a conjugate or pharmaceutical composition described herein to treat, improve or prevent hereditary bone marrow failure syndrome in the subject.

[0073]

[0073] In some embodiments, hereditary bone marrow failure syndromes include amegakaryocytic thrombocytopenia (Amega), Diamond-Blackfan anemia (DBA), congenital dyskeratosis (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachmann-Diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostman syndrome, refractory cytopenia, or radial dystolic thrombocytopenia (TAR).

[0074]

[0074] In some embodiments, what is provided herein are conjugates, cells, and pharmaceutical compositions described herein for use in the treatment of diseases in subjects requiring treatment.

[0075]

[0075] In some embodiments, what is provided herein is a conjugate, a cell, or a pharmaceutical composition as described herein, for use as a pharmaceutical.

[0076]

[0076] In one embodiment, what is provided herein is the use of the conjugate, the cell, or the pharmaceutical composition described herein for the manufacture of a pharmaceutical for the treatment of a disease in an object requiring treatment. [Brief explanation of the drawing]

[0077] [Figure 1A-B]

[0077] Figures 1A to 1D are line graphs showing the binding of recombinant expression anti-TFR1 monoclonal antibody (mAb) (shown in Example 1) (or isotype control) to soluble TFR1 (using Octet BLI). Figure 1A is a line graph showing the binding of the isotype control antibody to soluble TFR1 (showing that it does not bind). Figure 1B is a line graph showing the binding of anti-TFR1 mAb2 to soluble TFR1. [Figure 1C-D] Figure 1C is a line graph showing the binding of anti-TFR1 mAb3 to soluble TFR1. Figure 1D is a line graph showing the binding of anti-TFR1 Fab to soluble TFR1. [Figure 2A-B]

[0078] Figures 2A to 2D are line graphs showing the binding of anti-TFR1 mAb (shown in Example 1) to the surface of erythroid progenitor cells. Figure 2A is a line graph showing the percentage of positive cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentrations). Figure 2B is a line graph showing the MFI of cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentrations). [Figure 2C-D]Figure 2C is a line graph showing the percentage of positive cells treated with anti-TFR1 Fab (at the indicated concentrations). Figure 2D is a line graph showing the MFI of cells treated with anti-TFR1 Fab (at the indicated concentrations). [Figure 3A-B]

[0079] Figures 3A to 3D are graphs showing the internalization of anti-TFR1 mAb (described in Example 1) or isotype control erythroid progenitor cells. Figure 3A is a graph showing the percentage of positive cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentrations). Figure 3B is a graph showing the MFI of cells treated with anti-TFR1 mAb2 or anti-TFR1 mAb3 (at the indicated concentrations). [Figure 3C-D] Figure 3C is a graph showing the percentage of positive cells treated with anti-TFR1 Fab (at the indicated concentrations). Figure 3D is a graph showing the MFI of cells treated with anti-TFR1 Fab (at the indicated concentrations). [Figure 4A-B]

[0080] Figures 4A and 4B are line graphs showing the degree of mRNA knockdown of the indicated target genes by the indicated siRNAs in erythroid progenitor cells. Figure 4A is a line graph showing the knockdown of HPRT1 mRNA by HPRT1-2 siRNA (at the indicated siRNA concentration). Figure 4B is a line graph showing the knockdown of BCL11A mRNA by BCL11A-11 siRNA or BCL11A-5 siRNA (at the indicated siRNA concentration). [Figure 5A-B]

[0081] Figures 5A and 5B are bar graphs showing the degree of reduction in the levels of the indicated target proteins in erythroid progenitor cells by the indicated siRNAs. Figure 5A is a bar graph showing the reduction in BCL11A protein levels (at the indicated siRNA concentrations) by BCL11A-11 siRNA or BCA11A-5 siRNA. Figure 5B is a bar graph showing the reduction in HPRT1 protein levels (at the indicated siRNA concentrations) by HPRT1-2 siRNA. [Figure 6A-B]

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

[0083] Figures 7A and 7B are line graphs showing the survival rate (%) of erythroid precursor cells 48 hours and 72 hours after the initial administration of the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. Figure 7A is a line graph showing the survival rate (%) of erythroid precursor cells 48 hours after the initial administration of the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. Figure 7B is a line graph showing the survival rate (%) of erythroid precursor cells 72 hours after the initial administration of the indicated AOC at the indicated concentration. [Figure 7C-D]

[0084] Figures 7C and 7D are line graphs showing the knockdown of HPRT1 transcripts in erythroid progenitor cells 48 hours and 72 hours after initial administration of the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. Figure 7C is a line graph showing the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells 48 hours after initial administration of the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. Figure 7D is a line graph showing the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells 72 hours after initial administration of the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. [Figure 8A-B]

[0085] Figures 8A and 8B are line graphs showing the survival rate (%) of erythroid precursor cells 48 hours and 72 hours after the initial administration of the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. Figure 8A is a line graph showing the survival rate (%) of erythroid precursor cells 48 hours after the initial administration of the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. Figure 8B is a line graph showing the survival rate (%) of erythroid precursor cells 72 hours after the initial administration of the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. [Figure 8C-D]

[0086] Figures 8C and 8D are line graphs showing the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells 48 hours and 72 hours after initial administration of the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. Figure 8C is a line graph showing the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells 48 hours after initial administration of the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. Figure 8D is a line graph showing the knockdown of HPRT1 mRNA transcripts in erythroid progenitor cells 72 hours after initial administration of the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. [Figure 9]

[0087] This line graph shows the knockdown of the HPRT1 protein in erythroid progenitor cells 72 hours after the initial administration of the indicated AOC (AOC#2 or AOC#4). [Figure 10A-B]

[0088] Figure 10A is a line graph showing the survival rate (%) of erythroid precursor cells 72 hours after initial administration of the indicated AOC (AOC#5) at the indicated concentration. Figure 10B is a line graph showing the knockdown of HPRT1 mRNA transcript in erythroid progenitor cells 72 hours after initial administration of the indicated AOC (AOC#5) at the indicated concentration. [Figure 11A-B]

[0089] Figure 11A is a line graph showing the survival rate (%) of erythroid precursor cells 72 hours after initial administration of the indicated AOC (AOC#10 or AOC#11) at the indicated concentration. Figure 11B is a line graph showing the knockdown of BCL11A mRNA transcript in erythroid progenitor cells 72 hours after initial administration of the indicated AOC (AOC#10 or AOC#11) at the indicated concentration. [Figure 12A-B]

[0090] Figure 12A is a line graph showing the survival rate (%) of erythroid precursor cells 72 hours after initial administration of the indicated AOC (AOC#9) at the indicated concentration. Figure 12B is a line graph showing the knockdown of BCL11A transcript in erythroid progenitor cells 72 hours after initial administration of the indicated AOC (AOC#9) at the indicated concentration. [Figure 13A-B]

[0091] Figure 13A is a line graph showing the survival rate (%) of erythroid precursor cells 72 hours after initial administration of the indicated AOC (AOC#7 or AOC#8) at the indicated concentration. Figure 13B is a line graph showing the knockdown of BCL11A mRNA transcript in erythroid progenitor cells 72 hours after initial administration of the indicated AOC (AOC#7 or AOC#8) at the indicated concentration. [Modes for carrying out the invention]

[0078]

[0092] The inventors have found that molecular payloads (e.g., oligonucleotides) capable of regulating (e.g., inhibiting) the expression and / or activity of genes capable of regulating (e.g., suppressing) fetal hemoglobin production can be specifically targeted to hematopoietic cells (e.g., erythroid precursor cells) through conjugation with a targeting agent (e.g., an anti-TFR antibody). In this sense, the conjugates described herein are particularly useful in the treatment of hemoglobin disorders (e.g., sickle cell disease and thalassemia). Accordingly, this disclosure provides conjugates, as well as their use in pharmaceutical compositions and in methods for treating diseases (e.g., hemoglobin disorders).

[0079] table of contents TIFF2026524970000001.tif242170TIFF2026524970000002.tif242170TIFF2026524970000003.tif5170

[0080] 5.1 Definition

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

[0081]

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the claimed subject matter belongs. It should be understood that the above general description and the following detailed description are illustrative and descriptive only and do not limit any claimed subject matter.

[0082]

[0095] In this disclosure, unless otherwise specified, the use of the singular form includes the plural form. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limited.

[0083]

[0096] Where the term “comprising” is used to describe an aspect in this specification, it is understood that other similar aspects described using the terms “consisting of” and “consisting essentially of” are also provided herein.

[0084]

[0097] Where the term “and / or” is used herein, it is construed to mean that each of the two identified features or components is disclosed separately for both the case with and without the other. Accordingly, the term “and / or” as used herein in phrases such as “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 include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and; B and C; A (alone); B (alone); C (alone).

[0085]

[0098] Any concentration range, percentage range, ratio range, or integer range described herein shall be understood to include all integer values ​​within the range described, and, where appropriate, fractions thereof (such as one-tenth or one-hundredth of an integer), unless otherwise indicated.

[0086]

[0099] The term “approximately” means a value or composition that is within the acceptable margin of error for a particular value or composition as determined by those skilled in the art, and such margin of error depends at least in part on the method by which the value or composition is measured or determined, i.e., on the limits of the measuring system. Where a particular value or composition is shown in this disclosure, unless otherwise specified, the meaning of “approximately” is assumed to be within the acceptable margin of error for that particular value or composition.

[0087]

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

[0088]

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

[0089]

[0102] Where proteins, nucleic acid molecules, etc. are described in this specification, it is understood that recombinant forms of such proteins, nucleic acid molecules, etc. are also provided in this specification.

[0090]

[0103] Where a polypeptide or a set of polypeptides is described herein, it is understood that a protein obtained by folding such polypeptide or set of polypeptides into its three-dimensional structure (i.e., tertiary or quaternary structure) is also provided herein, and vice versa.

[0091]

[0104] Where a protein is described herein, polypeptides containing the same amino acid sequence folded into a linear or three-dimensional structure (i.e., a tertiary or quaternary structure) are also provided herein.

[0092]

[0105] In this specification, the term “administering” means physically introducing an agent (e.g., a conjugate as described herein) (or a precursor of an agent that is metabolized or converted in the subject's body to produce the agent in vivo (e.g., a precursor of a therapeutic agent)) into a subject using any of the various methods and delivery systems known to those skilled in the art. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods. Administration includes self-administration.

[0093]

[0106] The terms “agent” and “moiety” are used interchangeably herein and are used as comprehensive terms to describe any macromolecule or micromolecule (and any combination thereof). Exemplary agents 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. An agent may comprise one or more individual agents (these individual agents may be identical or different). For example, an agent may comprise antibodies (e.g., targeting agents described herein) and oligonucleotides (e.g., oligonucleotides described herein). Agents as defined herein include, for example, conjugates described herein.

[0094]

[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, which is defined as [antibody] x [antigen] / [antibody-antigen] (where [antibody-antigen] is the molar concentration of the antibody-antigen complex, [antibody] is the molar concentration of the unbound antibody, and [ligand] is the molar concentration of the unbound antigen). The affinity constant Ka is defined 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), a common method known in the art (e.g., BIAcore®-based assays) (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patents 5,283,173 and 5,468,614, the entire contents of which are incorporated herein by reference).

[0095]

[0108] As used herein, the terms “antibody” or “antibodies” are used in their broadest sense and encompass a variety of immunoglobulin (Ig) structures (e.g., human Ig (hIg), mouse Ig (mIg)). Such immunoglobulin structures include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies), and antibody fragments (i.e., antigen-binding fragments or their variants) insofar as they exhibit the desired antigen-binding activity. Therefore, the term antibody includes, for example, full-length antibodies, antigen-binding fragments of full-length antibodies, antibody CDRs, molecules containing VH and / or VL regions, 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, camelized antibodies, intrabodies, variable domains of novel antigen receptor beta-lactamases (VNAR fragments), afibodies, diabodies, tribodies, 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( The antibody comprises an ab')2 fragment, a disulfide-linked Fv(sdFv), an Fc fusion (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc), and any of the above antigen-binding fragments, and a conjugate or fusion protein containing any of the above. The antibody is an Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class of Ig (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ) may be. In certain embodiments, the antibody described herein is an IgG antibody, or a class of IgG (e.g., human IgG1 or IgG4) or a subclass thereof. In certain embodiments, the antibody described herein is an mIgG antibody, or a class of mIgG (e.g., mIgG1 or mIgG2a ) or a subclass thereof. 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, e.g., recombinant production in host cells (see, e.g., §5.4) or synthetic production.

[0096]

[0109] As used herein, the term “antibody-like scaffold” refers to an antigen-binding domain that is not based on Ig. Various antibody-like scaffolds are known in the art. For example, fibronectin (e.g., AdNectins®) and engineered ankyrin repeat proteins (DARPins®) are used as alternative scaffolds for antigen-binding domains. See, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics, Curr Opin Chem Biol 13:245-255 (2009); and Stumpp et al., Darpins: A new generation of protein therapeutics, Drug Discovery Today 13:695-701 (2008). The contents of these references are incorporated herein by reference in their entirety.Exemplary antibody-like scaffolds include, but are not limited to, lipocalin (e.g., see U.S. Patent No. 7,250,297) (e.g., Anticalin®), protein A-derived molecules, such as the Z domain of protein A (e.g., see U.S. Patent No. 5,831,012) (e.g., Affibody®), and the A domain of membrane receptors stabilized by disulfide bonds and Ca2+ (e.g., see U.S. Patent No. 7,803,907) (e.g., Avimer / Maxibody®). , serum transferrin (see, e.g., U.S. Patent Publication No. 2004 / 023334) (e.g., Transbody®), engineered ankyrin repeat protein (see, e.g., U.S. Patent No. 7417130) (e.g., DARPin®), fibronectin (see, e.g., U.S. Patent No. 6818418) (e.g., AdNectin®), C-type lectin domain (see, e.g., U.S. Patent Application Publication No. 2004 / 132094) (e.g., Tetrecantin®), Human gamma crystallin or ubiquitin (e.g., see U.S. Patent No. 7,838,629) (e.g., Affilin®), Knitz-type domains of human protease inhibitors (e.g., see U.S. Patent Publication No. 2004 / 209243), type C lectins (e.g., see U.S. Patent Publication No. 2004 / 132094) (e.g., Tetranectin®), cysteine ​​knot or notchin (e.g., see U.S. Patent No. 7,186,524) (e.g., Micr This includes obodies®, nucleic acid aptamers (e.g., see U.S. Patent No. 5,475,096), thioredoxin A scaffolds (e.g., see U.S. Patent No. 6,004,746) (peptide aptamers), and fibronectin type 10 III domains (e.g., see U.S. Patent No. 6,818,418) (e.g., AdNectins®), and cystine-packed peptides (e.g., see International Publication No. 2023,023,031).Additional 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. 2011;3(3):310-317. doi:10.4161 / mabs.3.3.15530. The entire contents of each of the aforementioned references are incorporated herein by reference. Antibody-like scaffolds include, for example, naturally occurring antigen conjugates, variants of naturally occurring antigen conjugates (e.g., functional variants), fragments of naturally occurring antigen conjugates (e.g., functional fragments), and synthetic antigen conjugates (i.e., non-natural antigen conjugates).

[0097]

[0110] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to the immune mechanism that leads to the lysis of target cells coated with an antibody (or Fc region-containing protein) (e.g., the Ig Fc-containing fusion protein described herein) by immune effector cells (e.g., NK cells). As used herein, the terms “decreased ADCC” and similar terms refer to a decrease in the number of target cells lysed within a given time by the ADCC mechanism as defined above at a given concentration of the antibody (or Ig Fc region-containing protein) (e.g., the Fc region-containing fusion protein described herein) in the culture medium surrounding the target cells, and / or an increase in the concentration of the antibody (or Ig Fc region-containing protein) (e.g., the Fc region-containing fusion protein described herein) in the culture medium surrounding the target cells, which is required to achieve the lysis of a given number of target cells within a given time by the ADCC mechanism as defined above. The reduction in ADCC is compared to ADCC mediated by the same antibody (or Fc-region-containing protein) (e.g., the Fc-containing fusion protein described herein) produced by the same type of host cells using the same standard manufacturing, purification, formulation, and storage methods (known to those skilled in the art), and which has not been modified (e.g., does not contain one or more amino acid mutations (e.g., amino acid substitutions) that mediate the reduction in ADCC). For example, the reduction in ADCC mediated by an antibody (or Fc-region-containing protein) (e.g., the Fc-containing fusion protein described herein) that contains an amino acid substitution in its Fc region that reduces ADCC is compared to ADCC mediated by the same antibody (or Fc-region-containing protein) (e.g., the Fc-containing fusion protein described herein) that does not have the amino acid substitution in its Fc region.

[0098]

[0111] As used herein, the terms “antisense oligonucleotide” or “ASO” mean standard single-stranded oligonucleotides known to those skilled in the art, which are capable of regulating the expression of a target gene (or protein) by, for example, hybridizing to a target nucleic acid (e.g., mRNA encoded by a target gene and encoding a target protein), particularly to a contiguous sequence on the target nucleic acid. Antisense oligonucleotides include DNA, RNA, and hybrid DNA / RNA oligonucleotides.

[0099]

[0112] As used herein, the term "antisense strand," when used in relation to the oligonucleotides described herein (e.g., RNAi agents (e.g., siRNA agents), antisense oligonucleotides), means an oligonucleotide that includes a complementary region containing a nucleotide sequence that is at least partially (e.g., substantially, or completely) complementary to the target nucleic acid sequence (e.g., target mRNA (e.g., a portion of target mRNA)). In the case of a single-stranded oligonucleotide (e.g., an antisense oligonucleotide), the antisense strand is the sole strand. In the case of a double-stranded oligonucleotide (e.g., siRNA), the antisense strand (the sense strand) is usually paired with the sense strand (as described herein).

[0100]

[0113] As used herein, the terms "BCL11 transcription factor A" or "BCL11A" refer to a C2H2-type zinc finger transcription factor that functions particularly in the suppression 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 Sequence ID No. 291 (NCBI Ref.: Np_075044.2).

[0101]

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

[0102]

[0115] As used herein, the term “bicyclic nucleoside” (“BNA”) refers to a nucleoside containing a bicyclic sugar.

[0103]

[0116] As used herein, the term “blunt end” refers to a double-stranded oligonucleotide that does not contain an unpaired nucleotide at its ends (e.g., 3' end, 5' end) (i.e., no nucleotide overhang). A double-stranded oligonucleotide may have blunt ends at, for example, the 3' end, the 5' end, or both the 3' and 5' ends of the molecule.

[0104]

[0117] As used herein, the terms “CDR” or “complementarity-determining region” refer to discontinuous antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These specific regions are 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 reference are incorporated herein by reference. Unless otherwise specified, the term “CDR” refers to the CDR as defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977). Those skilled in the art can determine CDRs as defined by other schemes (e.g., Chothia, IMGT) using methods commonly known to those skilled in the art.

[0105]

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

[0106]

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

[0107]

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

[0108]

[0121] As used herein, “complementary” in relation to a second nucleotide sequence (e.g., an antisense strand or antisense oligonucleotide) means the ability of a nucleic acid molecule containing a first nucleotide sequence to hybridize with a nucleic acid molecule containing a second nucleotide sequence to form a double-stranded region (via base-hydrogen bonding) under appropriate in vivo or in vitro conditions (e.g., specific standard conditions, mammalian (e.g., human) physiological conditions). A person skilled in the art can select the set of conditions most suitable for hybridization testing. Complementary sequences include, for example, Watson-Crick base pairs. For example, complementary nuclear base pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), and cytosine (C) and guanine (G). Complementary nucleic acid base pairs include natural and modified nucleotides, as well as nucleotide mimetic compounds, to the extent that at least the hybridization requirements described above are met. In this sense, the determination of complementarity (as described herein) does not depend on the chemical modification of the nucleotide (e.g., as described herein). For example, both (C) and 5-methylcytosine (mC) are complementary to (G).

[0109]

[0122] As used herein, the term “conjugation” means an operable linkage (e.g., chemical conjugation) of at least a first agent (e.g., oligonucleotides (e.g., oligonucleotides as described herein)) and a second agent (e.g., a protein (e.g., a targeting agent (e.g., a targeting agent as described herein (e.g., a hematopoietic cell targeting agent (e.g., an erythroid precursor cell targeting agent as described herein (e.g., an anti-TFR (e.g., anti-hTFR (e.g., anti-hTFR1)) antibody as 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 operable linking of two agents (e.g., chemical conjugation methods) are well known in the art and are commercially available. Available conjugation reagents and kits are also well known, and detailed instructions for their use are readily available from commercial suppliers. Operable conjugations (e.g., chemical conjugations) include both covalent and non-covalent conjugations. In some embodiments, the operable conjugation (e.g., chemical conjugation) includes an operable conjugation of a first agent (e.g., an oligonucleotide (e.g., the oligonucleotides described herein)) and a second agent (e.g., a protein (e.g., a targeting agent (e.g., the targeting agents described herein (e.g., hematopoietic cell targeting agents (e.g., erythroid precursor cell targeting agents (e.g., the anti-TFR (e.g., anti-hTFR (e.g., anti-hTFR1)) antibody)))).

[0110]

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

[0111]

[0124] As used herein, the term “disease” refers to any abnormal condition that impairs physiological function. This term is used broadly to encompass all disorders, diseases, abnormalities, conditions, illnesses, states, and syndromes in which physiological function is impaired, regardless of the nature of the etiology. The term “disease” includes infections (e.g., viral infections, bacterial infections, fungal infections, protozoan infections).

[0112]

[0125] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules containing multiple deoxyribonucleotides polymerized via phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0113]

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

[0114]

[0127] The term "effector function," when used in relation to the Ig Fc region or proteins containing the Ig Fc region (e.g., full-length antibodies), refers to the biological activity attributable to the Ig Fc region of a typical full-length antibody, and therefore varies depending on the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell 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.

[0115]

[0128] As used herein, the term “erythroid precursor cell” refers to any precursor of mature erythrocytes (i.e., mature enucleated erythrocytes). In this sense, erythroid precursor cells include, but are not limited to, megakaryocyte-erythroid progenitor cells, proerythroids, early erythroids, metaphase erythroids, late erythroids, and reticulocytes. In some embodiments, erythroid precursor cells include megakaryocyte-erythroid progenitor cells, proerythroids, early erythroids, metaphase erythroids, and late erythroids. In some embodiments, erythroid precursor cells include proerythroids, early erythroids, metaphase erythroids, and late erythroids.

[0116]

[0129] As used herein, the term “erythroid precursor cell targeting agent” refers to an agent that specifically binds to an antigen expressed on erythroid precursor cells (or a subpopulation thereof). For example, the antigen expressed inside or on erythroid precursor cells may be a membrane protein, e.g., an endogenous membrane protein or a superficial membrane protein. Typically, erythroid precursor cell targeting agents specifically bind to an antigen on erythroid precursor cells and facilitate the internalization of the erythroid precursor cell targeting agent (and associated molecular payload) into the erythroid precursor cells. In some embodiments, erythroid precursor cell targeting agents can specifically bind to internalizing cell surface receptors on erythroid precursor cells and be internalized into the erythroid precursor cells by receptor-mediated internalization. In some embodiments, the erythroid precursor cell targeting agent is a protein (e.g., an antibody), a peptide, a nucleic acid (e.g., an aptamer), or a small molecule. In some embodiments, the erythroid precursor cell targeting agent is linked to the molecular payload.

[0117]

[0130] As used herein, the term “EU numbering system” refers to the EU numbering rules for the constant region of antibodies, as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991. The full contents of each reference are incorporated herein by reference.

[0118]

[0131] As used herein, the term "Fab" refers to an antigen-binding domain, which comprises a Fab heavy chain containing a VH region and a CH1 region from the N-terminus to the C-terminus, and a light chain containing a VL region and a CL region from the N-terminus to the C-terminus, and the Fab heavy chain and light chain associate to form an antigen-binding domain.

[0119]

[0132] As used herein, the term "Fab-Fc" refers to an antibody containing a Fab operably linked to an Fc region.

[0120]

[0133] As used herein, the term “Fc region” refers to the C-terminal region of an Ig (e.g., human Ig) heavy chain containing at least one CH2 region operably linked 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 said Ig hinge region operably linked to the N-terminus of the CH2 region. In some embodiments, the Fc region is modified compared 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, and 5.3.2.4. Additional examples of proteins having modified Fc regions can be found in Saunders 2019 (KO Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” 2019, Frontiers in Immunology, V.10, Art.1296, pp.1~20). The entire contents of this document are incorporated herein by reference.

[0121]

[0134] In this specification, the terms "first" and "second" used in relation to Fc regions, etc., are used for convenience to distinguish between multiple types of each part. Unless otherwise specified, the use of these terms is not intended to confer any particular order or orientation in the protein. For example, an antibody described herein (e.g., a full-length antibody) may include two Fc regions associated via one or more covalent (e.g., disulfide) bonds.

[0122]

[0135] As used herein, the terms “framework region” or “FR region” refer to amino acid residues that are part of the variable region of an antibody but are not part of the CDR (for example, using the Kabat definition of CDR).

[0123]

[0136] In this specification, the term "full-length antibody" means an antibody having a structure substantially similar to that of a natural antibody, wherein the antibody comprises (i) a first Ig light chain including 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 including 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 including 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 including 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 associate to form a first antigen-binding domain, the second light chain and the second heavy chain associate to form a second antigen-binding domain, and the first heavy chain and the second heavy chain associate to form a dimer. In some embodiments, these two heavy chains contain substantially identical amino acid sequences, and these two light chains contain substantially identical amino acid sequences. In some embodiments, these two heavy chains contain substantially identical amino acid sequences, except for one or more amino acid modifications (e.g., those described herein) that promote appropriate heterodimerization between heavy chains, and these two light chains contain substantially identical amino acid sequences. Even if the antibody chains are substantially identical, they may not be completely identical if they differ due to post-translational modifications such as C-terminal cleavage of lysine residues or alternative glycosylation patterns.

[0124]

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

[0125]

[0138] In this specification, the term “functional variant” as used in relation to a protein means a protein that has at least one amino acid mutation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference protein, but which is less than or equal to 20%, 15%, 12%, 10%, or 8%, and which retains at least one specific function of the reference protein. Not all functions of the reference protein (e.g., wild type) are to be retained by the functional variant of the protein. In some cases, one or more functions are selectively reduced or eliminated. In some cases, the reference protein is the wild type protein.

[0126]

[0139] In this specification, the term “functional fragment” as used in relation to proteins refers to a fragment of a reference protein that retains at least one specific function. Not all functions of the reference polypeptide are necessarily retained by the functional fragment of that polypeptide. In some cases, one or more functions are selectively reduced or removed. In some cases, the reference protein is a wild-type protein.

[0127]

[0140] As used herein, the term “fuse” and its grammatical equivalent refer to the operable linkage of at least one polypeptide derived from a first polypeptide with another polypeptide derived from a second polypeptide, where the first and second polypeptides are distinct. The term “fuse” encompasses both the direct linkage of at least two polypeptides via peptide bonds and the indirect linkage via linkers (e.g., peptide linkers).

[0128]

[0141] As used herein, the term “fusion protein” and its grammatical equivalent refer to a protein comprising at least one polypeptide derived from a first polypeptide operably linked to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are not operably linked in nature. At least two polypeptides of such a fusion protein may be directly operably linked via peptide bonds, or indirectly operably linked via a linker (e.g., a peptide linker). Thus, for example, the term fusion protein encompasses embodiments in which polypeptide A is directly operably linked to polypeptide B via peptide bonds (polypeptide A-polypeptide B), and embodiments in which polypeptide A is operably linked to polypeptide B via a peptide linker (polypeptide A-peptide linker-polypeptide B). In some embodiments, the first and second polypeptides are different.

[0129]

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

[0130]

[0143] As used herein, the term "half-life extension polypeptide" refers to a polypeptide that, when operably ligated to another polypeptide (the polypeptide of interest), extends the half-life of the polypeptide of interest in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of such polypeptide or protein can be evaluated using in vitro models known in the art.

[0131]

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

[0132]

[0145] As used herein, the term “hematopoietic cells” refers to all blood cells. Therefore, the term hematopoietic cells include, but are not limited to, hematopoietic pluripotent stem cells (HSPCs), myeloid common progenitor cells, megakaryocyte-erythroid progenitor cells, erythroid progenitor cells, proerythroid cells, early erythroid cells, metaphase erythroid cells, late erythroid cells, reticulocytes, megakaryocytes, platelets, granulocyte-monocyte progenitor cells, monoblasts, promonocytes, monocytes, macrophages, myeloblasts, promyelocytes, myelocytes, eosinophils, basophils, neutrophils, lymphoid common progenitor cells, proNK lymphoblasts, NK cells, proB lymphoblasts, B lymphocytes, proT lymphoblasts, T lymphoblasts, and plasma cells.

[0133]

[0146] As used herein, the term “hematopoietic cell targeting agent” refers to an agent that specifically binds to an antigen expressed on hematopoietic cells (or a subpopulation thereof). For example, the antigen expressed inside or on hematopoietic cells may be a membrane protein, e.g., an endogenous membrane protein or a superficial membrane protein. Typically, a hematopoietic cell targeting agent specifically binds to an antigen on hematopoietic cells and facilitates the internalization of the hematopoietic cell targeting agent (and associated molecular payload) into the hematopoietic cells. In some embodiments, a hematopoietic cell targeting agent can specifically bind to an internalizing cell surface receptor on hematopoietic cells and be internalized into the hematopoietic cells by receptor-mediated internalization. In some embodiments, the hematopoietic cell targeting agent 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 targeting agent is linked to a molecular payload.

[0134]

[0147] As used herein, the term "heterogeneous" means that, when a first element is described in relation to a second element, the first and second elements do not exist in nature in the described arrangement. For example, a nucleic acid molecule containing a "heterogeneous portion" means a nucleic acid molecule to which a portion not naturally bound to the nucleic acid molecule (e.g., carbohydrates, small molecules, polypeptides, polynucleotides, lipids, synthetic polymers (e.g., PEG polymers), etc.) is bound.

[0135]

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

[0136]

[0149] In this specification, the term “isolated” as used in relation to an agent (e.g., a protein, a nucleic acid molecule, etc.) refers to an agent (e.g., a protein, a nucleic acid molecule, etc.) that is substantially free of other relevant cellular components in its natural state.

[0137]

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

[0138]

[0151] As used herein, the terms “modified nucleotide,” “nucleotide modification,” or “modification” as used in relation to a nucleotide or nucleic acid sequence refer to a nucleotide that includes chemical modifications, such as modified sugar moieties, modified nucleic acid bases, and / or modified internucleoside bonds, or any combination thereof. Exemplary modifications are provided herein; see, for example, §5.5.1.5. In certain embodiments of this disclosure, the inclusion of a deoxynucleotide (recognized as a naturally occurring form of nucleotide) within an RNA oligonucleotide is considered to constitute a modified nucleotide.

[0139]

[0152] As used herein, the term “molecular payload” refers to an agent that functions to modulate a biological outcome. In some embodiments, the molecular payload is operably linked to a targeting agent (e.g., a targeting agent described herein (e.g., 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 e.g., §5.5.1). In some practical embodiments, the molecular payload functions to modulate (e.g., inhibit) the transcription of a DNA molecule, to modulate (e.g., inhibit) the translation of an RNA (e.g., mRNA) molecule, to modulate (e.g., inhibit) the expression of a protein, or to modulate (e.g., inhibit) the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide containing a chain having a complementary region to a target nucleic acid molecule (e.g., an RNA molecule encoded by a target gene (e.g., an mRNA molecule encoded by a target gene)).

[0140]

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

[0141]

[0154] The terms “nucleic acid molecule,” “polynucleotide,” and “oligonucleotide” are used interchangeably herein and refer to polymers of DNA or RNA. Such nucleic acid molecules may be single-stranded or double-stranded and include natural, unnatural, or modified nucleotides, and include natural, unnatural, or modified internucleoside bonds, such as phosphoramidate or phosphorothioate bonds, instead of phosphodiester bonds found between nucleotides in unmodified nucleic acid molecules. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules obtainable by any means available in the art. This includes, but is not limited to, all nucleic acid molecules obtainable by recombinant means (e.g., cloning of nucleic acid molecules from recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reactions, etc.) and synthetic means. Those skilled in the art will understand that, unless otherwise noted, the nucleic acid sequences shown in this application use thymidine (T) as a representative DNA sequence, but when such sequence represents RNA (e.g., mRNA), thymidine (T) is replaced with uracil (U). Therefore, any RNA polynucleotide encoded by DNA identified by a specific sequence identification number may contain a corresponding RNA (e.g., mRNA) sequence encoded by DNA, and each thymidine (T) in the DNA sequence is substituted with uracil (U).

[0142]

[0155] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that extends from the double-stranded region of a double-stranded nucleic acid molecule. For example, 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.

[0143]

[0156] As used herein, the term “operably linked” refers to the linkage of two functionally related agents. For example, polypeptides are operably linked to another polypeptide if they are linked in-frame (directly or indirectly via a peptide linker) so that both polypeptides are functional (e.g., a fusion protein as described herein). Alternatively, for example, a transcriptional regulatory polynucleotide, such as a promoter, enhancer, or other expression regulatory element, is operably linked to a protein-coding polynucleotide if it affects the transcription of the protein-coding polynucleotide. The term “operably linked” also refers, for example, to the conjugation of a first agent (e.g., a protein (e.g., an antibody)) and a second agent (e.g., an oligonucleotide), where the first and second agents can mediate each other’s functions.

[0144]

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

[0145]

[0158] The determination of "percent identity" between two sequences (e.g., protein (amino acid sequence) or oligonucleotide (nucleic acid sequence)) can be achieved using mathematical algorithms. The identity determination (as described herein) does not depend on chemical modifications of nucleotides (e.g., those described herein). For example, in the identity determination, (mC) is identical to (C). A specific and non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, which is modified as found in Karlin S & Altschul SF (1993) PNAS 90:5873-5877 (each of these references is incorporated herein by reference in its entirety). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403. This document is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed by setting the NBLAST nucleotide program parameters to, for example, score=100 and word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed by setting the XBLAST program parameters to, for example, score0 and word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. Gapped BLAST can be used to obtain gapped alignments for comparison. This is described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402). This document is incorporated herein by reference in its entirety. Alternatively, it is possible to perform iterative searches using PSI BLAST to detect distant relationships between molecules (ibid.).When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters for each program (e.g., XBLAST, NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov)). Another specific, non-exclusive example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. This document is incorporated herein by reference in its entirety. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, the PAM120 score matrix (weight residue table), gap length penalty 12, and gap penalty 4 can be used. Percentage identity between two sequences can be determined using methods similar to those described above, with or without allowing gaps. In the calculation of percentage identity, only perfect matches are typically counted.

[0146]

[0159] As used herein, the term “pharmaceutical composition” means a composition suitable for administration to animals, such as human subjects, comprising a therapeutic agent (e.g., a conjugate as described herein) and a pharmaceutically acceptable carrier or diluent. “Pharmaceutically acceptable carrier or diluent” means a substance intended for use in contact with human and / or non-human animal tissues, free from excessive toxicity, irritation, allergic reactions, or other problems or complications, and commensurate with a reasonable therapeutic benefit / risk ratio.

[0147]

[0160] In this specification, the term “multiple” means two or more (for example, three or more, four or more, five or more, six or more, seven or more, nine or more, or ten or more).

[0148]

[0161] In this specification, the terms “protein,” “polypeptide,” and “peptide” refer to polymers consisting 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 the amino acid polymer chain. In the art, it is common to refer to shorter amino acid polymers (e.g., about 2 to 50 amino acids) as peptides and longer amino acid polymers (e.g., more than about 50 amino acids) as polypeptides. However, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably in this specification. In some embodiments, proteins are folded into their three-dimensional structure. Even when polypeptides (e.g., linear (i.e., primary) structures) are assumed in this specification, proteins folded into three-dimensional structures (i.e., tertiary or quaternary structures) 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., proteins not found in nature).

[0149]

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

[0150]

[0163] The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules containing multiple ribonucleotides polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may contain modified nucleotides, as well as natural, unnatural, or modified internucleoside bonds, such as phosphoramidate bonds or phosphorothioate bonds, instead of the phosphodiester bonds found between nucleotides in unmodified nucleic acid molecules.

[0151]

[0164] As used herein, the term “RNAi agent” refers to an agent comprising one or more RNA molecules capable of mediating targeted cleavage of RNA molecules (e.g., mRNA molecules) via the RNA-induced silencing complex (RISC) pathway. This allows RNAi agents to, for example, regulate (e.g., inhibit) the expression of a target gene or protein in cells (e.g., cells in a subject such as a mammalian subject). Examples of RNAi agents include siRNA, miRNA, and shRNA.

[0152]

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

[0153]

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

[0154]

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

[0155]

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

[0156]

[0169] As used herein, the term “sense strand” refers to an RNA molecule (e.g., some RNAi agents and some dsRNA agents described herein) that contains at least partially (e.g., substantially or completely) a region complementary to the antisense strand (as defined herein). Sense strands are often so named because their sequence orientation is the same as that of the target RNA (e.g., mRNA sequence).

[0157]

[0170] As used herein, the terms “single-domain antibody” or “sdAb” refer to an antibody having a single monomeric variable antibody domain. sdAbs can specifically bind to a particular antigen. VHH (as defined herein) is an example of an sdAb.

[0158]

[0171] As used herein, the term “specifically binds” refers to a preferential interaction between a first protein (e.g., an antibody) and a second protein (e.g., an antigen) compared to other amino acid sequences, i.e., a significantly higher binding affinity. Here, when a first protein is said to “specifically bind” to a second protein, it is understood that the first protein specifically binds to an epitope of the second protein. An “epitope” is the portion of the second protein that the first protein specifically recognizes. The term “specifically binds” also includes molecules that are cross-reactive to the same epitope of different species. For example, an antibody that specifically binds to human TFR is considered to specifically bind to human TFR, even if it is cross-reactive to TFR of other species (e.g., cynomolgus monkeys, mice, etc.). A protein can specifically bind to multiple different proteins. Specific binding can be measured, for example, by measuring binding affinity (e.g., standard methods known in the art and described herein, such as surface plasmon resonance (SPR) (e.g., BIAcore®-based assays), which are common methods known in the art (e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patents 5,283,173 and 5,468,614, the entire contents of which are incorporated herein by reference).

[0159]

[0172] As used herein, the term “subject” includes any animal, including humans or other animals. In some embodiments, the subject is a vertebrate (e.g., mammals, birds, fish, reptiles, or amphibians). In some embodiments, the subject is a human. In some embodiments, the subject of the method is a non-human mammal. In some embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), an even-toed ungulate (e.g., cattle, buffalo, sheep, goats, pigs, camels, llamas, alpacas, deer, horses, donkeys), a carnivore (e.g., dogs, cats), a rodent (e.g., rats, mice), or a rabbit (e.g., rabbits). In some embodiments, the subjects are birds belonging to the bird taxonomy order Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).

[0160]

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

[0161]

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

[0162]

[0175] As used herein, the term “therapeutic agent” refers to an agent (e.g., a conjugate described herein) that, when administered in a therapeutically effective dose, can achieve a desired therapeutic outcome in a subject or ex vivo (e.g., can treat a disease as defined herein).

[0163]

[0176] In this specification, the term “therapeutic dose” of a therapeutic agent means any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, can improve a disease state. Here, improvement of a disease state includes, for example, protecting a subject from the onset of a disease (or infection), improving the symptoms of a disease or infection (e.g., reducing the severity of symptoms of a disease or infection, reducing the frequency or duration of symptoms of a disease or infection, or increasing the asymptomatic period of a disease or infection), preventing or mitigating functional or physical impairment caused by a disease or infection, or promoting the regression of a disease (or infection). The ability of a therapeutic agent to improve a disease state can be evaluated using various methods known to those skilled in the art (e.g., in human subjects in clinical trials, in animal model systems to predict efficacy in humans, or by assaying the activity of the agent in in vitro assays).

[0164]

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

[0165]

[0178] As used herein, the terms “transferrin” or “TF” refer to transferrin, a plasma glycoprotein that functions 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 P02787).

[0166]

[0179] As used herein, the term “transferrin receptor” or “TFR” refers to a transmembrane homodimeric glycoprotein that functions in the intracellular uptake of iron from the plasma glycoprotein transferrin. The term TFR also includes multiple isoforms and homologs as appropriate. For example, human TFR (hTFR) includes homologs of hTFR1 and hTFR2. The amino acid sequence of the reference hTFR1 protein is shown in SEQ ID NO: 1 (UniProt Accession P02786|). TFR1 is also commonly known 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 Q9UP52).

[0167]

[0180] In this specification, terms such as “treat,” “treating,” and “treatment” refer to reducing or improving a disease and / or its associated symptoms, or obtaining a desired pharmacological and / or physiological effect. In the treatment of a disease, it is understood that complete elimination of the disease or its associated symptoms is not prevented, but is not necessarily required. In some embodiments, the effect is a therapeutic effect, i.e., the effect is to partially or completely reduce, decrease, eliminate, alleviate, mitigate, reduce the intensity of or cure a disease and / or its associated adverse symptoms. In some embodiments, the effect is a preventive effect, i.e., the effect is to protect or prevent the onset or recurrence of a disease. For this purpose, the methods of this disclosure include, for example, administering a therapeutically effective amount of a conjugate (or a carrier, pharmaceutical composition, etc., containing the same) as described herein.

[0168]

[0181] In this specification, when terms such as "mutation" or "variant" are used in relation to nucleotides or nucleic acid sequences, they refer to nucleic acid molecules that have at least one substitution, addition, deletion, or inversion of one or more nucleotides compared to a reference nucleic acid molecule. Similarly, in this specification, when terms such as "mutation" or "variant" are used in relation to peptides or proteins, they refer to peptides or proteins that have at least one substitution, addition, deletion, or inversion of amino acid residues compared to a reference peptide or protein.

[0169]

[0182] "Mutations that promote heterodimerization of the first and second Fc regions" (or similar expressions) refer to operations performed on the peptide backbone or post-translational modifications of an Fc region to reduce or prevent a polypeptide containing an Fc region from associating with the same polypeptide to form a homodimer. As used herein, "association-promoting modifications" include modifications performed separately on each of two Fc regions that are desirable to associate (i.e., the first and second Fc regions) and that are complementary in order to promote the association of the two Fc regions. For example, association-promoting modifications may alter the structure or charge of one or both Fc regions so that they are sterically or electrostatically more favorable to the association of the two Fc regions. Thus, heterodimerization occurs between a polypeptide containing the first Fc region and a polypeptide containing the second Fc region, but these two polypeptides may be non-identical in that the further components fused to each Fc region (e.g., antigen-binding domains) are not identical. In some embodiments, the association-promoting modification includes amino acid mutations, specifically amino acid substitutions, within the Fc region. In certain embodiments, the association-promoting modification includes distinct amino acid mutations, specifically one or more amino acid substitutions, in the first Fc region and the second Fc region, respectively. See, for example, §5.3.2.2.

[0170]

[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 the amino-terminus of the mature heavy chain (approximately 110–120 or 110–125 amino acids) or the mature light chain (approximately 90–115 amino acids), which vary widely in sequence between antibodies and are used to determine the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while more highly conserved regions within the variable domain are called the framework region (FR). While we do not wish to be constrained by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily involved in the interaction and specificity of antibodies and antigens. In certain embodiments, the variable region is the human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is the primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes rodent or mouse CDR and primate (e.g., non-human primate) framework regions (FR).

[0171]

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

[0172]

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

[0173]

[0186] As used herein, the term "VHH" refers to a type of single-domain antibody (sdAb) having a single monomeric weight-chain variable antibody domain (VH). Such antibodies are found in or can be produced from camelid mammals (e.g., camels, llamas) that naturally lack light chains, or they can be produced synthetically.

[0174]

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

[0175]

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

[0176]

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

[0177]

[0190] As used herein, the terms “ZBTB7A” or “Zinc Finger and BTB Domain-Containing 7A” refer specifically to the transcription factor that functions in the suppression 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 Ref.: NP_056982.1). 5.2 Conjugate

[0178]

[0191] This specification provides, in particular, conjugates (e.g., antibody-oligonucleotide conjugates) that are useful for regulating (e.g., inhibiting, reducing, enhancing) the expression and / or activity of target genes or proteins (e.g., within cells (e.g., erythroid precursor cells), e.g., within cells of a subject, e.g., a mammalian subject (e.g., a human subject) (e.g., via binding to target nucleic acid molecules (e.g., mRNA molecules)).

[0179]

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

[0180]

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

[0181]

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

[0182]

[0195] In some embodiments, when the conjugate binds 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 cell.

[0183]

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

[0184]

[0197] In some embodiments, the conjugate exhibits one or more of the following properties: (a) When it binds to TFRs (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the conjugate is internalized into the erythroid precursor cells; (b) When it binds to TFRs (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells, the conjugate does not induce target cell death; (c) the erythroid precursor cells (d) When bound to TFRs expressed on the surface (e.g., hTFR (e.g., TFR1)), the erythroid precursor cells remain alive; (f) even when internalized into the erythroid precursor cells, the conjugate does not induce the death of the erythroid precursor cells; and / or (e) when bound to TFRs expressed on the surface of erythroid precursor cells (e.g., hTFR (e.g., TFR1)), the conjugate does not induce the degradation of the TFRs (e.g., hTFR (e.g., TFR1)).

[0185]

[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 conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the erythroid precursor cell; (b) 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 conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) does not induce target cell death; (c) When a protein (e.g., an antibody) or conjugate that specifically binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains alive; (d) When internalized by an erythroid precursor cell, a protein (e.g., an antibody) or conjugate that specifically binds to a TFR (e.g., hTFR (e.g., hTFR1)) does not induce the death of the erythroid precursor cell; and / or (e) when a protein (e.g., an antibody) or conjugate that specifically binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, it does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)).

[0186]

[0199] In some embodiments, the conjugates described herein exhibit one or more of the following properties: (a) When bound 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 cell), the conjugate is internalized into the target cell; (b) When bound 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 cell), the conjugate does not induce target cell death; (c) When bound to a target cell (e.g., erythroid precursor cell), the conjugate is internalized into the target cell; (d) When the conjugate binds to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1))) expressed on the surface of a target cell, the target cell remains alive; (e) when internalized by a target cell (e.g., erythroid precursor cell), the conjugate does not induce the death of the target cell; and / or (e) when the conjugate binds 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 cell), the conjugate does not induce the degradation of the target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1))).

[0187] 5.3 Hematopoietic cell targeting agents

[0200] As described above, the conjugates described herein include a targeting agent (e.g., for targeting a molecular payload (e.g., an oligonucleotide described herein) to specific cells (e.g., within the subject). In some embodiments, the targeting agent is a hematopoietic cell (e.g., erythroid precursor cell) targeting agent. In some embodiments, the targeting agent can target a molecular payload (e.g., an oligonucleotide described herein) to hematopoietic cells (e.g., erythroid precursor cells). In some embodiments, the hematopoietic cells (e.g., erythroid precursor cells) are present in the bone marrow (e.g., within the subject). In some embodiments, the targeting agent can target the molecular payload to one or more cells in the bone marrow. In some embodiments, the targeting agent specifically targets erythroid precursor cells (e.g., in the bone marrow (e.g., within the subject)). In some embodiments, the targeting agent specifically targets erythroid precursor cells (e.g., TFRs (e.g., hTFRs (e.g., hTFR1))) expressed on the surface of erythroid precursor cells (e.g., in the bone marrow (e.g., within the subject)).

[0188]

[0201] It should be understood that various targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) may be used in accordance with this disclosure. For example, targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) include (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., one described herein). While exemplary targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) are described in more detail herein, it should be understood that the exemplary targeting agents provided herein (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) are not limiting.

[0189]

[0202] In some embodiments, hematopoietic cell (e.g., erythroid precursor cells) targeting agents specifically bind to antigens (e.g., TFRs (e.g., hTFRs (e.g., hTFR1))) expressed on the surface of hematopoietic cells (e.g., one or more subpopulations thereof) (e.g., erythroid precursor cells (e.g., in bone marrow)). In some embodiments, hematopoietic cell (e.g., erythroid precursor cells) targeting agents specifically bind to antigens (e.g., TFRs (e.g., hTFRs (e.g., hTFR1))) expressed on the surface of erythroid precursor cells. In some embodiments, hematopoietic cell (e.g., erythroid precursor cells) targeting agents specifically bind to antigens (e.g., TFRs (e.g., hTFRs (e.g., hTFR1))) expressed on the surface of erythroid precursor cells in bone marrow.

[0190]

[0203] By interacting with one or more molecules (e.g., proteins) expressed on the surface of target hematopoietic cells (e.g., erythroid precursor cells), both tissue (e.g., bone marrow) localization and selective or preferential uptake into hematopoietic cells (e.g., erythroid precursor cells) can be achieved. In some embodiments, molecules (e.g., proteins) that are substrates of the hematopoietic cell (e.g., erythroid precursor cell) uptake transporter are useful for delivering molecular payloads (e.g., oligonucleotides described herein) to hematopoietic cells (e.g., erythroid precursor cells). Large molecules such as antibodies can be taken up into hematopoietic cells (e.g., erythroid precursor cells) by binding to molecules (e.g., proteins) expressed on the surface of hematopoietic cells (e.g., erythroid precursor cells), followed by endocytosis. For example, as detailed below, a molecular payload (e.g., oligonucleotides described herein) conjugated to transferrin (or a functional fragment or functional variant thereof) or an anti-TFR (e.g., hTFR (e.g., hTFR1)) antibody may be taken up by hematopoietic cells (e.g., erythroid precursor cells) via binding to TFR (e.g., hTFR (e.g., hTFR1)). This TFT may then be endocytized, for example, via endocytosis, such as clathrin-dependent endocytosis.

[0191]

[0204] The use of hematopoietic cell (e.g., erythroid precursor cells) targeting agents may be useful for enriching molecular payloads (e.g., oligonucleotides described herein) in hematopoietic cells (e.g., erythroid precursor cells (e.g., in bone marrow (e.g., within the subject))) while reducing toxicity associated with effects in other cells or tissues. In some embodiments, hematopoietic cell (e.g., erythroid precursor cells) targeting agents enrich bound molecular payloads (e.g., oligonucleotides described herein) in hematopoietic cells (e.g., erythroid precursor cells (e.g., in bone marrow (e.g., within the subject))) compared to other tissues or cell types within the subject. In some embodiments, the hematopoietic cell (e.g., erythroid precursor cells) targeting agent concentrates the binding molecule payload (e.g., oligonucleotides as described herein) in hematopoietic cells (e.g., erythroid precursor cells) in amounts 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 in hematopoietic cells (e.g., non-erythroid precursor cells) compared to the amounts in non-hematopoietic cells (e.g., non-erythroid precursor cells). In some embodiments, the toxicity of a molecular payload (e.g., an oligonucleotide as described herein) in a subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when delivered to the subject bound to a hematopoietic cell (e.g., erythroid precursor cell) targeting agent.

[0192]

[0205] In some embodiments, a hematopoietic cell (e.g., erythroid precursor cell) targeting agent exhibits one or more of the following properties: (a) When it binds 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 cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent is internalized into the target cell; (b) When it binds 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 cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent does not induce target cell death; (c) (d) When the hematopoietic cell (e.g., erythroid precursor cells) targeting agent binds to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFR1))) expressed on the surface of a hematopoietic cell (e.g., erythroid precursor cells), the target cell remains viable; (e.g., when internalized by the target cell (e.g., erythroid precursor cells), the hematopoietic cell (e.g., erythroid precursor cells) targeting agent does not induce the death of the target cell (e.g., erythroid precursor cells).

[0193] 5.3.1 TFR-Targeting Agents

[0206] In some embodiments, the targeting agent (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to transferrin receptors (TFRs) (e.g., hTFRs (e.g., hTFR1)). In some embodiments, the targeting agent (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to hTFRs. In some embodiments, the targeting agent (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to hTFR1.

[0194]

[0207] TFR1 is a transmembrane homodimeric glycoprotein that functions particularly in the intracellular uptake of iron from the plasma glycoprotein transferrin (TF). Iron uptake from transferrin involves the binding of TF to TFRs (such as TFR1), internalization of TF within endocytic vesicles via receptor-mediated endocytosis, and release of iron induced by a decrease in endosome pH. TFR1 is expressed in placental syncytial cells, muscle cells, basal keratinocytes, hepatocytes, endocrine pancreatic cells, spermatocytes, and erythroid precursor cells. TFR expression is known to be high in erythroid precursor cells, but not in mature erythrocytes. TFR2 is known as a homolog of TFR1, but due to its higher affinity and expression pattern, TFR1 is considered the major protein responsible for iron uptake. For example, see 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 Cell Surface Antigen Expression During Hemopoietic Differentiation, Blood, Vol. 60(3), 1982, pp. 703-713, https: / / doi.org / 10.1182 / blood.V60.3.703.703. The entire contents of these references are incorporated herein by reference.

[0195]

[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 specification. TIFF2026524970000004.tif246170

[0196]

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

[0197]

[0210] In some embodiments, the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to hTFR1 and also specifically binds to one or more of the following: mouse TFR1, rat TFR1, and non-human primate TFR1 (e.g., cynomolgus monkey TFR1).

[0198]

[0211] In some embodiments, TFR (e.g., hTFR (e.g., hTFR1)) targeting agents enhance the distribution and / or uptake (e.g., intracellular, e.g., intracellular, e.g., intracellular, e.g., cells expressing TFR (e.g., hTFR (e.g., hTFR1)) (e.g., erythroid precursor cells (e.g., in bone marrow)) of the molecular payload (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)) targeting agents alter (e.g., extend) the lifetime (e.g., in vivo) of the molecular payload (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)) targeting agents provide enhanced affinity to selected targets, e.g., selected cell types, compartments (e.g., cell types, tissues, organs, or body regions) (e.g., compared to oligonucleotides lacking the targeting portion) (e.g., erythroid precursor cells (e.g., in bone marrow)).

[0199]

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

[0200]

[0213] In some embodiments, the TFR targeting agent is a protein. In some embodiments, the targeting agent is a transferrin protein. In some embodiments, the targeting agent is an antibody. In some embodiments, the targeting agent comprises an antibody-like scaffold (e.g., a cysteine ​​high-density peptide, see, for example, International Publication No. 2023023031; the entire contents of this document are incorporated herein by reference).

[0201] 5.3.1.1 TF Proteins

[0214] In some embodiments, the TFR targeting agent comprises a TF (e.g., hTF) (or a functional fragment or functional variant thereof). In some embodiments, the TFR targeting agent comprises the 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 Sequence ID No. 3.

[0202]

[0215] Mutant hTF proteins are known in the art; see, for example, International Publication Nos. 2009019314, 2008152140, European Patent Application No. 2216341, International Publication No. 2009149393, and U.S. Patent No. 8158579. The entire contents of these documents are incorporated herein by reference. In some embodiments, mutant hTF proteins exhibit increased stability and / or a longer plasma half-life (e.g., compared to a reference hTF protein) that do not contain one or more variants. In some embodiments, the mutant hTF protein is located at (e.g., positions V1, P2, D3, K4, T5, H14, Q20, S21, D24, K27, S28, V29, P31, S32, D33, A43, E89, D104, G106, G114, L122, 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, N 268, D277, K278, K280, E281, S287, P288, H289, K291, S298, P307, L326, T330, P335, T336, N413, S415 , D416, D420, K434, S435, A436, S437, D438, D442, N443, G446, N469, N472, G487, K489, D491, S501, G 502, L503, N510, T518, P539, Q540, G543, G544, K545, P547, D548, P549, K552, N553, N555, D558, D565 This includes substitution of non-cysteine ​​amino acid residues with cysteine ​​(in T567, P570, N576, A595, S610, N611, V612, T613, D614, S616, G617, T626, D634, D643, S666, T667, or S669 (according to the numbering in Sequence ID No. 3)) (for example, as described in International Publication No. 2009019314). In some embodiments, the mutant hTF protein includes the addition of a cysteine ​​residue (for example, as described in International Publication No. 2009019314).

[0203] (i) Examples of hTF mutant proteins

[0216] The amino acid sequences of the hTF mutant are shown in Table 2. TIFF2026524970000005.tif248170TIFF2026524970000006.tif253170TIFF2026524970000007.tif137170

[0204]

[0217] In some embodiments, the TFR targeting agent 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 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 Table 2, wherein the amino acid sequence of the TF variant contains at least one amino acid mutation compared to the amino acid sequence of the reference TF protein (e.g., SEQ ID NO: 3). In some embodiments, a TF variant (or its functional fragment or variant) contains or 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 shown in any one of SEQ ID NOs: 4-12, wherein the amino acid sequence of the TF variant contains at least one amino acid mutation compared to the amino acid sequence of the reference TF protein (e.g., SEQ ID NO: 3).

[0205] (ii) parts of different origins

[0218] In some embodiments, a TF (e.g., hTF) is operably linked to a heterogeneous portion (e.g., an Fc region (e.g., an Fc region as described herein (see, e.g., §5.3.2))). In some embodiments, the heterogeneous portion is a half-life extension portion. Examples of 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 heterogeneous polypeptide is a half-life extension polypeptide. Examples of half-life extension polypeptides include, but are not limited to, Ig, fragments of Ig, one or more Ig heavy chain constant regions, fragments of Ig constant regions, Ig Examples include Fc regions, hIg, hIg fragments, one or more hIg heavy chain constant regions, hIg constant region fragments, hIg Fc regions, human serum albumin (HSA), and HSA-binding proteins or peptides. The immunomodulatory proteins or polypeptides described herein, fused or conjugated to half-life extension regions, can be evaluated for their pharmacokinetic properties using standard in vivo methods known in the art.

[0206]

[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., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, Ig is IgG. In some embodiments, IgG is IgG1, IgG2, IgG3, or IgG4.

[0207]

[0220] In some embodiments, the heterologous polypeptide includes or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide includes or comprises a partial IgG4 hinge region, an IgG4 CH2 region, and an IgG CH3 region.

[0208]

[0221] In some embodiments, the heterologous polypeptide includes or consists of an Ig Fc region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region includes or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region includes or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region includes or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG4 hinge region, the IgG4 CH2 region, and the IgG4 CH3 region.

[0209]

[0222] In some embodiments, the heterologous polypeptide includes 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.

[0210]

[0223] In some embodiments, the heterologous polypeptide includes or consists of an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the heterologous polypeptide includes or comprises a partial hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0211]

[0224] In some embodiments, the heterologous polypeptide includes or consists of an hIg Fc region. In some embodiments, the hIg Fc region includes or consists of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region includes or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region includes or consists of at least a portion of the hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region includes or consists of an hIg hinge region, an hIg CH2 region, and an hIg CH3 region. In some embodiments, the hIg Fc region includes or consists of at least a portion of the hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region includes or comprises an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region includes or comprises at least a portion of the hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the hIg Fc region includes or comprises an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0212] 5.3.1.2 TFR-binding peptides and antibody-like scaffolds

[0225] In some embodiments, the TFR targeting agent comprises a peptide that specifically binds 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, U.S. Patent No. 6,743,893 and U.S. Patent No. 8,399,653. The entire contents of each document are incorporated herein by reference.

[0213]

[0226] In some embodiments, the TFR targeting agent 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, International Publication No. 2023023031 (describes an anti-TFR cysteine ​​high-density peptide) and International Publication No. 2021076546 (describes a TFR-binding fibronectin type III domain). The entire contents of each document are incorporated herein by reference.

[0214] (i) Examples of TFR-binding peptides and antibody-like scaffolds

[0227] Table 3 shows the amino acid sequences of exemplary TFR-specific antibody-like scaffolds. TIFF2026524970000008.tif247170TIFF2026524970000009.tif198170

[0215]

[0228] In some embodiments, the TFR targeting agent comprises a TFR-specific peptide or antibody-like scaffold (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 shown in Table 3. In some embodiments, the TFR-specific peptide (or its functional fragment or variant) contains, or 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 shown in any one of sequences 13-41. In some embodiments, the TFR targeting agent comprises an antibody-like scaffold (or its functional fragment or variant). In some embodiments, the antibody-like scaffold (or its functional fragment or variant) contains, or 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 antibody-like scaffold shown in Table 3. In some embodiments, the antibody-like scaffold (or its functional fragment or variant) contains 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 NOs.

[0216] (ii) parts of different origins

[0229] In some embodiments, the TFR-binding peptide or antibody-like scaffold is operably linked to a heterologous portion (e.g., an Fc region (e.g., an Fc region as described herein (see, e.g., §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 extension polypeptides include, but are not limited to, Ig, fragments of Ig, one or more Ig heavy chain constant regions, fragments of Ig constant regions, Ig Examples include Fc regions, hIg, hIg fragments, one or more hIg heavy chain constant regions, hIg constant region fragments, hIg Fc regions, human serum albumin (HSA), and HSA-binding proteins or peptides. The immunomodulatory proteins or polypeptides described herein, fused or conjugated to half-life extension regions, can be evaluated for their pharmacokinetic properties using standard in vivo methods known in the art.

[0217]

[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., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, Ig is IgG. In some embodiments, IgG is IgG1, IgG2, IgG3, or IgG4.

[0218]

[0231] In some embodiments, the heterologous polypeptide includes or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide includes or comprises a partial IgG4 hinge region, an IgG4 CH2 region, and an IgG CH3 region.

[0219]

[0232] In some embodiments, the heterologous polypeptide includes or consists of an Ig Fc region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region includes or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region includes or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region includes or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG4 hinge region, the IgG4 CH2 region, and the IgG4 CH3 region.

[0220]

[0233] In some embodiments, the heterologous polypeptide includes 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.

[0221]

[0234] In some embodiments, the heterologous polypeptide includes or consists of an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of a partial hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide includes or consists of an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the heterologous polypeptide includes or comprises a partial hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0222]

[0235] In some embodiments, the heterologous polypeptide includes or consists of an hIg Fc region. In some embodiments, the hIg Fc region includes or consists of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region includes or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region includes or consists of at least a portion of the hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the hIg Fc region includes or consists of an hIg hinge region, an hIg CH2 region, and an hIg CH3 region. In some embodiments, the hIg Fc region includes or consists of at least a portion of the hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region includes or comprises an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the hIg Fc region includes or comprises at least a portion of the hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the hIg Fc region includes or comprises an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0223] 5.3.1.3 Anti-TFR (e.g. anti-TFR1) antibodies

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

[0224]

[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.

[0225] (i) Examples of anti-TFR (e.g., anti-TFR1) antibodies

[0238] In some embodiments, the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) comprises an anti-TFR1 antibody. Anti-hTFR1 antibodies that can be used in the conjugates described herein are known in the art.

[0226]

[0239] Exemplary anti-TFR1 antibodies known in the industry that can be used in the conjugates described herein include, but are not limited to, OKT9 (see, e.g., U.S. Patent No. 4,364,934); M11, M23, M27, B84 (see, e.g., International Publication No. 2015 / 098989 and U.S. Patent No. 9,994,641); 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4 (see, e.g., International Publication No. 2016 / 081643 and U.S. Patent No. 9,708,406); 8D3 (see, e.g., U.S. Patent Application Publication No. 2010 / 0077498 and Lee et al. "Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain"). See "Barrier in Mouse" 2000, J Pharmacol. Exp. Ther., 292:1048-1052); OX26 (e.g., 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 (e.g., 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)); and also the following commercially available clones (e.g., Novus Biologicals)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, e.g., U.S. Patent Application Publication No. 2011 / 0311544 and U.S. Patent No. 7572895); B3 / 25 and T58 / 30 (see, e.g., Trowbridge, I. Set al. "Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumor cells." Nature, 1981, Vol. 294, pp. 171-173); and the following commercially available clones (e.g., BioXcell): R17 217.1.3, 5E9C11, OKT9 (BE0023 clone), BK19.9, B3 / 25, T56 / 14 and T58 / 1 (see, e.g., Gatter, K.C. et al. "Transferrin receptors in human tissues: their distribution and possible clinical relevance." J Clin). See Pathol. 1983 May;36(5):539-45); 5E9C11;R17 217.1.3 (available from BioXcell), BE0175 (available from BioXcell). The entire contents of these documents are incorporated herein by reference.

[0227]

[0240] Exemplary anti-TFR (e.g., hTFR (e.g., hTFR1)) antibodies that can be used in the conjugates described herein are described, for example, in the following publications: International Publication No. 2023283531; International Publication No. 2021154477; International Publication No. 2020132584; International Publication No. 2021154476; International Publication No. 2021150382; International Publication No. 2023023031; International Publication No. 2021146256; International Publication No. 2021142275; U.S. Patent Application Publication No. 20220017635; International Publication No. 2016207 No. 240; US Patent No. 11267896; US Patent Application Publication No. 20220143206, US Patent No. 11028179; US Patent No. 11286305; International Publication No. 2023087017; International Publication No. 2023086864; International Publication No. 2023044398; International Publication No. 2023039611; International Publication No. 2023034409; International Publication No. 2023283623; International Publication No. 2023283624; International Publication No. 2023283619; International Publication No. 2023283620; International Publication No. 2023283615; International Publication Patent No. 2023283613; International Publication No. 2023283614; Portuguese Patent No. 11672872; Portuguese Patent No. 11648318; International Publication No. 2022271549; International Publication No. 2022201122; International Publication No. 2022174114; International Publication No. 2022026152; International Publication No. 2022020107; International Publication No. 2022020106; International Publication No. 2022020105; International Publication No. 2022020108; International Publication No. 2022020109; International Publication No. 2021205358; US Patent Application Publication No. 202301 74646; U.S. Patent Application Publication No. 20210299266; International Publication No. 2021195469; U.S. Patent No. 11446387; U.S. Patent Application Publication No. 20220409735; U.S. Patent Application Publication No. 20210301290; U.S. Patent Application Publication No. 20210369762; U.S. Patent No. 11525137; U.S. Patent No. 11555190; U.S. Patent No. 11111308; U.S. Patent No. 10550188; U.S. Patent No. 10508151; U.S. Patent Application Publication No. 20160208008; U.S. Patent Application Publication No. 20150291697;U.S. Patent No. 20130171061; U.S. Patent No. 9562230; U.S. Patent No. 7976841; U.S. Patent No. 4364934; International Publication No. 2015098989; U.S. Patent No. 9994641; International Publication No. 2016081643; U.S. Patent No. 9708406; U.S. Patent Publication No. 2010077498; U.S. Patent Publication No. 20110311544; U.S. Patent Publication No. 7572895; International Publication No. 2019075417; U.S. Patent Publication No. 20060286030; U.S. Patent Publication No. 20190240346; U.S. Patent Publication No. 20130 No. 216476; International Publication No. 2023283531; U.S. Patent Publication No. 20130177579; U.S. Patent No. 9598496; U.S. Patent No. U.S. Patent Publication No. 20130045206; U.S. Patent Publication No. 20060039908; U.S. Patent No. 6015555; U.S. Patent No. 60083 No. 26; US Patent No. 5648469; European Patent No. 79696; International Publication No. 2023034409; US Patent No. 4364934; US Patent No. 8409573; US Patent No. 9708406; US Patent No. 9611323; International Publication No. 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, pages 8516-8522; Lee et al. "Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse" 2000, J Pharmacol. Exp. Ther., 292: 1048-1052; Lee et al. "Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse" 2000, J Pharmacol. Exp. Ther., 292: 1048-1052;Haobam,B.et al.2014.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, ISet al. "Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumor cells." Nature, 1981, vol. 294, pp. 171-173; Gatter, KCet al. "Transferrin receptors in human tissues: their distribution and possible clinical relevance." J Clin Pathol. 1983 May; 36(5):539-4. The entire contents of each of these documents are incorporated herein by reference.

[0228]

[0241] Table 4 shows 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 expressed according to Kabat notation. Those skilled in the art can determine the CDRs defined by other schemes (e.g., Chothia, IMGT) using methods commonly known to those skilled in the art. TIFF2026524970000010.tif239170TIFF2026524970000011.tif252170TIFF2026524970000012.tif25317 0TIFF2026524970000013.tif253170TIFF2026524970000014.tif253170TIFF2026524970000015.tif76170

[0229]

[0242] Table 26 shows the amino acid sequences of additional exemplary anti-hTFR1 antibodies that can be used in the conjugates described herein. The CDRs of the anti-hTFR antibodies in Table 26 are expressed according to Kabat notation. Those skilled in the art can determine the CDRs defined by other schemes (e.g., Chothia, IMGT) using methods commonly known to those skilled in the art. TIFF2026524970000016.tif251170TIFF2026524970000017.tif250170TIFF2026524970000018.tif152170

[0230]

[0243] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFR1) antibody includes the anti-TFR (e.g., hTFR, e.g., hTFR1) antibody specified herein and / or incorporated herein by reference.

[0231]

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

[0232]

[0245] In some embodiments, the amino acid sequence of VH CDR1 includes, or consists of, the amino acid sequence of VH CDR1 of VH of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 includes, or consists of, the amino acid sequence of VH CDR2 of VH of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 includes, or consists of, the amino acid sequence of VH CDR3 of VH of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR3 of VH CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0233]

[0246] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 of the anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of the anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 of the anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of the anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); VH The amino acid sequence of CDR3 is the amino acid sequence of VH CDR3 of VH anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR3 of VH anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of an amino acid sequence containing one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0234]

[0247] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 includes, or consists of, the amino acid sequence of VH CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0235]

[0248] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference The amino acid sequence of CDR3, or the amino acid sequence of VH CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, comprising or consisting of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0236]

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

[0237]

[0250] In some embodiments, the amino acid sequence of VL CDR1 includes, or consists of, the amino acid sequence of VL CDR1 of VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR1 of VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 includes, or consists of, the amino acid sequence of VL CDR2 of VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR2 of VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 is the amino acid sequence of VL CDR3 of the anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR3 of the anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two or three amino acid mutations (e.g., substitutions, deletions, additions, etc.).

[0238]

[0251] In some embodiments, the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR1 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, comprising or consisting of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR2 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, comprising or consisting of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); VL The amino acid sequence of CDR3 is the amino acid sequence of VL CDR3 of the VL anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR3 of the VL anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0239]

[0252] In some embodiments, the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 includes, or consists of, the amino acid sequence of VL CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0240]

[0253] In some embodiments, the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3 is the amino acid sequence of VL CDR3 of an anti-TFR antibody specified herein 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 specified herein and / or incorporated herein by reference, comprising or consisting of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0241]

[0254] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFR1) comprises VH including VH CDR1, VH CDR2, and VH CDR3, and VL including VL CDR1, VL CDR2, and VL CDR3.

[0242]

[0255] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 of the anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of the anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 of the anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of the anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); VH The amino acid sequence of CDR3 is the amino acid sequence of VH CDR3 of VH anti-TFR antibodies specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR3 of VH anti-TFR antibodies specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 of VL anti-TFR antibodies specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR1 of VL anti-TFR antibodies specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.);The amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR2 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, which comprises or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3 is the amino acid sequence of VL CDR3 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR3 of the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference, which comprises or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).;

[0243]

[0256] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference The amino acid sequence of CDR3, or the amino acid sequence of VH CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR1, or the amino acid sequence of VL CDR1 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which includes or consists of one, two, or three amino acid mutations (e.g., substitutions, deletions, additions, etc.);The amino acid sequence of VL CDR2 includes, or consists of, the amino acid sequence of VL CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR2 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3 includes, or consists of, the amino acid sequence of VL CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, or the amino acid sequence of VL CDR3 of an anti-TFR antibody specified herein and / or incorporated herein by reference, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.);

[0244]

[0257] In some embodiments, the amino acid sequence of the 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 the VH of an anti-TFR antibody specified herein and / or incorporated herein by reference. In some embodiments, the amino acid sequence of the 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 the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference. In some embodiments, the amino acid sequence of the 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 the VH of an anti-TFR antibody specified herein and / or incorporated herein by reference; and the amino acid sequence of the 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 the VL of an anti-TFR antibody specified herein and / or incorporated herein by reference.

[0245]

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

[0246]

[0259] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFR1) comprises a VH that comprises VH CDR1, VH CDR2, and VH CDR3.

[0247]

[0260] In some embodiments, the amino acid sequence of VH CDR1 includes, or consists of, the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 includes, or consists of, the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 includes, or consists of, the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0248]

[0261] In some embodiments, the amino acid sequence of VH CDR1 includes, or consists of, the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 includes, or consists of, the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 includes, or consists of, the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0249]

[0262] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0250]

[0263] In some embodiments, the amino acid sequence of VH CDR1 includes, or consists of, the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 includes, or consists of, the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 includes, or consists of, the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0251]

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

[0252]

[0265] In some embodiments, the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 shown in Table 4, or the amino acid sequence of VL CDR1 shown in Table 4, which includes or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown in Table 4, which includes or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 is the amino acid sequence of VL CDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown in Table 4, which includes or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0253]

[0266] In some embodiments, the amino acid sequence of VL CDR1 includes, or consists of, the amino acid sequence of VL CDR1 shown in Table 4, or the amino acid sequence of VL CDR1 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 includes, or consists of, the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 includes, or consists of, the amino acid sequence of VL CDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown in Table 4, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0254]

[0267] In some embodiments, the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 shown in Table 4, or the amino acid sequence of VL CDR1 shown in Table 4 which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown in Table 4 which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 is the amino acid sequence of VL CDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown in Table 4 which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0255]

[0268] In some embodiments, the amino acid sequence of VL CDR1 includes, or consists of, the amino acid sequence of VL CDR1 shown in Table 4, or the amino acid sequence of VL CDR1 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 includes, or consists of, the amino acid sequence of VL CDR2 shown in Table 4, or the amino acid sequence of VL CDR2 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 includes, or consists of, the amino acid sequence of VL CDR3 shown in Table 4, or the amino acid sequence of VL CDR3 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0256]

[0269] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFR1) comprises VH including VH CDR1, VH CDR2, and VH CDR3, and VL including VL CDR1, VL CDR2, and VL CDR3.

[0257]

[0270] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 is 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, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2, or the amino acid sequence of VL CDR2 shown in Table 4, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3, or the amino acid sequence of VL CDR3 shown in Table 4, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0258]

[0271] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 shown in Table 4, or the VL CDR1 shown in Table 4 The amino acid sequence of CDR1 includes, or consists of, an amino acid sequence containing, or comprising, one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 includes, or consists of, the amino acid sequence of VL CDR2 shown in Table 4, or an amino acid sequence of VL CDR2 shown in Table 4 that contains, or consists of, one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3 includes, or consists of, the amino acid sequence of VL CDR3 shown in Table 4, or an amino acid sequence of VL CDR3 shown in Table 4 that contains, or consists of, one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0259]

[0272] In some embodiments, the amino acid sequence of VH contains 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 contains 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 contains 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; the amino acid sequence of VL contains 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.

[0260]

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

[0261]

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

[0262]

[0275] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown in Table 26, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown in Table 26, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26, which includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0263]

[0276] In some embodiments, the amino acid sequence of VH CDR1 includes, or consists of, the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown in Table 26, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 includes, or consists of, the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown in Table 26, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 includes, or consists of, the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0264]

[0277] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown in Table 26, which contains or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown in Table 26, which contains or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26, which contains or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0265]

[0278] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown in Table 26, which contains or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown in Table 26, which contains or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26, which contains or consists of an amino acid sequence containing 1, 2 or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0266]

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

[0267]

[0280] In some embodiments, the amino acid sequence of VL CDR1 includes, or consists of, the amino acid sequence of VL CDR1 shown in Table 26, or the amino acid sequence of VL CDR1 shown in Table 26, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 includes, 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, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 includes, 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, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0268]

[0281] In some embodiments, the amino acid sequence of VL CDR1 includes, or consists of, the amino acid sequence of VL CDR1 shown in Table 26, or the amino acid sequence of VL CDR1 shown in Table 26, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR2 includes, 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, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 includes, 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, which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitutions, deletions, additions, etc.).

[0269]

[0282] In some embodiments, the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 shown in Table 26, or the amino acid sequence of VL CDR1 shown in Table 26 that includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 is the amino acid sequence of VL CDR2 shown in Table 26, or the amino acid sequence of VL CDR2 shown in Table 26 that includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 is the amino acid sequence of VL CDR3 shown in Table 26, or the amino acid sequence of VL CDR3 shown in Table 26 that includes or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0270]

[0283] In some embodiments, the amino acid sequence of VL CDR1 includes, or consists of, the amino acid sequence of VL CDR1 shown in Table 26, or the amino acid sequence of VL CDR1 shown in Table 26 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 includes, 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 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 includes, 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 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0271]

[0284] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFR1) comprises VH including VH CDR1, VH CDR2, and VH CDR3, and VL including VL CDR1, VL CDR2, and VL CDR3.

[0272]

[0285] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown in Table 26, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown in Table 26, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); VL The amino acid sequence of CDR1 is the amino acid sequence of VL VL CDR1 shown in Table 26, or the amino acid sequence of VL VL CDR1 shown in Table 26, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 is the amino acid sequence of VL VL CDR2 shown in Table 26, or the amino acid sequence of VL VL CDR2 shown in Table 26, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3 is the amino acid sequence of VL VL CDR3 shown in Table 26, or the amino acid sequence of VL VL CDR3 shown in Table 26, which contains or consists of one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0273]

[0286] In some embodiments, the amino acid sequence of VH CDR1 is the amino acid sequence of VH CDR1 shown in Table 26, or the amino acid sequence of VH CDR1 shown in Table 26 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 is the amino acid sequence of VH CDR2 shown in Table 26, or the amino acid sequence of VH CDR2 shown in Table 26 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 is the amino acid sequence of VH CDR3 shown in Table 26, or the amino acid sequence of VH CDR3 shown in Table 26 which contains or consists of 1, 2, or 3 amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 is the amino acid sequence of VL CDR1 shown in Table 26, or the VL CDR1 shown in Table 26 The amino acid sequence of CDR1 includes, or consists of, an amino acid sequence containing, or comprising, one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 includes, or consists of, the amino acid sequence of VL CDR2 shown in Table 26, or an amino acid sequence of VL CDR2 shown in Table 26 that contains, or consists of, one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR3 includes, or consists of, the amino acid sequence of VL CDR3 shown in Table 26, or an amino acid sequence of VL CDR3 shown in Table 26 that contains, or consists of, one, two, or three amino acid mutations (e.g., substitution, deletion, addition, etc.).

[0274]

[0287] In some embodiments, the amino acid sequence of VH contains 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 contains 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 contains 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; the amino acid sequence of VL contains 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.

[0275] 5.3.2 Ig Steady-State Regions

[0288] In some embodiments, the antibody (or heterologous polypeptide (e.g., operably linked to a protein that specifically binds to TFRs (e.g., hTF) (see, e.g., §5.3.1.3))) includes an IgG CH2 region and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes a partial IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes a partial IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0276]

[0289] In some embodiments, the antibody (or heterologous polypeptide) includes an Ig Fc region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region includes or consists of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region includes or consists of the IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region includes or consists of the IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region includes or consists of at least a portion of the IgG4 hinge region, the IgG4 CH2 region, and the IgG4 CH3 region.

[0277]

[0290] In some embodiments, the antibody (or heterologous polypeptide) comprises a first Ig Fc region and a second Ig Fc region. In some embodiments, the first and / or second Ig Fc regions comprise or consist of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second Ig Fc regions comprise or consist of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second Ig Fc regions comprise or consist of at least a portion of the IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the first and / or second Ig Fc regions comprise or consist of at least a portion of the IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the first and / or second Ig Fc regions comprise at least a portion of the IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the first and / or second Ig Fc region includes or comprises an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the first and / or second Ig Fc region includes or comprises at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the first and / or second Ig Fc region includes or comprises an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0278]

[0291] In some embodiments, the antibody (or heterologous polypeptide) 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.

[0279]

[0292] In some embodiments, the antibody (or heterologous polypeptide) includes an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes a partial hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes a partial hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) includes an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0280]

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

[0281]

[0294] In some embodiments, the antibody (or heterologous polypeptide) comprises a first hIg Fc region and a second hIg Fc region. In some embodiments, the first and / or second hIg Fc regions comprise or consist of at least a portion of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second hIg Fc regions comprise or consist of the hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and / or second hIg Fc regions comprise or consist of at least a portion of the hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the first and / or second hIg Fc regions comprise or consist of the hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the first and / or second hIg Fc region includes or comprises at least a portion of the hIgG1 hinge region, the hIgG1 CH2 region, and the hIgG1 CH3 region. In some embodiments, the first and / or second hIg Fc region includes or comprises the hIgG1 hinge region, the hIgG1 CH2 region, and the hIgG1 CH3 region. In some embodiments, the first and / or second hIg Fc region includes or comprises at least a portion of the hIgG4 hinge region, the hIgG4 CH2 region, and the hIgG4 CH3 region. In some embodiments, the first and / or second hIg Fc region includes or comprises the hIgG4 hinge region, the hIgG4 CH2 region, and the hIgG4 CH3 region.

[0282]

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

[0283]

[0296] Table 5 shows the amino acid sequences of the heavy chain constant region and light chain constant region of exemplary reference hIgG1 and hIgG4 that can be incorporated into one or more embodiments described herein (e.g., anti-TFR (e.g., hTFR (e.g., hTFR1)) antibodies and heterologous polypeptides). TIFF2026524970000019.tif250170TIFF2026524970000020.tif251170TIFF2026524970000021.tif196170

[0284]

[0297] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more hIg constant regions, and the amino acid sequences of one or more hIg constant regions contain or consist of 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 amino acid sequences of the polypeptides shown in Table 5. In some embodiments, the amino acid sequences of one or more hIg constant regions contain or consist of the amino acid sequences shown in Table 5.

[0285]

[0298] In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of the amino acid sequences shown in Table 5, further including one or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions) that make up less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of amino acid sequences that include or consist of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions) as shown in Table 5. In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of amino acid sequences that include or consist of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions) as shown in Table 5. In some embodiments, the amino acid sequences of one or more hIg constant regions include, or consist of, amino acid sequences containing or consisting of approximately 1 or fewer, approximately 2 or fewer, approximately 3 or fewer, approximately 4 or fewer, approximately 5 or fewer, approximately 6 or fewer, approximately 7 or fewer, approximately 8 or fewer, approximately 9 or fewer, approximately 10 or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions), as shown in Table 5.

[0286]

[0299] In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of the amino acid sequences shown in Table 5, further including one or more amino acid substitutions that make up less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of amino acid sequences that include or consist of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, as shown in Table 5. In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of amino acid sequences that include or consist of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, as shown in Table 5. In some embodiments, the amino acid sequence of one or more hIg constant regions includes or consists of an amino acid sequence containing or consisting of approximately 1 or fewer, approximately 2 or fewer, approximately 3 or fewer, approximately 4 or fewer, approximately 5 or fewer, approximately 6 or fewer, approximately 7 or fewer, approximately 8 or fewer, approximately 9 or fewer, approximately 10 or more amino acid substitutions, as shown in Table 5.

[0287]

[0300] In some embodiments, the amino acid sequences of one or more hIg constant regions include, or consist 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 any one of the amino acid sequences of SEQ ID NOs.

[0288]

[0301] In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of one of the amino acid sequences of SEQ ID NOs: 170-198, further including one or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions) that make up less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of one of the amino acid sequences of SEQ ID NOs: 170-198, which includes or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of one of the amino acid sequences of SEQ ID NOs: 171-198, which includes or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions is one of the amino acid sequences of SEQ ID NOs. 171 to 198, which includes or consists of an amino acid sequence containing or comprising about 1 or less, about 2 or less, about 3 or less, about 4 or less, about 5 or less, about 6 or less, about 7 or less, about 8 or less, about 9 or less, or 10 or less amino acid mutations (e.g., amino acid substitutions, deletions, or additions).

[0289]

[0302] In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of one of the amino acid sequences of SEQ ID NOs: 170-198, further including one or more amino substitutions in less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequences of one or more hIg constant regions include or consist of one of the amino acid sequences of SEQ ID NOs: 170-198, which includes 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 sequences of one or more hIg constant regions include or consist of one of the amino acid sequences of SEQ ID NOs: 171-198, which includes 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 one or more hIg constant regions is one of the amino acid sequences of SEQ ID NOs. 171 to 198, which includes or consists of an amino acid sequence containing or consisting of about 1 or less, about 2 or less, about 3 or less, about 4 or less, about 5 or less, about 6 or less, about 7 or less, about 8 or less, about 9 or less, or 10 or less amino acid substitutions.

[0290] 5.3.2.1 Ig Effector Function

[0303] As described herein, in some embodiments, the antibody (or heterologous polypeptide) includes an Fc region (see, for example, §5.3.2). In some embodiments, the Fc region of the antibody (or heterologous polypeptide) described herein exhibits reduced Fc effector function in one or more ways compared to a reference (e.g., wild-type) Fc region. Exemplary Fc effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), and binding affinity to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγRIIa, and / or FcγIIIa))).

[0291]

[0304] Standard in vitro and / or in vivo assays known in the art can be performed to evaluate Fc effector function, including one or more of the following: ADCC, CDC, ADCP, Fc receptor (e.g., Fcγ receptor) binding affinity, and C1q binding affinity.

[0292]

[0305] For example, ADCC activity can be evaluated using standard (radioactive and non-radioactive) methods known in the art (see, for example, International Publication Nos. 2006 / 082515 and 2012 / 130831). The entire contents of these publications are incorporated herein by reference. For example, ADCC activity is chromium-5 ( 51 It can be evaluated using a Cr assay. In short, 51 Cr is preloaded onto target cells, NK cells are added to the culture, and the radioactivity of the cell culture supernatant is evaluated (showing lysation of target cells by NK cells). Similar non-radioactive assay methods are also available, but target cells are 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 vol. 4, 3(2012):310-8. Doi:10.4161 / mabs.19873. The entire contents of this document are incorporated herein by reference. An example of a commercially available non-radioactive assay is, for example, ACTI for flow cytometry. TMThis includes non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, Calif.) and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, Wis.). Additional 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 U.S. Patent Nos. 5500362 and 5821337; Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063; Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA 82 (1985) 1499-1502; and Bruggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361. The entire contents of each of these documents are incorporated herein by reference. Alternatively, the ADCC activity of the fusion proteins described herein can be evaluated in vivo in animal models, such as those disclosed in Clynes, et al., Proc. Nat'l Acad. Sci. USA 95(1998) 652-6566. The entire contents of this document are incorporated herein by reference.

[0293]

[0306] C1q binding assays can be used to evaluate the ability of the antibodies (or heterologous polypeptides) described herein to bind to C1q (or to bind with lower affinity than the reference fusion protein), thereby lacking (or reducing) CDC activity. Binding of the antibodies (or heterologous polypeptides) described herein to C1q can be determined by various in vitro assays known in the art for determining Fc-C1q interactions (e.g., biochemical or immunological assays), including, for example, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling of one or more components of the object being tested, and / or may employ a variety of detection methods, including but not limited to chromogenic labeling, fluorescent labeling, luminescence labeling, or isotopic labeling. For detailed descriptions of binding affinity and kinetics, see, for example, Paul, WE, ed., Fundamental Immunology, 4. th This can be found in Ed., Lippincott-Raven, Philadelphia (1999). The entire contents of this document are incorporated herein by reference. For example, see the C1q and C3c binding ELISAs described in International Publication Nos. 2006 / 029879 and 2005 / 100402. The entire contents of each of these documents are incorporated herein by reference. Additional CDC activity assays include, for example, the assays described in 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 entire contents of each of these documents are incorporated herein by reference.

[0294]

[0307] ADCP activity can also be measured by in vitro or in vivo methods known in the art, as well as by 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 entire contents of this document are incorporated herein by reference). For example, a primary cell-based ADCP assay can be used, in which fresh human peripheral blood mononuclear cells (PBMCs) are isolated, monocytes are separated, and then differentiated into macrophages in culture using standard methods. The macrophages are fluorescently labeled and added to a culture containing fluorescently labeled target cells. Phagocytosis events can be analyzed by FACS screening and / or microscopic observation. Furthermore, a modified version of the above assay (reporter version) can be used, employing a modified cell line that stably expresses FcγRIIa(CD32a) (e.g., a modified T cell line, e.g., THP-1) as an effector cell line, thereby eliminating the need for primary cells. Exemplary ADCPs 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, E Get al. Determining the phagocytic activity of clinical antibody samples. J.Vis.Exp.3588(2011).Doi:10.3791 / 3588 (each of these references is incorporated herein by reference).

[0295]

[0308] The binding of antibodies (or heterologous polypeptides) described herein to Fc receptors can be determined by various in vitro assays known in the art (e.g., biochemical or immunological assays) to determine 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 may utilize labeling of one or more components of the substance being tested and / or employ various detection methods including, but not limited to, chromogenic labeling, fluorescent labeling, luminescence labeling, or isotopic labeling. For detailed descriptions of binding affinity and kinetics, see, for example, Paul, WE, ed., Fundamental Immunology, 4 th This can be found in Ed., Lippincott-Raven, Philadelphia (1999). The entire contents of this document are incorporated herein by reference.

[0296]

[0309] In some embodiments, the Fc region of the antibody (or heterologous polypeptide) described herein is altered (e.g., including one or more amino acid mutations (e.g., one or more amino acid substitutions, deletions, additions, etc.)) compared to the amino acid sequence of a reference Fc region (e.g., wild-type Fc region, e.g., Table 5 herein (e.g., SEQ ID NOs. 178, 180, 191, or 195)) (hereinafter referred to as the "altered Fc region"). In some embodiments, one or more amino acid mutations (e.g., one or more amino acid substitutions, deletions, additions, etc.) reduce or eliminate one or more Fc effector functions compared to a reference Fc that does not contain the mutations (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, additions, etc.)).

[0297]

[0310] In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region is undetectable or shows reduced ADCC compared to a reference antibody (or heterologous polypeptide) that does not contain the Fc region mutation (e.g., one or more amino acid mutations (e.g., substitution, deletion, or addition of one or more amino acids)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region is undetectable or shows reduced CDC compared to a reference antibody (or heterologous polypeptide) that does not contain the Fc region mutation (e.g., one or more amino acid mutations (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region is undetectable or shows reduced ADCP compared to a reference antibody (or heterologous polypeptide) that does not contain the Fc region mutation (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region exhibits reduced 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))) or is undetectable compared to a reference antibody (or heterologous polypeptide) that does not contain a mutation in the Fc region (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region exhibits reduced specific binding affinity to FcγRI, FcγIIa, and / or FcγIIIa, and is undetectable compared to an antibody (or heterologous polypeptide) that does not contain a mutation in the Fc region (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)).In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region exhibits reduced specific binding affinity to FcγRI or is undetectable compared to a reference antibody (or heterologous polypeptide) that does not contain an Fc mutation (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region exhibits reduced specific binding affinity to FcγIIa or is undetectable compared to a reference antibody (or heterologous polypeptide) that does not contain an Fc region mutation (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region exhibits reduced specific binding affinity to FcγIIIa or is undetectable compared to an antibody (or heterologous polypeptide) that does not contain an Fc region mutation (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, an antibody (or heterologous polypeptide) containing a modified Fc region exhibits reduced specific binding affinity to C1q or is undetectable compared to a reference antibody (or heterologous polypeptide) that does not contain the Fc region mutation (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)).

[0298]

[0311] In some embodiments, antibodies (or heterologous polypeptides) containing an Fc region are undetectable for ADCC. In some embodiments, antibodies (or heterologous polypeptides) containing an Fc region are undetectable for CDC. In some embodiments, antibodies (or heterologous polypeptides) containing an Fc region are undetectable for ADCP. In some embodiments, antibodies (or heterologous polypeptides) containing an Fc region have undetectable 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 heterologous polypeptides) containing an Fc region have undetectable specific binding affinity to FcγRI, FcγIIa, and / or. In some embodiments, an antibody (or heterologous polypeptide) containing Fc has an undetectable specific binding affinity to FcγRI. In some embodiments, an antibody (or heterologous polypeptide) containing Fc has an undetectable specific binding affinity to FcγIIa. In some embodiments, an antibody (or heterologous polypeptide) containing the Fc region has an undetectable specific binding affinity to FcγIIIa. In some embodiments, an antibody (or heterologous polypeptide) containing the Fc region has an undetectable specific binding affinity to C1q.

[0299]

[0312] Amino acid substitutions that reduce or eliminate one or more Fc effector functions are known in the art. For example, see 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 this document are incorporated herein by reference. More specifically, see, for example, Table 4 in Saunders.

[0300]

[0313] In some embodiments, the altered Fc includes an hIgG1 Fc region containing one or more amino acid mutations (e.g., one or more amino acid substitutions). In some embodiments, the hIgG1 Fc region includes amino acid substitutions at the amino acid positions L234, L235 and / or P329 (Kabat EU numbering). In some embodiments, the hIgG1 Fc region includes amino acid substitutions L234A and / or L235A (Kabat EU numbering). In some embodiments, the hIgG1 Fc region includes amino acid substitutions L234A, L235A, and P329G (Kabat EU numbering). In some embodiments, the hIgG1 Fc region includes amino acid substitutions L234A, L235A, and P329A (Kabat EU numbering).

[0301]

[0314] In some embodiments, the altered Fc region includes an hIgG4 Fc region containing one or more amino acid mutations (e.g., one or more amino acid substitutions). In some embodiments, the hIgG4 Fc region includes amino acid substitutions at the amino acid positions S228, F234, and / or L235 (Kabat EU numbering). In some embodiments, the hIgG4 Fc region includes amino acid substitutions S228P, F234A, and / or L235A (Kabat EU numbering). In some embodiments, the hIgG4 Fc region includes amino acid substitutions S228P, F234A, and / or L235E (Kabat EU numbering). In some embodiments, the hIgG4 Fc includes amino acid substitutions S228P and / or L235E (Kabat EU numbering).

[0302]

[0315] Table 6 shows examples of altered Fc region amino acid sequences that are known in the art to exhibit another (one more) reduction in effector function. TIFF2026524970000022.tif245170TIFF2026524970000023.tif249170TIFF2026524970000024.tif249170TIFF2026524970000025.tif41170

[0303]

[0316] In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing 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 polypeptide shown in Table 6.

[0304]

[0317] In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further includes one or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions) that make up less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further includes or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further includes or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region comprising or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further comprises or consists of about 1 or fewer, about 2 or fewer, about 3 or fewer, about 4 or fewer, about 5 or fewer, about 6 or fewer, about 7 or fewer, about 8 or fewer, about 9 or fewer, or 10 or fewer amino acid mutations (e.g., substitutions, deletions, additions, etc.).

[0305]

[0318] In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further includes one or more amino acid substitutions making up less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further includes 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 mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further includes 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 mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region comprising or consisting of the amino acid sequence of the polypeptide shown in Table 6, and further comprises or consists of about 1 or fewer, about 2 or fewer, about 3 or fewer, about 4 or fewer, about 5 or fewer, about 6 or fewer, about 7 or fewer, about 8 or fewer, about 9 or fewer, or 10 or fewer amino acid substitutions.

[0306]

[0319] In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region comprising 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 any one of the amino acid sequences of SEQ ID NOs. 199-224.

[0307]

[0320] In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199 to 224, and further comprises one or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions) accounting for less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199 to 224, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of a mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199 to 224, and further includes or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of a mutant hIg Fc fusion protein or polypeptide includes an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199 to 224, and further includes or consists of about 1 or fewer, about 2 or fewer, about 3 or fewer, about 4 or fewer, about 5 or fewer, about 6 or fewer, about 7 or fewer, about 8 or fewer, about 9 or fewer, or 10 or fewer amino acid mutations (e.g., substitutions, deletions, additions, etc.).

[0308]

[0321] In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199-224, and further comprises one or more amino acid substitutions making up less than 15% (less than 12%, less than 10%, less than 8%). In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199-224, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide comprises an hIg Fc region containing or consisting of one of the amino acid sequences of SEQ ID NOs. 199-224, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide comprises an hIg Fc region comprising or consisting of one of the amino acid sequences of SEQ ID NOs. 199 to 224, and further comprises or consists of about 1 or fewer, about 2 or fewer, about 3 or fewer, about 4 or fewer, about 5 or fewer, about 6 or fewer, about 7 or fewer, about 8 or fewer, about 9 or fewer, or 10 or fewer amino acid substitutions.

[0309]

[0322] In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and / or alanine at position L235 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and alanine at position L235 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine, glycine, or serine at position P329 (Kabat EU numbering).

[0310]

[0323] In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and alanine, glycine, or serine at position P329 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and alanine at position P329 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and glycine amino acids at position P329 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and serine amino acids at position P329 (Kabat EU numbering).

[0311]

[0324] In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and / or alanine, glycine, or serine at position P329 (EU numbering by Kabat); and contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the polypeptide shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and / or alanine at position L235 (Kabat EU numbering); and contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the polypeptide shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and alanine, glycine, or serine at position P329 (EU numbering by Kabat); and contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the polypeptides shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and alanine at position P329 (EU numbering by Kabat); and contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the polypeptide shown in Table 6.In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and glycine at position P329 (EU numbering by Kabat); and contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the polypeptide shown in Table 6. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and serine at position P329 (EU numbering by Kabat); and contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the polypeptide shown in Table 6.

[0312]

[0325] In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and / or an alanine, glycine, or serine amino acid at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and / or alanine at position L235 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and alanine at position L235 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and an alanine, glycine, or serine amino acid at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224.In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and alanine at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes an alanine amino acid at position L234, an alanine amino acid at position L235, and a glycine amino acid at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and celinglysine amino acids at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224.

[0313]

[0326] In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and / or an alanine, glycine, or serine amino acid at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and / or alanine at position L235 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234 and alanine at position L235 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and an alanine, glycine, or serine amino acid at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224.In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and alanine at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and glycine at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG1 Fc region includes alanine at position L234, alanine at position L235, and serine at position P329 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224.

[0314]

[0327] In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234 and / or alanine at position L235 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234 and alanine at position L235 (Kabat EU numbering). In some embodiments, the amino acid sequence of the hIgG4 Fc region includes proline at position S228 (Kabat EU numbering).

[0315]

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

[0316]

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

[0317]

[0330] In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234, alanine at position L235, and / or proline at position S228 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234 and / or alanine at position L235 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234, alanine at position L235, and proline at position S228 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224.

[0318]

[0331] In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234, alanine at position L235, and / or proline at position S228 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234 and / or alanine at position L235 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region includes phenylalanine at position L234, alanine at position L235, and proline at position S228 (EU numbering by Kabat); and includes 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 any one of the amino acid sequences of Sequence ID No. 199-224.

[0319] 5.3.2.2 Promotion of heterodimerization

[0332] As described herein, in some embodiments, the antibody (or heterologous polypeptide) 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 contain one or more amino acid modifications to each other to promote heterodimerization. Heterodimeric forms derived from IgG can be generated by methods known in the art, such as forced heavy-chain heterodimerization. Forced heavy chain heterodimerization can be achieved using methods known in the art, such as knob-in-hole or strand exchange engineered domains (SEEDs), for example, see Ji-Hee et al., "Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins," Frontiersin Immunology, v7 (article 394) (2016) DOI=10.3389 / fimmu.2016.00394 (hereinafter, "Ji-Hee 2016"). The entire contents of this document are incorporated herein by reference.

[0320]

[0333] In some embodiments, the interface between the first IgFc region and the second IgFc region is modified, for example, by introducing amino acid substitutions, to enhance heterodimerization compared to, for example, an unmodified interface (e.g., a naturally occurring interface). For example, dimerization between the first IgFc region and the second IgFc region can be enhanced by providing one or more of the following at the IgFc interface between the first and second Fc regions: paired protrusion-cavity ("knob-in-hole"), electrostatic interaction, or chain exchange. As a result, for example, the ratio of heteromultimer formation to homomultimer formation is increased compared to an unmodified interface.

[0321]

[0334] Knob-in-hole amino acid pairing modifications are known in the art and are described, for example, in U.S. Patent No. 5,731,116; No. 7,476,724; 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 full contents of each of these documents are incorporated herein by reference. Generally, knob-in-holes involve 1) introducing one or more amino acid substitutions into one or both CH3 domains of a first and second target Ig Fc region to promote heterodimerization, and 2) combining the modified Ig Fc regions under conditions that promote heterodimerization. "Knobs" are typically created by substituting a smaller amino acid in the Fc region of the parent Ig with a larger amino acid (e.g., T366Y or T366W), while "holes" are created by substituting a larger residue in the parent Ig Fc region with a smaller amino acid (e.g., Y407T, T366S, L368A, or Y407V). Exemplary knob-in-hole mutations include S354C and T366W in the "knob" Ig Fc region, and Y349C, T366S, L368A, and Y407V in the "hole" Ig Fc region. Other exemplary knob-in-hole mutations that can be incorporated into one or more embodiments, along with further exemplary optional stabilizing Ig Fc cysteine ​​mutations, are shown in Table 7. TIFF2026524970000026.tif54170

[0322]

[0335] Table 8 shows exemplary amino acid sequences of Fc regions known in the art to promote heterodimerization. TIFF2026524970000027.tif247170TIFF2026524970000028.tif248170TIFF20265249700 00029.tif248170TIFF2026524970000030.tif244170TIFF2026524970000031.tif166170

[0323]

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

[0324]

[0337] In some embodiments, the amino acid sequence of the first Fc region includes the T366W amino acid substitution (Kabat EU numbering), and the amino acid sequence of the second Fc region includes the following amino acid substitutions: T366S, L368A, and Y407V (Kabat EU numbering), all of which are substitutions 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 includes the S354C amino acid substitution (Kabat EU numbering), and the amino acid sequence of the second Fc region includes the Y349C amino acid substitution (Kabat EU numbering), all of which are substitutions to the amino acid sequence of an exemplary reference Ig Fc region (e.g., the reference Ig Fc region shown in Table 5).

[0325]

[0338] In some embodiments, the amino acid sequence of the first Fc region includes the following amino acid substitutions: T366W and S354C (Kabat EU numbering), and the amino acid sequence of the second Fc region includes the following amino acid substitutions: T366S, L368A, Y407V and Y349C (Kabat EU numbering), all of which are substitutions to the amino acid sequence of an exemplary reference Ig Fc region (e.g., the reference Ig Fc region shown in Table 5).

[0326]

[0339] In some embodiments, the amino acid sequence of the second Fc region includes the T366W amino acid substitution (Kabat EU numbering), and the amino acid sequence of the second Fc region includes the following amino acid substitutions: T366S, L368A, and Y407V (Kabat EU numbering), all of which are substitutions 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 second hIg further includes the S354C amino acid substitution (Kabat EU numbering), and the amino acid sequence of the second Fc region includes the Y349C amino acid substitution (Kabat EU numbering), all of which are substitutions to the amino acid sequence of an exemplary reference Ig Fc region (e.g., the reference Ig Fc region shown in Table 5).

[0327]

[0340] In some embodiments, the amino acid sequence of the second Fc region includes the following amino acid substitutions: T366W and S354C (EU numbering by Kabat), and the amino acid sequence of the second Fc region includes the following amino acid substitutions: T366S, L368A, Y407V and Y349C (EU numbering by Kabat), all of which are substitutions to the amino acid sequence of an exemplary reference Ig Fc region (e.g., the reference Ig Fc region shown in Table 5).

[0328]

[0341] In some embodiments, the amino acid sequence of the first IgFc region includes a W amino acid at position T366, and the amino acid sequence of the second IgFc region includes an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all assigned EU numbers by Kabat).

[0329]

[0342] In some embodiments, the amino acid sequence of the first IgFc region includes a W amino acid at position T366, and the amino acid sequence of the second IgFc region includes an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all assigned EU numbers by Kabat).

[0330]

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

[0331]

[0344] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by Kabat), and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of sequence numbers 233-240, and the second Ig The amino acid sequence of the Fc region contains an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 sequence numbers 249-256.

[0332]

[0345] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 225, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 241.

[0333]

[0346] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 226, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 242.

[0334]

[0347] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 227, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 243.

[0335]

[0348] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 228, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 244.

[0336]

[0349] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 229, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 245.

[0337]

[0350] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 230, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 246.

[0338]

[0351] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 231, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 247.

[0339]

[0352] In some embodiments, the amino acid sequence of the first Fc region contains a W amino acid at position T366 (Kabat EU numbering) 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 SEQ ID NO: 232, and the amino acid sequence of the second Ig Fc region contains an S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407 (all Kabat EU numbering) 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 SEQ ID NO: 248.

[0340]

[0353] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 233, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 249.

[0341]

[0354] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 234, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 250.

[0342]

[0355] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 235, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 251.

[0343]

[0356] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 236, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 252.

[0344]

[0357] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 237, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 253.

[0345]

[0358] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 238, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 254.

[0346]

[0359] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 239, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 255.

[0347]

[0360] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366 and a C amino acid at position S354 (both EU numbered by 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 SEQ ID NO: 240, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349 (all assigned EU numbers by 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 Sequence ID No. 256.

[0348] 5.3.2.3 Ig constant region mutations for site-directed conjugation

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

[0349]

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

[0350]

[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 (for example, 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 (for example, to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region includes the addition of one or more cysteine ​​amino acid residues (for example, 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 (for example, to mediate conjugation).

[0351] 5.3.2.4 Examples of mutated Fc regions

[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, e.g., §5.3.2). In some embodiments, the first Ig Fc region and the second Ig Fc region each comprise one or more amino acid mutations 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, e.g., §5.3.2.1), as well as one or more amino acid modifications that promote heterodimerization of the first and second Fc regions (see, e.g., §5.3.2.2).

[0352]

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

[0353]

[0366] Table 9 shows an example of the amino acid sequence of a mutant Fc region. TIFF2026524970000032.tif247170TIFF2026524970000033.tif248170TIFF2026524970000034.tif24817 0TIFF2026524970000035.tif248170TIFF2026524970000036.tif248170TIFF2026524970000037.tif80170

[0354]

[0367] In some embodiments, the amino acid sequence of the first Fc region includes 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 (all assigned EU numbers by Kabat), and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 257-264, and the second Ig The amino acid sequence of the Fc region includes 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 (all assigned EU numbers by 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 sequence numbers 265-272.

[0355]

[0368] In some embodiments, the amino acid sequence of the first Fc region includes a tryptophan amino acid at position T366, a cysteine ​​amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329 (all assigned EU numbers by Kabat), and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 273-280, and the second Ig The amino acid sequence of the Fc region includes 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 at position Y349, a cysteine ​​amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329 (all assigned EU numbers by 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 sequence numbers 281-288.

[0356]

[0369] In some embodiments, the amino acid sequence of the first Fc region includes a tryptophan amino acid at position T366, a cysteine ​​amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329 (all assigned EU numbers by 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 SEQ ID NO: 257, and the second Ig The amino acid sequence of the Fc region includes 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 (all assigned EU numbers by 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 Sequence ID No. 265.

[0357]

[0370] In some embodiments, the amino acid sequence of the first Fc region includes a tryptophan amino acid at position T366, a cysteine ​​amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329 (all assigned EU numbers by 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 SEQ ID NO: 258, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all assigned EU numbers by 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 Sequence ID No. 266.

[0358]

[0371] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 259, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all assigned EU numbers by 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 Sequence ID No. 267.

[0359]

[0372] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 260, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, an A amino acid at position L368, and a V amino acid at position Y407 (all assigned EU numbers by 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 Sequence ID No. 268.

[0360]

[0373] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 261, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all assigned EU numbers by 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 Sequence ID No. 269.

[0361]

[0374] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 262, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all assigned EU numbers by 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 Sequence ID No. 270.

[0362]

[0375] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 263, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all assigned EU numbers by 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 Sequence ID No. 271.

[0363]

[0376] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 264, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329 (all assigned EU numbers by 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 Sequence ID No. 272.

[0364]

[0377] In some embodiments, the amino acid sequence of the first Fc region includes a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and a G amino acid residue at position P329 (all EU numbered by 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 SEQ ID NO: 273, and the second Ig The amino acid sequence of the Fc region includes an S amino acid at position T366, an A amino acid at position L368, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and a G amino acid residue at position P329, a V amino acid at position Y407, and a C amino acid at position Y349 (all...

Claims

1. It is a conjugate, (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)), (b) A hematopoietic cell targeting agent operably linked to at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA), and / or target protein expressed by hematopoietic cells. A conjugate that includes this.

2. A conjugate according to claim 1, which, when bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, is internalized into the hematopoietic cell.

3. A conjugate according to claim 1 or 2, having the following properties: (a) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce the death of the target cell; (b) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell continues to survive; (c) When internalized by a hematopoietic cell, the conjugate does not induce the death of the hematopoietic cell; and / or (d) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)); A conjugate that indicates one or more of the following.

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

5. A conjugate according to any one of claims 1 to 4, wherein the protein (e.g., antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) has the following properties: (a) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the hematopoietic cell; (b) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) (c) When it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell continues to survive; (d) Even when internalized into a hematopoietic cell, 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 cell; and / or (e) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, 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)). A conjugate that indicates one or more of the following.

6. It is a conjugate, (a) A erythroid precursor 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)), (b) Erythroid precursor cell targeting agents operably linked to at least one oligonucleotide that modulates (e.g., inhibits) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA), and / or target protein expressed by erythroid precursor cells. A conjugate that includes this.

7. A conjugate according to claim 6, wherein when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

8. A conjugate according to claim 6 or 7, having the following properties: (a) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce the death of the target cell; (b) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains alive; (c) When internalized by an erythroid precursor cell, the conjugate does not induce the death of the erythroid precursor cell; and / or (d) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)); A conjugate that indicates one or more of the following.

9. A conjugate according to any one of claims 6 to 9, having the following characteristics: (a) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) When bound to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce target cell death; (c) the surface of an erythroid precursor cell (d) When the conjugate binds to a TFR expressed on the surface of an erythroid precursor cell (e.g., hTFR (e.g., TFR1)), the erythroid precursor cell continues to survive; (e) when internalized into the erythroid precursor cell, the conjugate does not induce the death of the erythroid precursor cell; and / or (e) when the conjugate binds to a TFR expressed on the surface of an erythroid precursor cell (e.g., hTFR (e.g., TFR1)), the conjugate does not induce the degradation of the TFR (e.g., hTFR (e.g., TFR1)). A conjugate that indicates one or more of the following.

10. A conjugate according to any one of claims 6 to 10, wherein the protein (e.g., antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) has the following properties: (a) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., antibody) or conjugate that specifically binds to the TFR (e.g., hTFR (e.g., hTFR1)) is internalized into the erythroid precursor cell; (b) when it binds to a TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, 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 target cell. (c) When the TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells is bound, the erythroid precursor cells continue to survive; (d) Even when internalized by 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) when the TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of erythroid precursor cells is bound, 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)). A conjugate that indicates one or more of the following.

11. The conjugate according to any one of claims 1 to 10, 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. Antibodies include full-length antibodies, 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 (Example: (VHH) ２ ) or (sdAb) ２ -Fc (e.g. (VHH) ２ The conjugate according to claim 11, comprising or consisting of -Fc.

13. The conjugate according to claim 11 or 12, wherein the antibody is an IgG (e.g., human IgG (hIgG)) antibody.

14. The conjugate according to any one of claims 11 to 13, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody (e.g., an IgG1 or IgG4 antibody).

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

16. Antibodies include full-length antibodies, Fab-Fc, scFv-Fc, and (scFv). ２ -Fc, sdAb-Fc (e.g., VHH-Fc) or (sdAb) ２ -Fc (e.g. (VHH) ２ A conjugate according to any one of claims 11 to 15, comprising or consisting of -Fc.

17. The conjugate according to claim 15 or 16, wherein the Ig (e.g., hIg)Fc region includes 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 to 17, wherein the Ig (e.g., hIg)Fc region includes a hinge region, a CH2 region, and a CH3 region.

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

20. The conjugate according to claim 19, wherein hIg is human IgG (hIgG).

21. The conjugate according to claim 20, wherein IgG is IgG1 or IgG4.

22. A conjugate according to any one of claims 15 to 21, wherein the Fc region of Ig (e.g., hIg) comprises one or more amino acid substitutions compared to the Fc region of reference Ig (e.g., hIg), and the Fc region of reference Ig reduces or eliminates one or more of the following effector functions compared to the Fc region of reference hIg: 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, FcγRIIIIa, and / or FcγRIIIIb) (e.g., FcγRI, FcγIIa, and / or FcγIIIa)).

23. A conjugate according to any one of claims 15 to 22, wherein the Fc region of Ig (e.g., hIg) 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, FcγRIIIIa, and / or FcγRIIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

24. A conjugate according to any one of claims 15 to 23, wherein Ig is hIgG1, and the amino acid sequence of the Fc region includes an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, the amino acid positions conform to the EU index of Kabat.

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

26. A conjugate according to any one of claims 15 to 25, wherein 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 amino acid position P329, the amino acid positions according to the EU index of Kabat.

27. A conjugate according to any one of claims 15 to 26, wherein 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 amino acid position P329, the amino acid positions according to the EU index of Kabat.

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

29. A conjugate according to any one of claims 15 to 23, wherein Ig is hIgG4, and the amino acid sequence of the Fc region includes 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, wherein the amino acid positions follow the EU index of Kabat.

30. A conjugate according to any one of claims 1 to 23 or 29, wherein 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, and the amino acid positions follow the EU index of Kabat.

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

32. A conjugate according to any one of claims 11 to 31, wherein the antibody comprises a first Fc region and a second Fc region associated via at least one covalent bond (e.g., a disulfide bond).

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

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

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

36. A conjugate according to any one of claims 11 to 35, wherein the amino acid sequence of the first Fc region includes an amino acid substitution at amino acid position Y349, and the amino acid positions follow the EU index of Kabat.

37. A conjugate according to any one of claims 11 to 36, wherein the amino acid sequence of the first Fc region includes cysteine ​​at amino acid position Y349, and the amino acid positions follow the EU index of Kabat.

38. A conjugate according to any one of claims 11 to 37, wherein the amino acid sequence of the second Fc region includes an amino acid substitution at amino acid position T366, and the amino acid positions follow the EU index of Kabat.

39. A conjugate according to any one of claims 11 to 38, wherein the amino acid sequence of the second Fc region includes tryptophan at amino acid position T366, and the amino acid positions follow the EU index of Kabat.

40. A conjugate according to any one of claims 11 to 39, wherein the amino acid sequence of the second Fc region of the antibody includes an amino acid substitution at amino acid position S354, and the amino acid position follows the EU index of Kabat.

41. A conjugate according to any one of claims 11 to 40, wherein the amino acid sequence of the second Fc region of the antibody includes cysteine ​​at amino acid position S354, and the amino acid positions follow the EU index of Kabat.

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

43. A conjugate according to any one of claims 11 to 42, 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. A conjugate according to claim 43, wherein the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) or a functional fragment or functional variant thereof.

45. A conjugate according to any one of claims 1 to 44, wherein the oligonucleotide enhances the expression and / or activity of a target gene, a target nucleic acid (e.g., mRNA), and / or a target protein.

46. A conjugate according to any one of claims 1 to 45, wherein the oligonucleotide inhibits the expression and / or activity of a target gene, a target nucleic acid (e.g., mRNA), and / or a target protein.

47. A conjugate according to any one of claims 1 to 46, wherein an oligonucleotide binds to a target gene, a target nucleic acid (e.g., mRNA), and / or a target protein, thereby regulating the expression and / or activity of the target gene, the target nucleic acid (e.g., mRNA), and / or the target protein by binding to the target nucleic acid molecule (e.g., a target mRNA molecule (e.g., a portion of the target mRNA molecule)).

48. A conjugate according to any one of claims 1 to 47, wherein the target nucleic acid molecule is a target mRNA molecule (for example, a part of a target mRNA molecule).

49. A conjugate according to any one of claims 1 to 48, wherein the oligonucleotide mediates one or more of the following conjugates: degradation of a target nucleic acid molecule (e.g., mRNA), dysfunction of a target nucleic acid molecule (e.g., mRNA), modification of a target nucleic acid molecule (e.g., mRNA), alteration of the 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 blockage of the translation of a target nucleic acid molecule (e.g., mRNA), or any combination thereof.

50. A conjugate according to any one of claims 1 to 49, wherein the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

51. A conjugate according to any one of claims 1 to 50, wherein the oligonucleotide comprises or comprises an antisense strand having a region complementary to a target sequence (e.g., a target gene, a target nucleic acid (e.g., mRNA), and / or an mRNA sequence encoded by a target protein).

52. The conjugate according to any one of claims 1 to 51, wherein the oligonucleotide is single-stranded or double-stranded.

53. The conjugate according to any one of claims 1 to 52, wherein the oligonucleotide is DNA, RNA, or an RNA-RNA hybrid molecule.

54. The conjugate according to any one of claims 1 to 53, wherein the oligonucleotide comprises a sense strand and an antisense strand that form a double-stranded region.

55. The conjugate according to any one of claims 1 to 54, wherein the sense strand and antisense strand are parts of a single nucleic acid molecule, and for example, the hairpin loop is located between the sense strand and antisense strand of a single nucleic acid molecule.

56. The conjugate according to any one of claims 1 to 55, wherein the sense strand and the antisense strand are separate nucleic acid molecules, i.e., linked only via a double-stranded region.

57. The conjugate according to any one of claims 1 to 56, wherein 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.

58. The conjugate according to any one of claims 1 to 57, wherein the oligonucleotide is at least one modified nucleotide.

59. The conjugate according to any one of claims 1 to 58, wherein at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides constituting the oligonucleotide are modified.

60. The conjugate according to any one of claims 1 to 59, wherein all or substantially all of the nucleotides constituting the oligonucleotide are modified.

61. The conjugate according to any one of claims 1 to 60, wherein at least one of the modified nucleotides comprises a modified sugar (e.g., a ribose moiety).

62. The conjugate according to any one of claims 1 to 61, wherein at least one of the modified nucleotides comprises a modified nucleic acid base.

63. The conjugate according to any one of claims 1 to 62, wherein the oligonucleotide comprises at least one modified internucleoside bond (e.g., at least one phosphorothioate internucleoside bond).

64. A conjugate according to any one of claims 1 to 63, wherein at least one modified nucleotide is a 2'-modified nucleotide, for example, 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-M0E), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMA0E), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), 2'-O-N-methylacetamide(2'-O-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)-restricted ethyl-bridged nucleic acid (cEt), for example, the 2'-modified nucleotide is 2'-O-methyl or 2'-fluoro(2'-F).

65. The conjugate according to any one of claims 1 to 64, wherein a protein encoded by a target gene, a target nucleic acid (e.g., mRNA), and / or a target protein is associated with one or more abnormal hemoglobin disorders.

66. A conjugate according to any one of claims 1 to 65, wherein inhibition or reduction of the expression and / or activity of a protein encoded by a target gene, target nucleic acid (e.g., mRNA) and / or target protein 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. A conjugate according to any one of claims 1 to 66, wherein the expression and / or activity of a target gene, a target nucleic acid (e.g., mRNA), and / or a target protein is associated with the suppression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the level of adult hemoglobin, and / or an increase in the ratio of adult hemoglobin to fetal hemoglobin.

68. The conjugate according to any one of claims 1 to 67, wherein the target gene, target nucleic acid (e.g., mRNA), and / or target protein are transcription factors.

69. The conjugate according to any one of claims 1 to 68, wherein the target gene, target nucleic acid (e.g., mRNA), and / or target protein are highly expressed in erythroid precursor cells compared to other non-erythroid precursor cell types.

70. A conjugate according to any one of claims 1 to 69, wherein the target gene is B-cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCL11A)), zinc finger and BTB domain-containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF transcription factor 1 (KLF1) (e.g., hKLF1), FA complement group A (FANCA) (e.g., human FANCA), diskelin pseudouridine synthase 1 (DKC1) (e.g., human DKC1), telomere elongation helicase 1 (RTEL1) (e.g., human R TEL1), 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 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-derived elastase (ELA2) (e.g., human ELA2), HCLS1-related protein X-1 (HAX1 ) (e.g., human HAX1), glucose-6-phosphatase catalytic subunit 3 (G6PC3) (e.g., human G6PC3), growth factor-independent 1 transcription repressor (GFI1) (e.g., human GFI1), WASP actin nucleation promoter (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), SAM (Sterile AlphaA conjugate that is Motif domain-containing 9 (SAMD9) (e.g., human SAMD9), SAM domain-containing 9-like (SAMD9L) (e.g., human SAMD9L), or MDS1-EVI1 complex locus (MECOM) (e.g., human MECOM).

71. The conjugate according to any one of claims 1 to 70, wherein the target gene is BCL11A (e.g., hBCL11A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLF1).

72. A conjugate according to any one of claims 1 to 71, wherein (a) a protein that specifically binds to TFR is (b) noncovalently conjugated to at least one oligonucleotide.

73. A conjugate according to any one of claims 1 to 72, wherein (a) a protein that specifically binds to TFR is covalently conjugated to at least one oligonucleotide.

74. A conjugate according to any one of claims 1 to 73, wherein (a) a protein that specifically binds to TFR is directly conjugated to (b) at least one oligonucleotide.

75. A conjugate according to any one of claims 1 to 74, wherein (a) a protein that specifically binds to TFR is (b) indirectly conjugated to at least one oligonucleotide via a linker.

76. The conjugate according to any one of claims 1 to 75, wherein the linker is cleavable or non-cleavable.

77. The conjugate according to any one of claims 1 to 76, wherein (b) comprises at least two, three, four, five, six or more nucleotides.

78. The conjugate according to any one of claims 1 to 77, wherein each of at least two, three, four, five, or six or more oligonucleotides is individually conjugated to a protein (e.g., an antibody) that specifically binds to a TFR (e.g., as described herein).

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

80. The cell according to claim 79, which is in vitro, ex vivo, or in vivo.

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

82. A kit comprising a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to claim 81.

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

84. The method according to claim 83, wherein the cells are in vitro, ex vivo, or in vivo.

85. The method according to claim 83 or 84, wherein the target is cells (for example, human subjects).

86. A method for delivering a conjugate, cells, or pharmaceutical composition to a target, comprising administering to the target a conjugate according to any one of claims 1 to 78, cells according to claim 79 or 80, or a pharmaceutical composition according to claim 81, thereby delivering the conjugate, cells, or pharmaceutical composition to the target.

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

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

89. The method according to claim 87 or 88, wherein the cells are in vitro, ex vivo, or in vivo.

90. The method according to any one of claims 87 to 89, wherein the target is cells (for example, human subjects).

91. A method for regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA), and / or target protein expressed by hematopoietic cells within a cell, comprising administering a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to claim 81 to a target, thereby regulating (e.g., inhibiting or enhancing) the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA), and / or target protein.

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

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

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

95. The method according to claim 93 or 94, wherein the cells are in vitro, ex vivo, or in vivo.

96. The method according to any one of claims 93 to 95, wherein the target is cells (for example, human subjects).

97. The method according to any one of claims 93 to 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, target nucleic acid (e.g., mRNA), and / or target protein expressed by hematopoietic cells within a target cell, comprising administering to the target a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to claim 81, thereby reducing or inhibiting the expression and / or activity of a target gene, target nucleic acid (e.g., mRNA), and / or target protein in the target.

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

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

101. A method for increasing the level of fetal hemoglobin in a subject, comprising administering to the subject a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to 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, comprising administering to the subject a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to 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, comprising administering a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to claim 81 to the subject to treat, improve, or prevent a hereditary blood disorder.

104. The method according to claim 103, wherein the hereditary blood disorder is a hemoglobin disorder or a hereditary bone marrow failure syndrome.

105. A method for treating, improving, or preventing hemoglobin disorders in a subject, comprising administering a conjugate according to any one of claims 1 to 78 or a pharmaceutical composition according to claim 81 to the subject to treat, improve, or prevent hemoglobin disorders.

106. The method according to claim 105, wherein the hemoglobin disorder 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 hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobinopathy, methemoglobinemia, or any combination thereof.

107. The method according to claim 105 or 106, wherein the hemoglobin disorder is sickle cell disease or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

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

109. The method according to any one of claims 105 to 108, wherein the subject is suspected of having 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 hemoglobinopathy, methemoglobinemia, or any combination thereof, or has been diagnosed with any of these.

110. The method according to any one of claims 105 to 109, wherein the subject is suspected of having sickle cell disease or thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia) or has been diagnosed with sickle cell disease or thalassemia.

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

112. The method according to claim 111, wherein the hereditary bone marrow failure syndrome is amegakaryotic thrombocytopenia (amega), Diamond-Blackfan anemia (DBA), congenital dyskeratosis (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachmann-Diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostman syndrome, refractory cytopenia, or radial dysthrombocytopenia (TAR).

113. A conjugate according to any one of claims 1 to 78, a cell according to claim 79 or 80, or a pharmaceutical composition according to claim 81, for use in the treatment of a target disease requiring treatment.

114. A conjugate according to any one of claims 1 to 78, a cell according to claim 79 or 80, or a pharmaceutical composition according to claim 81, for use as a pharmaceutical.

115. Use of a conjugate according to any one of claims 1 to 78, a cell according to claim 79 or 80, or a pharmaceutical composition according to claim 81 for use in the manufacture of a pharmaceutical for the treatment of a target disease requiring treatment.

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