Anti-transferrin receptor antibodies, conjugates thereof, and uses thereof

Abstract

The present invention provides transferrin receptor (TfR1) antibodies for conjugation with a therapeutic payload. The TfR1 antibodies can be used to deliver a therapeutic payload (e.g., an oligonucleotide-based agent) to CNS tissue, skeletal muscle tissue, or cardiac tissue in vivo.

Description

[0001] Cross-references to related applications This application claims priority to the following U.S. Provisional Patent Application Serial No. 63 / 624,417, filed January 24, 2024; U.S. Provisional Patent Application Serial No. 63 / 573,151, filed April 2, 2024; U.S. Provisional Patent Application Serial No. 63 / 662,243, filed June 20, 2024; and U.S. Provisional Patent Application Serial No. 63 / 718,062, filed November 8, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to compositions comprising anti-transferrin receptor (TfR1) antibodies conjugated to oligonucleotide-based reagents, and related methods of using said conjugations or compositions thereof to treat various diseases in the CNS, skeletal muscle, heart, and other tissues.

[0003] sequence list This application contains a sequence list (compliant with standard ST26), which has been submitted in XML format and is incorporated herein by reference in its entirety. The XML sequence list file is named 30728-WO_SeqListing.xml, was created on January 22, 2025, and has a size of 906 kb. Background Technology

[0004] RNA interference (RNAi) is the process by which RNA is degraded to silence gene expression. One type of RNAi molecule is a short (i.e., <30 nucleotides) double-stranded RNA (“dsRNA”) molecule present in the cell (Fire et al., 1998, ). Nature 391:806-811). These short dsRNA molecules, called “short interfering RNA” or “siRNA”, cause disruption of messenger RNA (“mRNA”) that shares sequence homology with the siRNA (Elbashir et al., 2001, ). Genes Dev, (15:188-200). It is believed that one strand of siRNA is incorporated into a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). RISC uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves these target mRNAs or inhibits their translation. siRNA is apparently recycled very much like a multiplexer, where a single siRNA molecule can induce the cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi degradation of mRNA is a safe and effective technique for suppressing target gene expression.

[0005] RNAi offers a very exciting approach to treating and / or preventing diseases. Some of the key benefits of RNAi compared to various traditional treatments include: RNAi's ability to target very specific genes involved in disease processes with high specificity, thereby reducing or eliminating off-target effects; RNAi is a normal cellular process that leads to highly specific RNA degradation; and the interfering RNA can be modified so that it does not trigger a host immune response.

[0006] Several methods for delivering interfering RNA (siRNA) are being tested / developed for in vivo use. For example, siRNA can be delivered: “naked” in saline solution; complexed with polycations, cationic lipids / lipid transfection reagents, or cationic peptides; as a component of defined molecular conjugates (e.g., cholesterol-modified siRNA, TAT-DRBD / siRNA complexes); as a component of liposomes; and as a component of nanoparticles. These methods have shown varying degrees of success. However, for RNAi to achieve therapeutic success through gene knockdown, it should be delivered to the affected cell type. Therefore, new and improved methods for delivering targeted RNAi molecules in vivo are needed to realize and enhance the therapeutic potential of RNAi. Summary of the Invention

[0007] This disclosure provides novel anti-transferrin receptor (TfR1) antibodies that exhibit high binding affinity and / or high expression yield against the target antigen TfR1. The TfR1 antibodies provided herein are suitable for conjugation with oligonucleotide-based reagents such as small interfering RNA (MIRNA), wherein the antibody binds to TfR1 to facilitate crossing the blood-brain barrier (BBB) ​​and delivery to desired cell types, including but not limited to cells in the CNS, skeletal muscle, and heart. Therefore, the interfering RNA conjugated with the TfR1 antibody of this disclosure can be used to deliver interfering RNA molecules to the central nervous system (CNS) of a patient. This disclosure also provides therapeutic methods using TfR1 antibodies conjugated with interfering RNA for in vivo delivery of interfering RNA molecules.

[0008] Therefore, one aspect of this disclosure is characterized by an antibody that binds to the transferrin receptor (TfR1), said antibody comprising a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL).

[0009] VH includes: (a) a heavy chain complementarity determination region 1 (HC CDR1) shown as GX1TFX2SYWX3H, where X1 is F or Y; X2 is K, N or T; and X3 is M or V; (b) a heavy chain complementarity determination region 2 (HC CDR2) shown as EINPTNGRX4NYIEKFKS (SEQ ID NO: 23), where X4 is F, S, T or V; and (c) a heavy chain complementarity determination region 3 (HC CDR3) shown as GTRAYHY (SEQ ID NO: 24). VL includes: (a) a light chain complementarity determination region 1 (LC CDR1) shown as RASDX5LYX6NLA (SEQ ID NO: 5), wherein X5 is G, K, or N, and X6 is R or S; (b) a light chain complementarity determination region 2 (LC CDR2) shown as DAX7X8LAS, wherein X7 is F, K, R, or T; and X8 is K, L, N, or R; and (c) a light chain complementarity determination region 3 (LC CDR3) shown as QHFWGTPLT (SEQ ID NO: 13).

[0010] In some implementations, X1 is F or Y; X2 is T; X3 is M; X4 is T or V; X5 is G or K; X6 is S; X7 is T; and X8 is L or K.

[0011] In some instances, the antibody comprises HC CDR1, HC CDR2, and HC CDR3 as shown in Table 2. Alternatively or additionally, the antibody comprises LC CDR1, LC CDR2, and LC CDR3 as shown in Table 1.

[0012] In some instances, the antibody comprises: HC CDR1, HC CDR2 and HC CDR3 shown as SEQ ID NO: 16, 19 and 24 respectively, and LC CDR1, LC CDR2 and LC CDR3 shown as SEQ ID NO: 4, 11 and 13 respectively (i.e., the same HC and LC CDRs as clone Fab0070).

[0013] In other examples, the antibodies comprise: HC CDR1, HC CDR2 and HC CDR3 shown as SEQ ID NO: 14, 20 and 24 respectively, and LC CDR1, LC CDR2 and LC CDR3 shown as SEQ ID NO: 2, 7 and 13 respectively (i.e., the same HC and LC CDRs as clone Fab0061).

[0014] In some embodiments, the anti-TfR1 antibodies disclosed herein are humanized antibodies. Such humanized antibodies may comprise the light chain framework regions shown in Table 3. Alternatively or additionally, the antibodies comprise the heavy chain framework regions shown in Table 4. In some instances, the anti-TfR1 antibodies provided herein may comprise the VH regions shown in Table 5; and / or wherein said antibodies comprise the VL regions shown in Table 5.

[0015] In some cases, the anti-TfR1 antibodies provided in this article are full-length antibodies, their antigen-binding fragments, or single-chain variable region fragment (scFv) antibodies.

[0016] In specific examples, antibodies are Fab molecules comprising: (a) a heavy chain containing a VH (e.g., any of the VHs provided in Table 5) and a CH1 heavy chain constant region fragment; and (b) a light chain containing a VL (e.g., any of the VLs provided in Table 5) and a light chain constant region (CL). In some cases, CH1 may have a human IgG heavy chain, which, for example, contains the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 34). Alternatively or additionally, CL may have a human light chain constant region (e.g., κ or λ chain) containing, for example, the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29).

[0017] In one specific example, the anti-Tf1R antibody provided herein may comprise: a heavy chain comprising (e.g., composed of) the amino acid sequence of SEQ ID NO: 61, and a light chain comprising (e.g., composed of) the amino acid sequence of SEQ ID NO: 62. In another specific example, the anti-TfR1 antibody may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO: 45, and a light chain comprising the amino acid sequence of SEQ ID NO: 46 (e.g., Fab0061).

[0018] In some cases, the anti-Tf1R antibodies provided herein may contain an Fc fragment. For example, an anti-Tf1R antibody may be a Fab-Fc molecule containing a VH-CH1 chain, a VL-CL chain, and an Fc fragment linked to either the VH-CH1 chain or the VL-CL chain. An example is provided in Table 5 (xAb000293).

[0019] In another aspect, this document provides conjugates comprising an antibody that binds to the transferrin receptor (TfR1) (anti-TfR1 antibody) and an oligonucleotide-based reagent. In some cases, the anti-TfR1 antibody is any of the antibodies provided herein (e.g., those listed in Tables 1, 2, and 5, such as clones Fab0061 and Fab0070, or their functional equivalents, which may contain the same heavy and light chain CDRs as Fab0061 or Fab0070).

[0020] In some embodiments, oligonucleotide-based reagents can be directly conjugated to anti-TfR1 antibodies via covalent or non-covalent bonds (e.g., ionic bonds, hydrogen bonds, or hydrophobic interactions). In some cases, oligonucleotide-based reagents can be conjugated to antibodies via, for example, peptides, polymers, or nucleic acid-binding proteins. In some instances, oligonucleotide-based reagents can be conjugated to anti-TfR1 antibodies via linkers. Examples include, but are not limited to, chemical linkers, peptide linkers, polymer linkers, or nucleic acid-binding peptides. In one instance, the linker can be a peptide linker, such as a glycine spacer having 1-4 glycine residues.

[0021] The oligonucleotide-based reagent in any conjugate disclosed herein may comprise one or more chemically modified nucleotides. In some embodiments, the nucleotide may be chemically modified by: substitution with alkyl, alkoxy, amino, denitrogenated, halogenated, hydroxyl, and / or thiol groups; sugar modification; one or more non-nucleotides; one or more deoxyribonucleotides; one or more non-phosphodiester bonds; or combinations thereof. In some instances, the oligonucleotide-based reagent may comprise one or more sugar modifications, which may comprise a 2′ OH group replaced by a 2′ amino group, a 2′ O-methyl group, a 2′ methoxyethyl group, or a 2′-O,4′-C methylene bridge. In some instances, the oligonucleotide-based reagent may comprise one or more non-nucleotides, which may comprise xanthine, hypoxanthine, azapurine, meththioadenine, 7-deadenine, or O- and N-modified nucleotides. Alternatively or additionally, the oligonucleotide-based reagent may comprise one or more non-phosphodiester bonds, which may comprise oxygen in the phosphate group substituted with one or more nitrogen or sulfur groups.

[0022] In some instances, the oligonucleotide-based reagent in any of the conjugates provided herein may be an RNAi reagent comprising a sense strand and an antisense strand forming a double strand. In some cases, the sense strand is conjugated to an anti-TfR1 antibody, which may be a Fab molecule.

[0023] In some cases, oligonucleotide-based reagents, such as RNAi reagents, can attenuate the expression of target genes of interest. Examples include, but are not limited to, APP, ataxin-1, ataxin-2, ataxin-3, ataxin-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich serine / threonine protein kinase 2, complement C3, microtubule-associated protein tau, α-2A adrenergic receptor, sodium channel protein type 9 subunit α, apolipoprotein E, α-synuclein, possible G protein-coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, and DYRK1A. In specific examples, the target genes are APP, ATXN2, ATXN3, AR, SOD1, HTT, C9ORF72, LRRK2, C3, MAPT, ADRA2A, SCN9A, APOE, SNCA, GPR75, or FUS.

[0024] In some cases, anti-TfR1 antibodies are Fab molecules conjugated to a cap portion. Oligonucleotide-based reagents are conjugated to linkers. Oligonucleotide-based reagents are conjugated to Fab molecules via linkers. In some instances, the cap portion has the following structure: (CP-1113). Alternatively or additionally, the connector has the following structure: In a specific example, oligonucleotide-based reagents are RNAi reagents containing a sense strand and an antisense strand, which form a double strand. The sense strand can be conjugated to a linker.

[0025] In another aspect, this disclosure provides pharmaceutical compositions comprising an anti-TfR1 antibody or a conjugate thereof (e.g., any of the antibodies or conjugates thereof provided herein) and a pharmaceutically acceptable carrier. The anti-TfR1 antibody may be any of the antibodies provided herein, such as those listed in Tables 1, 2, and 5, such as clones Fab0061 and Fab0070, or their functional equivalents, which may contain the same heavy and light chain CDRs as Fab0061 or Fab0070.

[0026] Furthermore, this document provides a method for attenuating the expression of target genes in the brain of a subject, the method comprising administering an effective amount of a conjugate or a pharmaceutical composition comprising such a conjugate to a subject in need. The conjugate comprises an anti-TfR1 antibody conjugated to an oligonucleotide-based reagent. The anti-TfR1 antibody can be any of the antibodies provided herein, such as those listed in Tables 1, 2, and 5, such as clones Fab0061 and Fab0070, or their functional equivalents, which may contain the same heavy and light chain CDRs as Fab0061 or Fab0070. The oligonucleotide-based reagent (e.g., an RNAi reagent) attenuates the expression of target genes of interest, such as those provided herein. Any conjugate provided herein can be used in the methods disclosed herein.

[0027] In some implementations, the subjects are patients with or suspected of having a central nervous system (CNS) disease. Examples include, but are not limited to, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau proteinosis, spinocerebellar ataxia, Alexander's disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinosis, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

[0028] In some embodiments, the conjugate or pharmaceutical composition comprising such conjugate is administered to the subject via intravenous injection, subcutaneous injection, intramuscular injection, intraparenchymal injection, or intrathecal injection.

[0029] Furthermore, this document provides methods for alleviating CNS diseases or conditions, skeletal muscle diseases, heart diseases or conditions, or diseases in other tissues, comprising administering to a subject in need an effective amount of any conjugate disclosed herein or a pharmaceutical composition comprising such conjugates. The conjugate comprises an anti-Tf1R antibody as disclosed herein (e.g., those listed in Tables 1, 2, and 5, such as clones Fab0061 and Fab0070, or their functional equivalents, which may contain the same heavy and light chain CDRs as Fab0061 or Fab0070) and an oligonucleotide-based reagent (e.g., an RNAi reagent) that attenuates the expression of a target gene of interest, such as APP, ataxia-1, ataxia-2, ataxia-3, ataxia-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, and leucine-rich filaments. Amino acid / threonine protein kinase 2, complement C3, microtubule-associated protein tau, α-2A adrenergic receptor, sodium channel protein type 9 subunit α, apolipoprotein E, α-synuclein, possible G protein-coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, DYRK1A, DUX4, DM1, ACVR2A, ACVR2B, MSTN, and GYS1. Subjects may be patients with or suspected of having a CNS disease selected from the following: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau disease, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinosis, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, hereditary small fiber neuropathy, or muscular dystrophy or atrophy (including but not limited to facioscapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy, and myotonic dystrophy type 1), spinal muscular atrophy, or metabolic myopathy (including but not limited to Pompe disease), or neuromuscular junction disease (including but not limited to myasthenia gravis (MG)), or metabolic syndrome.

[0030] Additionally, within the scope of this disclosure are any conjugates or pharmaceutical compositions comprising such conjugates provided herein for the treatment of diseases such as those disclosed herein, or for the use of the conjugates / pharmaceutical compositions in the manufacture of medicaments for the treatment of diseases.

[0031] Additionally, this disclosure provides nucleic acids or nucleic acid sets containing nucleotide sequences encoding the heavy and light chains of the anti-TfR1 antibodies disclosed herein, such as those listed in Tables 1, 2, and 5, such as clones Fab0061 and Fab0070, or their functional equivalents, which may contain the same heavy and light chain CDRs as Fab0061 or Fab0070. In some cases, the nucleic acid or nucleic acid set is a vector or vector set. In some instances, the vector or vector set is an expression vector or expression vector set. This document also provides host cells containing the nucleic acids or nucleic acid sets disclosed herein.

[0032] Furthermore, this disclosure is characterized by a method for producing anti-TfR1 antibodies, comprising: (i) culturing host cells carrying nucleic acids encoding any anti-TfR1 antibody provided herein (e.g., those listed in Tables 1, 2 and 5, such as clones Fab0061 and Fab0070, or their functional equivalents, which may contain the same heavy and light chain CDRs as Fab0061 or Fab0070) under conditions that allow for the expression of anti-TfR1 antibodies; and (ii) harvesting the anti-TfR1 antibodies thus produced.

[0033] The following provides alternative implementations of this disclosure.

[0034] Implementation Scheme 1. An antibody that binds to transferrin receptor (TfR1), said antibody comprising a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL); The VH includes: The heavy chain complementarity determination region 1 (HC CDR1) is shown as GX1TFX2SYWX3H, where X1 is F or Y; X2 is K, N or T; and X3 is M or V. The heavy chain complementarity determination region 2 (HC CDR2) is shown as EINPTNGRX4NYIEKFKS (SEQ ID NO: 23), where X4 is F, S, T, or V; and The heavy chain complementarity determination region 3 (HC CDR3) shown as GTRAYHY (SEQ ID NO: 24) is displayed; and The VL mentioned therein includes: The light chain complementarity determination region 1 (LC CDR1) is shown as RASDX5LYX6NLA (SEQ ID NO: 5), where X5 is G, K or N, and X6 is R or S; The light chain complementarity determination region 2 (LC CDR2) is shown as DAX7X8LAS, where X7 is F, K, R, or T; and X8 is K, L, N, or R; and The light chain complementarity determination region 3 (LC CDR3) is shown as QHFWGTPLT (SEQ ID NO: 13).

[0035] Implementation Scheme 2. The antibody of Implementation Scheme 1, wherein X1 is F or Y; X2 is T; X3 is M; X4 is T or V; X5 is G or K; X6 is S; X7 is T; and X8 is L or K.

[0036] Implementation Scheme 3. The antibody of Implementation Scheme 1, wherein the antibody comprises HC CDR1, HC CDR2 and HC CDR2 of any one of Fab0070, Fab0061, Fab0060, Fab0062, Fab0063, Fab0064, Fab0065, Fab0066, Fab0067, Fab0068, Fab0069, Fab0071, Fab0072, Fab0073, Fab0074, Fab0165, Fab0166, Fab0167 or Fab0168 as shown in Table 2. CDR3; and / or wherein the antibody comprises LC CDR1, LC CDR2 and LC CDR3 of any one of Fab0070, Fab0061, Fab0060, Fab0062, Fab0063, Fab0064, Fab0065, Fab0066, Fab0067, Fab0068, Fab0069, Fab0071, Fab0072, Fab0073, Fab0074, Fab0165, Fab0166, Fab0167 or Fab0168 shown in Table 1.

[0037] Implementation Scheme 4. The antibody of Implementation Scheme 3, wherein the antibody comprises: (a) HC CDR1, HC CDR2, and HC CDR3 as SEQ ID NO: 16, 19, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 as SEQ ID NO: 4, 11, and 13, respectively; or (b) HC CDR1, HC CDR2 and HC CDR3 shown as SEQ ID NO: 14, 20 and 24 respectively, and LC CDR1, LC CDR2 and LC CDR3 shown as SEQ ID NO: 2, 7 and 13 respectively.

[0038] Implementation Scheme 5. An antibody of any one of Implementation Schemes 1-4, wherein the antibody is a humanized antibody; optionally, wherein the antibody comprises the light chain framework region shown in Table 3 and / or the heavy chain framework region shown in Table 4.

[0039] Implementation Scheme 6. The antibody of Implementation Scheme 5, wherein the antibody comprises any one of the VH sequences shown in Table 5; and / or wherein the antibody comprises any one of the VL sequences shown in Table 5.

[0040] Implementation Scheme 7. An antibody of any one of Implementation Schemes 1-6, wherein the antibody is a full-length antibody, its antigen-binding fragment, or a single-chain variable region fragment (scFv) antibody.

[0041] Implementation Scheme 8. An antibody of any one of Implementation Schemes 1-7, wherein the antibody is a Fab molecule; wherein the heavy chain comprises VH and CH1 heavy chain constant region fragments; and wherein the light chain comprises VL and light chain constant region (CL).

[0042] Implementation Scheme 9. The antibody of Implementation Scheme 8, wherein CH1 comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 34); and / or The CL contains the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29).

[0043] Implementation Scheme 10. An antibody of Implementation Scheme 8 or 9, wherein the VH comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTLVTVSS (SEQ ID NO: 59), and / or wherein the VL comprises the amino acid sequence of DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 60).

[0044] Implementation Plan 11. The antibody of Implementation Plan 8 or 9, wherein: (a) The heavy chain contains the amino acid sequence of SEQ ID NO: 61, and the light chain contains the amino acid sequence of SEQ ID NO: 62; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO: 45, and the light chain contains the amino acid sequence of SEQ ID NO: 46.

[0045] Implementation Scheme 12. An antibody of any one of Implementation Schemes 1-11, wherein the antibody comprises an Fc fragment, optionally wherein the antibody is a Fab-Fc molecule.

[0046] Implementation Scheme 13. A conjugate comprising an antibody (anti-TfR1 antibody) that binds to the transferrin receptor (TfR1) and an oligonucleotide-based reagent, wherein the anti-TfR1 antibody is described in any one of Implementation Schemes 1-12.

[0047] Implementation Scheme 14. The conjugate of Implementation Scheme 13, wherein the oligonucleotide-based reagent is directly conjugated to the anti-TfR1 antibody via a covalent or non-covalent bond.

[0048] Implementation Scheme 15. The conjugate of Implementation Scheme 13, wherein the oligonucleotide-based reagent is conjugated to an anti-TfR1 antibody via a linker, the linker optionally being a chemical linker, peptide linker, polymer linker, or nucleic acid-binding peptide.

[0049] Implementation Scheme 16. The conjugate of Implementation Scheme 13, wherein the linker is a peptide linker, which is a glycine spacer having 1-4 glycine residues.

[0050] Implementation Scheme 17. A conjugate of any one of Implementation Schemes 13-16, wherein the oligonucleotide-based reagent comprises one or more chemically modified nucleotides.

[0051] Implementation Scheme 18. The conjugate of Implementation Scheme 17, wherein the nucleotide is chemically modified by: substitution with alkyl, alkoxy, amino, denitrogenated, halogenated, hydroxyl and / or thiol groups; sugar modification, one or more non-nucleotides; one or more deoxyribonucleotides; one or more non-phosphodiester bonds; or combinations thereof.

[0052] Implementation Scheme 19. The conjugate of Implementation Scheme 18, wherein: The sugar modification includes a 2′ OH group replaced by a 2′ amino group, a 2′ O-methyl group, a 2′ fluorine group, a 2′ methoxyethyl group, or a 2′-O, 4′-C methylene bridge; The one or more non-nucleotides comprise xanthine, hypoxanthine, azapurine, methionine, 7-deadenosine, or O- and N-modified nucleotides; and / or The one or more non-phosphodiester bonds involve the oxygen in the phosphate group being replaced by one or more nitrogen or sulfur atoms.

[0053] Implementation Scheme 20. A conjugate of any one of Implementation Schemes 13-19, wherein the oligonucleotide-based reagent is an RNAi reagent comprising a sense strand and an antisense strand, the sense strand and the antisense strand forming a double strand.

[0054] Implementation Scheme 21. The conjugate of Implementation Scheme 20, wherein the RNAi reagent attenuates the expression of target genes selected from the following: APP, ataxia-1, ataxia-2, ataxia-3, ataxia-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich serine / threonine protein kinase 2, complement C3, microtubule-associated protein tau, α-2A adrenergic receptor, sodium channel protein type 9 subunit α, apolipoprotein E, α-synuclein, possible G protein-coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, and DYRK1A.

[0055] Implementation Scheme 22. The conjugate of Implementation Scheme 21, wherein the target gene is selected from APP, ATXN2, ATXN3, AR, SOD1, HTT, C9ORF72, LRRK2, C3, MAPT, ADRA2A, SCN9A, APOE, SNCA, GPR75 and FUS.

[0056] Implementation Scheme 23. A conjugate of any one of Implementation Schemes 20-22, wherein the sense chain is conjugated to an anti-TfR1 antibody, wherein the anti-TfR1 antibody is a Fab molecule.

[0057] Implementation Scheme 24. A conjugate of any one of Implementation Schemes 13-23, wherein the anti-TfR1 antibody is a Fab molecule; wherein the oligonucleotide-based reagent is conjugated with a linker having the following formula: , in A Represents the attachment point to the Fab molecule, and R This represents the attachment point of the oligonucleotide-based reagent moiety in the linker and conjugate.

[0058] Implementation Scheme 25. A conjugate of any one of Implementation Schemes 13-23, wherein the anti-TfR1 antibody is a Fab molecule comprising a cap portion; wherein the oligonucleotide-based reagent is conjugated to a linker, and wherein the oligonucleotide-based reagent is conjugated to the Fab molecule via a linker.

[0059] Implementation Scheme 26. The conjugate of Implementation Scheme 25, wherein the cap portion has the following formula: (CP-1113); and / or said connector has The structure, in which A Represents the attachment point to the Fab molecule, and R This represents the attachment point of the oligonucleotide-based reagent moiety in the linker and conjugate.

[0060] Implementation Scheme 27. A conjugate of Implementation Scheme 25 or Implementation Scheme 26, wherein the oligonucleotide-based reagent is an RNAi reagent comprising a sense strand and an antisense strand, the sense strand and the antisense strand forming a double strand; and wherein the sense strand is conjugated to a linker.

[0061] Implementation Scheme 28. A pharmaceutical composition comprising an anti-TfR1 antibody or a conjugate thereof and a pharmaceutically acceptable carrier, wherein the anti-TfR1 antibody is described in any one of Implementation Schemes 1-12.

[0062] Implementation Scheme 29. The pharmaceutical composition of Implementation Scheme 27, comprising a conjugate of an anti-TfR1 antibody, wherein the conjugate is described in any of Implementation Schemes 13-27.

[0063] Implementation Scheme 30. A method for attenuating the expression of a target gene in the brain of a subject, comprising administering to the subject in need an effective amount of a conjugate or a pharmaceutical composition comprising such a conjugate, The conjugate comprises an anti-TfR1 antibody conjugated with an oligonucleotide-based reagent. The anti-TfR1 antibody described in any one of embodiments 1-12; and The oligonucleotide-based reagents described therein attenuate the expression of the target gene.

[0064] Implementation Scheme 31. The method of Implementation Scheme 30, wherein the conjugate is described in any one of Implementation Schemes 12-27.

[0065] Implementation Scheme 32. The method of Implementation Scheme 30 or Implementation Scheme 31, wherein the subject is a person who has or is suspected of having a central nervous system (CNS) disease.

[0066] Implementation Scheme 33. The method of Implementation Scheme 32, wherein the CNS disease is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau protein disease, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 protein disease, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

[0067] Implementation Scheme 34. The method of any one of Implementation Schemes 30-33, wherein the conjugate or pharmaceutical composition comprising such conjugate is administered to the subject by intravenous injection, subcutaneous injection, intramuscular injection, intraparenchymal injection or intrathecal injection.

[0068] Implementation Scheme 35. A method for alleviating CNS disease, comprising administering to a subject in need an effective amount of the conjugate shown in any one of Implementation Schemes 12-26 or a pharmaceutical composition comprising such conjugate; The conjugates described herein contain oligonucleotide-based reagents that attenuate the expression of target genes selected from: APP, ataxia-1, ataxia-2, ataxia-3, ataxia-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich serine / threonine protein kinase 2, complement C3, microtubule-associated protein tau, α-2A adrenergic receptor, sodium channel protein type 9 subunit α, apolipoprotein E, α-synuclein, possible G protein-coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, and DYRK1A; and The subjects mentioned therein had or were suspected of having a CNS disease selected from the following: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau proteinosis, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinosis, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

[0069] Implementation Scheme 36. A method for attenuating the expression of a target gene in the skeletal muscle of a subject, comprising administering to the subject in need an effective amount of a conjugate or a pharmaceutical composition comprising such a conjugate, The conjugate comprises an anti-TfR1 antibody conjugated with an oligonucleotide-based reagent. The anti-TfR1 antibody described in any one of embodiments 1-12; and The oligonucleotide-based reagents described therein attenuate the expression of the target gene.

[0070] Implementation Scheme 37. The method of Implementation Scheme 36, wherein the target gene is selected from DUX4, DM1, ACVR2A, ACVR2B, MSTN and GYS1.

[0071] Implementation scheme 38. The method of implementation scheme 36 or 37, wherein the subject is a patient who has or is suspected of having a skeletal muscle disease.

[0072] Implementation Scheme 39. The method of Implementation Scheme 38, wherein the skeletal muscle disease is selected from facioscapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy and myotonic dystrophy type 1, spinal muscular atrophy, or metabolic myopathy (including but not limited to Pompe disease), or neuromuscular junction disease (including but not limited to myasthenia gravis (MG)), or metabolic syndrome.

[0073] Implementation Scheme 40. A nucleic acid or nucleic acid group comprising nucleotide sequences encoding the heavy and light chains of the anti-TfR1 antibody shown in any one of Implementation Schemes 1-12.

[0074] Implementation Scheme 41. The nucleic acid or nucleic acid group of Implementation Scheme 40, which is a vector or vector group.

[0075] Implementation Scheme 42. The nucleic acid or nucleic acid group of Implementation Scheme 41, wherein the vector or vector group is an expression vector or expression vector group.

[0076] Implementation Scheme 43. A host cell comprising the nucleic acid or nucleic acid group shown in any one of Implementation Schemes 40-42.

[0077] Implementation Scheme 44. A method for producing anti-TfR1 antibodies, comprising: (i) Culture the host cells of implementation scheme 43 under conditions that allow for the expression of anti-TfR1 antibodies; and (ii) Harvest the anti-TfR1 antibodies produced as a result.

[0078] Specific preferred embodiments of the invention will become apparent from the following more detailed description of certain preferred embodiments and the claims. Attached Figure Description

[0079] These and other features, aspects, and advantages of this disclosure can be better understood when the following detailed description is read in conjunction with the accompanying drawings.

[0080] Figure 1 The study showed that, in different brain regions, relative androgen receptor (AR) mRNA expression was knocked down after delivery of selected TfR1-targeted Fab conjugated to siRNA (with an antisense strand complementary to androgen receptor mRNA) compared to control siRNA.

[0081] Figure 2 The study showed that, in different brain regions, relative androgen receptor mRNA expression was knocked down after delivery of selected TfR1-targeting Fab conjugated to siRNA (with an antisense strand complementary to androgen receptor mRNA) compared to control siRNA.

[0082] Figure 3 The study showed that, in different brain regions, relative androgen receptor mRNA expression was knocked down after delivery of selected TfR1-targeting Fab conjugated to siRNA (with an antisense strand complementary to androgen receptor mRNA) compared to control siRNA.

[0083] Figure 4 Showing the standardization for Fab0002, in K D The binding affinity of the selected TfR1-targeting Fab was calculated.

[0084] Figure 5 The knockdown of MAPT protein in various tissues is shown in accordance with the description in Example 13. Detailed Implementation

[0085] The details shown herein are merely illustrative and are intended only for the purpose of explaining preferred embodiments of the invention, and are presented to provide the most useful and readily understood description of the principles and concepts of various embodiments of the invention. In this regard, no further details of the invention beyond what is necessary for a basic understanding of the invention are shown; the description, in conjunction with the drawings and / or embodiments, makes it apparent to those skilled in the art how various forms of the invention can be manifested in practice.

[0086] definition The definitions and descriptions below are intended and anticipated to be controlling in any future interpretation, unless explicitly and unambiguously modified in the embodiments described below, or when the application of their meaning renders any interpretation meaningless or substantially meaningless. Where the interpretation of a term renders it meaningless or substantially meaningless, the definition should be taken from Webster's Dictionary, Third Edition, or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (edited by Anthony Smith, Oxford University Press, Oxford, 2004).

[0087] As used herein, unless otherwise stated, all percentages are by weight.

[0088] As used herein and unless otherwise stated, the terms “an” and “a kind” are to be regarded as meaning “one / kind,” “at least one / kind,” or “one or more / a combination of.” Unless the context requires otherwise, singular terms used herein shall include the plural, and plural terms shall include the singular.

[0089] As used herein, the term "receptor" is intended to encompass the entire receptor or its ligand-binding portion. These portions of the receptor specifically include those regions sufficient for ligand-specific binding to occur, including those regions capable of being recognized by antibodies or antibody fragments.

[0090] Antibodies (used interchangeably in plural form) are immunoglobulin molecules that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, peptides, etc., by means of at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term “antibody,” such as an anti-TfR1 antibody, encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, biantibodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., VH-only antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site of desired specificity, including glycosylated variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies. Antibodies, such as anti-TfR1 antibodies, include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to belong to any particular class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the antibody's constant domain of its heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0091] As used herein, the term “Fab” refers to the antigen-binding fragment (Fab) region that binds to the TfR1 antigen and includes six complementarity-determining regions (CDRs), three of which are on the variable light chain (VL) and three of which are on the variable heavy chain (VH).

[0092] The term "ligand" is defined as any molecule or atom that binds to a receptor molecule, such as a protein, nucleotide, or fragment thereof, binding domain, or complement.

[0093] As used herein, the term “protein” includes peptides, polypeptides, common molecules, fusion proteins, purified naturally occurring proteins, artificially synthesized proteins, recombinant proteins, antibodies, antibody fragments and their analogues, derivatives or combinations thereof.

[0094] The term “antigen-binding fragment” (e.g., Fab) is further expected to encompass fully humanized and chimeric antibody fragments, bifunctional antibody fragments, etc., that contain parts from more than one species.

[0095] The term "HA2 peptide" refers to a peptide that contains the N-terminal 20 amino acids of the influenza virus hemagglutinin protein.

[0096] The term "conjugate" is defined as consisting of two or more molecules; or two or more entities coupled together. Preferably, the two molecules or entities are conjugated through nonspecific or specific protein-protein interactions, covalent bonding, non-covalent bonding, or coordination chemistry. In the context of this invention, the first molecule may be an interfering RNA molecule, and the second molecule may be a ligand of a receptor on a target cell as defined herein. In some embodiments, the term conjugate includes a TfR1 Fab coupled to an oligonucleotide-based reagent (e.g., siRNA), which may also include a linker and / or peptide or protein for releasing the oligonucleotide-based reagent from endosomes as described herein.

[0097] As used herein, the terms “interfering RNA” and “interfering RNA molecule” refer to all RNA or RNA-like molecules that can interact with RISC and participate in RISC-mediated changes in gene expression.

[0098] Unless otherwise stated, the term "siRNA" as used herein refers to double-stranded interfering RNA.

[0099] As used herein, the term "therapeuticly effective amount" refers to the amount of interfering RNA or a pharmaceutical composition containing interfering RNA determined to produce a therapeutic response in mammals. Such therapeutically effective amounts can be readily determined by those skilled in the art and using the methods described herein.

[0100] As used in this article, the phrase “attenuated expression” when referring to a gene or mRNA means the administration or expression of a certain amount of interfering RNA (e.g., siRNA) to reduce the translation of the target mRNA into a protein by means of mRNA cleavage or by direct inhibition of translation.

[0101] As used herein, the terms “suppress,” “silence,” and “attenuate” refer to a measurable reduction in the expression of the target mRNA or corresponding protein compared to the expression of the target mRNA or corresponding protein in the absence of the interfering RNA of the present invention.

[0102] The term "knockdown" refers to a reduction in the expression of a target mRNA or corresponding protein.

[0103] As used herein, the phrases “target sequence” and “target mRNA” refer to such mRNA or a portion of an mRNA sequence that can be recognized by the interfering RNA used in the method of the present invention, thereby allowing the interfering RNA to silence gene expression as described herein.

[0104] As used herein, the term “CNS condition” includes conditions or disorders relating to a patient’s central nervous system (CNS) (including, but not limited to, the brain and spinal cord). In some implementations, CNS conditions are selected from amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau proteinosis, spinocerebellar ataxia, Alexander disease, Kennedy’s disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinosis, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

[0105] As used herein, the term "musculoskeletal disorders" includes conditions or disorders related to skeletal muscle tissue, including but not limited to muscular dystrophy, including but not limited to FSHD, myotonic dystrophy type 1, and Duchenne muscular dystrophy.

[0106] As used herein, the term “patient” means a person or other mammal who has a CNS condition or is at risk of having a CNS condition.

[0107] As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size properties of the protein in which the substitution occurs. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those found in references compiling such methods, for example, *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., eds., 2nd ed., ColdSpring Harbor Laboratory Press, ColdSpring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conservative substitution of amino acids includes substitutions in amino acids within the following group: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0108] The "percentage of identity" between two amino acid sequences was determined using the algorithm described by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, which was modified as described by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This type of algorithm was incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of interest. When there are gaps between two sequences, methods such as those described by Altschul et al. can be used. Nucleic Acids Res As described in 25(17):3389-3402, 1997, the use of Gapped BLAST is permitted. When using the BLAST and Gapped BLAST procedures, the default parameters of their respective procedures (e.g., XBLAST and NBLAST) can be used.

[0109] Transferrin receptor (TfR1) is an integrated membrane glycoprotein that mediates iron uptake in individual cells. The number of receptors on the cell surface is correlated with cell proliferation, with the highest number found on actively growing cells and the lowest number on quiescent and terminally differentiated cells. Monoclonal antibody binding (Teh et al.) FEBS J. 272:6344-6353, 2005) and hydroxyl radical-mediated protein footprinting (Liu et al.) Biochemistry (42:12447-12454, 2003) suggests that the TfR1 binding domain of Tf is located in the C lobe of human Tf, within residues 334-679. Residues 365-401 (especially 381-401), 415-433, and 457-470 appear to be particularly important for receptor binding.

[0110] Targeting the TfR1 receptor is a promising approach to facilitate drug delivery across the blood-brain barrier (BBB) ​​to the brain. This article presents anti-TfR1 antibodies with high binding affinity for TfR1, along with other desirable characteristics such as high expression yield. This article reports how such anti-TfR1 antibodies successfully facilitated the delivery of exemplary siRNA molecules conjugated to them to brain cells, thereby attenuating the expression of genes targeted by the siRNA. Accordingly, the anti-TfR1 antibodies presented herein can be used to achieve efficient drug delivery to the brain, thus addressing one of the greatest challenges associated with developing therapeutics for treating central nervous system (CNS) diseases.

[0111] I. Anti-TfR1 Fab and anti-TfR1 antibody This disclosure provides antibodies that bind to TfR1, such as human TfR1. In some embodiments, the anti-TfR1 antibodies disclosed herein are capable of binding to TfR1 expressed on cell surfaces, such as brain endothelial cells. Therefore, the antibodies disclosed herein can be used to facilitate the delivery of antibody-conjugated therapeutic or diagnostic agents to the brain. In some cases, the anti-TfR1 antibodies provided herein can also be used to detect the presence of TfR1 receptors in vitro or in vivo. As used herein, the term "anti-TfR1 antibody" refers to any antibody capable of binding to a TfR1 peptide (e.g., a TfR1 peptide expressed on a cell surface, such as brain endothelial cells), which may have a suitable source, such as human or non-human mammals (e.g., mice, rats, rabbits, primates such as monkeys, etc.).

[0112] Typical antibody molecules contain a heavy chain variable region (V). H ) and light chain variable region (V L This typically involves antigen binding. H and V L The region can be further subdivided into high-variability regions, also known as "complementary determinant regions" ("CDRs"), interspersed with more conservative regions called "frame regions" ("FRs"). Each V H and V L Typically, it consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methods known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, for example, Kabat, E.A., et al. (1991). Sequences of Proteins of Immunological Interest, Fifth Edition US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Allazikani et al. (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0113] The anti-TfR1 antibody described herein may be a full-length antibody containing two heavy chains and two light chains, each comprising a variable domain and a constant domain. Alternatively, the anti-TfR1 antibody may be an antigen-binding fragment of a full-length antibody. Examples of binding fragments covered within the term "antigen-binding fragment" of a full-length antibody include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of an antibody arm; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) a functionally separate complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using a synthetic linker via recombination, enabling the preparation of a single protein chain in which the VL and VH regions pair to form a monovalent molecule called a single-stranded Fv (scFv). See, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0114] In some embodiments, the anti-TfR1 antibody provided herein is a TfR1 antigen-binding fragment region (Fab) that specifically binds to TfR1. In some instances, the TfR1-specific Fab binds to TfR1 on brain endothelial cells. In some embodiments, the TfR1-specific Fab is capable of recognizing and specifically binding to one or more epitopes on TfR1.

[0115] The anti-TfR1 antibodies disclosed in this paper, such as TfR1-specific Fab, can competitively bind to TfR1 with transferrin (Tf), a physiological TfR1 ligand. After Fab binds to TfR1 on the cell surface, Fab and the attached interfering RNA are transferred into the cell via constitutive clathrin-mediated endocytosis.

[0116] The antibodies described herein may have a suitable origin, such as mice, rats, or humans. Such antibodies are not naturally occurring, meaning they are not produced in animals without human intervention (e.g., immunizing such animals with a desired antigen or a fragment thereof, or isolating them from an antibody library). Any antibody described herein, such as an anti-TfR1 antibody, may be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogeneous population of antibodies, and "polyclonal antibody" refers to a heterogeneous population of antibodies. Neither term limits the source of the antibody or the manner in which it is prepared.

[0117] In some implementations, the anti-TfR1 antibody is a human antibody, which can be isolated from a human antibody library or generated in transgenic mice. For example, a fully human antibody can be obtained using commercially available mice (which have been engineered to express a specific human immunoglobulin). Transgenic animals designed to produce a more desired (e.g., fully human antibody) or more robust immune response can also be used to generate humanized antibodies or human antibodies. Examples of such technologies are Xenomouse™ from Amgen, Inc. (Fremont, Calif.), and HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, the antibody can be recombinantly prepared using phage display or yeast technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12: 433-455. Alternatively, antibody library display techniques known in the art, such as phage, yeast, mammalian cell, or mRNA display techniques, can be used to produce human antibodies and antibody fragments in vitro from a gene bank of immunoglobulin variable (V) domains derived from non-immunized donors.

[0118] In other embodiments, the anti-TfR1 antibody may be a humanized antibody. A humanized antibody refers to the form of a non-human (e.g., mouse) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the receptor's CDR are replaced with residues of a CDR from a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and capability. In some cases, one or more Fv frame region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not found in the receptor antibody or in the input CDR or frame sequence, but are included to further improve and optimize antibody performance. In some cases, humanized antibodies may contain at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the CDR regions correspond to those regions of non-human immunoglobulins, and all or substantially all of the FR regions are those regions of the common sequence of human immunoglobulins. Humanized antibodies preferably also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically a constant region or domain of a human immunoglobulin. The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, said CDRs also referred to as one or more CDRs “derived” from one or more CDRs of the original antibody. Humanized antibodies may also involve affinity maturation. Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989).

[0119] In some embodiments, the anti-TfR1 antibody described herein binds specifically to the corresponding target antigen (here, TfR1) or its epitope. The term "specifically binds" to an antigen or epitope is a well-known term in the art. It is said to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with a higher affinity than the molecule reacts with alternative targets. It binds "specifically" to a target antigen or epitope if it binds with higher affinity, stronger affinity, easier binding, and / or for a longer duration than the antibody binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (TfR1) or an epitope therein is an antibody that binds to that target antigen with higher affinity, stronger affinity, easier binding, and / or for a longer duration than it binds to other antigens or other epitopes of the same antigen. It should also be understood under this definition that, for example, an antibody that specifically binds to a first target antigen may bind specifically or preferentially to a second target antigen, or may not bind specifically or preferentially. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some instances, antibodies that "specifically bind" to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen (i.e., baseline binding activity can only be detected in conventional methods).

[0120] In some implementations, the anti-TfR1 antibody, as described herein, has a suitable binding affinity for the target antigen (e.g., TfR1) or its antigenic epitope. As used herein, "binding affinity" refers to the apparent binding constant or K0. A K A It is the dissociation constant (K) D The inverse of ) . The anti-TfR1 antibodies described herein may have a binding affinity (K) for TfR1 of at least 2 nM, 1 nM, 0.5 nM, 0.1 nM or lower. D Increased binding affinity corresponds to decreased K. D The higher affinity of an antibody for the first antigen compared to the second antigen can be achieved by binding to the K+ receptor of the second antigen. A (or numerical value K) D Compared to higher K binding to the first antigen A (or a smaller value for K) DThis is indicated by [the specific antigen]. In such cases, the antibody is specific to the first antigen (e.g., the first protein in the first conformation or its mimicry) relative to the second antigen (e.g., the same first protein in the second conformation or its mimicry; or the second protein). The difference in binding affinity (e.g., specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 90, 100, 500, 1000, 10,000, or 10 [units of measurement missing]. 5 In some implementations, any anti-TfR1 antibody can be further subjected to affinity maturation to increase the antibody's binding affinity to the target antigen or its epitope.

[0121] Binding affinity (or binding specificity) can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopic methods (e.g., using fluorescence assays). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of the bound protein relative to the target protein concentration. The concentration of the bound protein ([bound]) is generally correlated with the concentration of the free target protein ([free]) by the following equation: [bound] = [free] / (Kd + [free]).

[0122] However, it is not always necessary to make K. A The precise determination is necessary because sometimes a quantitative measurement of affinity is obtained (e.g., determination is made using methods such as ELISA or FACS analysis, with K...). A It is proportional and therefore can be used for comparison, such as determining whether the affinity is higher (e.g., 2 times higher), obtaining a qualitative measurement of affinity, or obtaining an inferred value of affinity (e.g., by functional assays such as activity in vitro or in vivo assays) is sufficient.

[0123] A. Example anti-TfR1 antibody In some embodiments, the anti-TfR1 antibodies provided herein are humanized antibodies derived from mouse monoclonal antibody clones. Briefly, the heavy and light chain complementarity-determining domains of a mouse parent are transplanted into the shared human VH and VL frames to generate a parental humanized antibody. A humanized library is generated using Kunkel mutagenesis to introduce variants at certain frame region locations, and CDR-FR linker sites are constructed and clones exhibiting high binding affinity for human TfR1 are screened. The selected mab1G clone showed the highest binding affinity and was therefore selected for further affinity maturation. See the examples below. Exemplary anti-TfR1 antibodies derived from affinity maturation, containing variants in certain CDRs relative to the mab1G parent, exhibit further enhanced binding affinity. Certain clones, such as Fab0061 and Fab0070, showed superior rodent and cynomolgus monkey cross-activity exceeding that of the mouse parent (Fab version). Furthermore, some clones, such as Fab0061 and Fab0070, showed an approximately 4-fold increase in expression yield compared to the mouse parents in Fab form.

[0124] Therefore, the anti-TfR1 antibody provided herein (e.g., in Fab form) may comprise a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL), each comprising three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. In some cases, the anti-TfR1 antibody contains a heavy chain CDR3 shown as GTRAYHY (SEQ ID NO: 24) and a light chain CDR3 shown as QHFWGTPLT (SEQ ID NO: 13). The anti-TfR1 antibody may further comprise a heavy chain CDR1 having a shared motif of GX1TFX2SYWX3H, where X1 is F or Y; X2 is K, N, or T; and X3 is M or V; and a heavy chain CDR2 having a shared motif of EINPTNGRX4NYIEKFKS (SEQ ID NO: 23), where X4 is F, S, T, or V. Alternatively or additionally, the anti-TfR1 antibody may further comprise a light chain CDR1 having a concordant motif of RASDX5LYX6NLA (SEQ ID NO: 5), wherein X5 is G, K, or N, and X6 is R or S; and a light chain CDR2 having a concordant motif of DAX7X8LAS, wherein X7 is F, K, R, or T; and X8 is K, L, N, or R. In some instances, X1 in the concordant motif is F or Y; X2 is T; X3 is M; X4 is T or V; X5 is G or K; X6 is S; X7 is T; and X8 is L or K. In some embodiments, X1 in the concordant motif is F; X2 is T; X3 is M; and X4 is T. In some embodiments, X5 in the concordant motif is K; X6 is S; X7 is T; and X8 is L. In some implementations, the common motifs are: X1 is F; X2 is T; X3 is M; X4 is T; X5 is K; X6 is S; X7 is T; and X8 is L.

[0125] In some implementations, X1 is F. In some implementations, X1 is Y.

[0126] In some implementations, X2 is K. In some implementations, X2 is N. In some implementations, X2 is T.

[0127] In some implementations, X3 is M. In some implementations, X3 is V.

[0128] In some implementations, X4 is F. In some implementations, X4 is S. In some implementations, X4 is T. In some implementations, X4 is V.

[0129] In some implementations, X5 is G. In some implementations, X5 is K. In some implementations, X5 is N.

[0130] In some implementations, X6 is R. In some implementations, X6 is S.

[0131] In some implementations, X7 is F. In some implementations, X7 is K. In some implementations, X7 is R. In some implementations, X7 is T.

[0132] In some implementations, X8 is K. In some implementations, X8 is L. In some implementations, X8 is N. In some implementations, X8 is R.

[0133] Exemplary combinations of light chain CDRs for anti-TfR1 antibodies provided herein are provided in Table 1 below. Exemplary combinations of heavy chain CDRs for anti-TfR1 antibodies provided herein are provided in Table 2 below. In some cases, anti-TfR1 antibodies may comprise one or more of the heavy and light chain framework regions provided in Tables 3 and 4 below.

[0134] In some embodiments, the anti-TfR1 antibody described herein binds to the same TfR1 polypeptide epitope as any of the exemplary antibodies described herein (e.g., those disclosed in Table 5, such as clones Fab0061 or Fab0070), or competes with the exemplary antibody for binding to the TfR1 antigen. In some instances, the exemplary antibody is Fab0061. In other instances, the exemplary antibody is Fab0070. An epitope refers to a site on a target antigen that is recognized and bound by an antibody. This site may consist entirely of an amino acid component, entirely of chemical modifications of amino acids in a protein (e.g., glycosyl moieties), or a combination thereof. Overlapping epitopes include at least one common amino acid residue. Epitopes may be linear and typically 6-15 amino acids in length. Alternatively, epitopes may be conformational. Epitopes to which the antibody binds can be determined using conventional techniques, such as epitope mapping (see, for example, described below). Antibodies that bind to the same epitopes as the exemplary antibodies described herein may bind to the exact same epitopes or substantially overlapping epitopes (e.g., containing fewer than 3 non-overlapping amino acid residues, fewer than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue). Whether two antibodies compete with each other to bind to a homologous antigen can be determined by a competitive assay, which is well known in the art.

[0135] In some instances, anti-TfR1 antibodies contain the same V as the exemplary antibodies described herein (e.g., those provided in Table 5 such as Fab0061 or Fab0070). H and / or V L CDR. Both antibodies have the same V. H and / or V LCDR means that when determined by the same method (e.g., the Kabat method, Chothia method, AbM method, contact method, or IMGT method known in the art; see, for example, bioinf.org.uk / abs / ), their CDRs are identical. Such anti-TfR1 antibodies may have the same V compared to the exemplary antibodies described herein. H The same V L Or both.

[0136] Also within the scope of this disclosure are functional variants of any exemplary anti-TfR1 antibodies as disclosed herein. Such functional variants are substantially similar to the exemplary antibodies in both structure and function. The functional variants contain a V that is substantially the same as that of the exemplary antibodies. H and V L CDR. For example, it can be a variation of up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residues contained only in the total CDR region of the antibody, and with substantially similar affinity (e.g., having K at the same order of magnitude). D The value) binds to the same epitope of TfR1. In some cases, the functional variant may have the same heavy chain CDR3 as the exemplary antibody, and optionally the same light chain CDR3 as the exemplary antibody. Alternatively or additionally, the functional variant may have the same heavy chain CDR2 as the exemplary antibody. (The last sentence appears to be incomplete and possibly refers to a different context.) H In contrast, such anti-TfR1 antibodies can contain V only with CDR amino acid residue variations in the heavy chain CDR1. H Fragment. In some instances, anti-TfR1 antibodies may further comprise V having the same V as the exemplary antibody. L CDR3, and optionally the same V L V of CDR1 or VL CDR2 L Excerpt.

[0137] Alternatively or additionally, amino acid residue variations can be conserved amino acid residue substitutions.

[0138] In some implementations, anti-TfR1 antibodies may contain such heavy chain CDRs as the V of the exemplary antibodies described herein. H Compared to the CDR, the heavy chain CDR individually or collectively has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. Alternatively or additionally, anti-TfR1 antibodies may comprise such light chain CDRs as the V of the exemplary antibodies described herein. LCompared to the CDR, the light chain CDR individually or collectively has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. As used herein, “individually” means that, relative to the corresponding CDR of the exemplary antibody, one CDR of the antibody shares the indicated sequence identity. “Collectively” means that, with respect to the three V of the combined antibody… H or V L CDR and the corresponding three V of the exemplary antibody in combination H or V L CDRs share the sequence identity indicated.

[0139] In some embodiments, the anti-TfR1 antibody may comprise a VH having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity with a VH of one of the exemplary antibodies provided in Table 5 (e.g., the VH of Fab0061 or Fab0070). Alternatively or additionally, the anti-TfR1 antibody may comprise a VL having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity with a VH of one of the exemplary antibodies provided in Table 5 (e.g., the VL of Fab0061 or Fab0070). Such variants may comprise the same heavy and light chain CDRs as the exemplary antibodies, wherein the variation is only in one or more of the FR regions.

[0140] In some embodiments, the heavy chain of any anti-TfR1 antibody as described herein may further include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). The heavy chain constant region may have any suitable origin, such as human, mouse, rat, or rabbit. Alternatively or additionally, the light chain of the anti-TfR1 antibody may further include a light chain constant region (CL), which may be any CL known in the art. In some instances, the CL is a κ light chain. In other instances, the CL is a λ light chain. Antibody heavy chains and light chain constant regions are well known in the art, such as those available in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0141] In some cases, anti-TfR1 antibodies are in Fab form, comprising a heavy chain containing any VH region and CH1 fragment disclosed herein, and a light chain containing any VL region and light chain constant region (LC) disclosed herein. The CH1 fragment may belong to any immunoglobulin (Ig) heavy chain constant region (e.g., human Ig heavy chain constant region). In some cases, CH1 may belong to an IgG (e.g., IgG1, IgG2, or IgG4) heavy chain. In some cases, the LC may be of human origin. It may be a κ chain. Alternatively, it may be a λ chain. Examples of anti-TfR1 Fab antibodies disclosed herein are provided in Table 5.

[0142] In some embodiments, the anti-TfR1 antibody disclosed herein may be a single-chain antibody (scFv). The scFv antibody may comprise a VH fragment and a VL fragment, which may be linked via a flexible peptide linker. In some cases, the scFv antibody may be VH→VL oriented (from the N-terminus to the C-terminus). In other cases, the scFv antibody may be VL→VH oriented (from the N-terminus to the C-terminus).

[0143] Any anti-TfR1 antibody disclosed herein may further include an Fc fragment, such as an Fc fragment derived from a human immunoglobulin (e.g., a human IgG molecule). In some instances, such anti-TfR1 antibodies may be Fab-Fc molecules, wherein the Fc fragment may be fused to a VH-CH1 chain or a VL-LC chain. An example of such a Fab-Fc molecule is provided in Table 5.

[0144] B. Preparation of anti-TfR1 antibody As described herein, antibodies capable of binding to TfR1 can be prepared by any method known in the art, such as via recombinant techniques. See, for example, Harlow and Lane (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0145] When needed, antibodies of interest (monoclonal or polyclonal) (e.g., produced by hybridoma cell lines or isolated from antibody libraries) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or amplification. The sequence encoding the antibody of interest can be maintained in a vector within host cells, and the host cells can then be amplified and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to, for example, humanize the antibody or improve affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is of a non-human origin and intended for use in clinical trials and treatments in humans, the constant region can be modified to more closely resemble the human constant region to avoid an immune response. Alternatively or additionally, it may be desirable to genetically manipulate the antibody sequence to obtain higher affinity and / or specificity for the target antigen, as well as greater efficacy in enhancing TfR1 activity. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to the antibody while still maintaining its binding specificity to the target antigen.

[0146] Alternatively, antibodies capable of binding to the target antigen (TfR1 molecule) as described herein can be isolated from a suitable antibody library via routine practice. The antibody library can be used via a routine screening process to identify proteins that bind to the target antigen (e.g., human TfR1, such as cell surface TfR1). During selection, peptide components are probed with the target antigen or fragments thereof, and if a peptide component binds to the target, it is typically identified by retention on a support. Retained display library members are recovered from the support and analyzed. Analysis may include amplification and subsequent selection under similar or different conditions. For example, positive and negative selection may alternate. Analysis may also include determining the amino acid sequence of the peptide component and purifying the peptide component for detailed characterization.

[0147] There are a variety of conventional methods known in the art for identifying and isolating antibodies that can bind to the target antigens described herein, including phage display, yeast display, ribosome display, or mammalian display techniques.

[0148] Genetically modified antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced via, for example, conventional recombinant technologies. In one instance, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a monoclonal antibody). Once isolated, the DNA can be placed in one or more expression vectors, which are then transfected into host cells to obtain monoclonal antibody synthesis in recombinant host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or otherwise non-immunoglobulin-producing myeloma cells. See, for example, PCT Publication WO 87 / 04462. DNA can then be modified, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking immunoglobulin coding sequences to all or part of the coding sequences of non-immunoglobulin polypeptides. In this way, genetically modified antibodies with binding specificity to target antigens, such as “chimeric” or “hybrid” antibodies, can be prepared.

[0149] Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one instance, following methods known in the art, the VH and VL variable regions of the parental nonhuman antibody were subjected to three-dimensional molecular modeling analysis. Next, using the same molecular modeling analysis, framework amino acid residues predicted to be important for the formation of the correct CDR structure were identified. In parallel, using the parental VH and VL sequences as search queries, human VH and VL chains with amino acid sequences homologous to those of the parental nonhuman antibody were identified from any antibody gene database. Human VH and VL receptor genes were then selected.

[0150] The CDR region within the selected human receptor gene can be replaced by a CDR region from a parental nonhuman antibody or a functional variant thereof. If necessary, residues predicted to be important in interacting with the CDR region within the framework region of the parental strand (see description above) can be used to replace the corresponding residues in the human receptor gene.

[0151] Single-chain antibodies can be prepared via recombinant technology by linking nucleotide sequences encoding the heavy chain variable region with nucleotide sequences encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the techniques described above for single-chain antibody production (US Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce scFv libraries that display phages, yeast, mammalian cells, or mRNA, and TfR1-specific scFv clones can be identified from the libraries following standard procedures. Positive clones can undergo further screening to identify those that enhance CD19 activity.

[0152] Antibodies obtained following the methods known in the art and described herein can be characterized using methods well-known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” Many methods known in the art exist for mapping and characterizing epitope localization on proteins, including resolving the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In another instance, epitope mapping can be used to determine the sequence to which the antibody binds. Epitopes can be linear epitopes, i.e., contained in a single amino acid segment, or conformational epitopes formed by the three-dimensional interactions of amino acids, which are not necessarily contained in a single segment (a linear sequence of primary structure). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for antibody binding assays. In another example, epitopes to which antibodies bind can be identified in systematic screening by using overlapping peptides derived from the target antigen sequence and determining antibody binding. Based on gene fragment expression assays, open reading frames encoding the target antigen are randomly or through specific genetic constructs, and the reactivity of the expressed antigen fragment with the antibody to be tested is determined. For example, gene fragments can be produced by PCR, then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Some epitopes can also be identified using large libraries (phage libraries) of random peptide sequences displayed on the surface of phage particles.

[0153] Alternatively, a defined library of overlapping peptide fragments can be used to test antibody binding in a simple binding assay. In another instance, mutagenesis of the antigen-binding domain, domain exchange assays, and alanine scan mutagenesis can be performed to identify sufficient and / or necessary residues required for epitope binding. For example, domain exchange assays can be performed using mutants of the target antigen, where various fragments of TfR1 have been replaced (exchanged) with sequences from closely related but antigenically different proteins (e.g., another member of the tumor necrosis factor receptor family). By evaluating antibody binding to mutant TfR1, the importance of specific antigen fragments for antibody binding can be assessed.

[0154] Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[0155] In some instances, anti-TfR1 antibodies (e.g., in Fab form) are prepared using recombinant techniques illustrated below.

[0156] Nucleic acids encoding the heavy and light chains of the anti-TfR1 antibody as described herein can be cloned into an expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one example, the nucleotide sequences encoding the heavy and light chains are operatively linked to different promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operatively linked to a single promoter, such that both the heavy and light chains are expressed by the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0157] In some instances, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each chain can be isolated from the host cell expressing that chain, and the isolated heavy and light chains can be mixed and incubated under suitable conditions that allow for antibody formation.

[0158] Generally, nucleic acid sequences encoding one or all strands of an antibody can be cloned into a suitable expression vector and operatively ligated to a suitable promoter using methods known in the art. For example, under suitable conditions, the nucleotide sequence and the vector can be contacted with a restriction endonuclease to generate complementary ends on each molecule, which can then pair with each other and be ligated together with a ligase. Alternatively, synthetic nucleic acid adapters can be ligated to the ends of genes. These synthetic adapters contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used for antibody production.

[0159] Various promoters can be used to express the antibodies described herein, including but not limited to the cytomegalovirus (CMV) immediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter, and herpes simplex virus TK promoter.

[0160] Adjustable promoters can also be used. Such adjustable promoters include those that use lac repressors from E. coli as transcription regulators to regulate transcription from mammalian cell promoters carrying lac operons [Brown, M. et al., Cell, 49: 603-612 (1987)], and those that use tetracycline repressors (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)]. Other systems include the FK506 dimer using estradiol, RU486, dihydroxymilosterone, or rapamycin, VP16, or p65. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0161] A tunable promoter comprising a repressor and an operon can be used. In one embodiment, a lac repressor from *E. coli* can act as a transcription regulator to regulate transcription from mammalian cell promoters carrying the lac operon [M. Brown et al., Cell, 49: 603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992)]. A tetracycline repressor (tetR) is combined with a transcription activator (VP 16) to produce a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), which is then combined with a minimal promoter carrying tetO derived from the major immediate early promoter of human cytomegalovirus (hCMV) to produce a tetR-tet operon system controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor alone (tetR), rather than a tetR-mammalian cell transcription factor fusion derivative, can act as a potent trans-regulator to control gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16): 1392-1399 (2003)). A particular advantage of this tetracycline-induced switch is that it does not require the use of a tetracycline repressor-mammalian cell trans-activator or a repressor fusion protein, which can be toxic to cells in some cases (Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)) to achieve its regulated effect.

[0162] Additionally, the vector may contain some or all of the following: selectable marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyomavirus origin of replication and ColE1 for appropriate episome replication; internal ribosome binding sites (IRESes), multifunctional multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing transgenic vectors are well known and available in the art.

[0163] Examples of polyadenylation signals that can be used to practice the methods described herein include, but are not limited to, human type I collagen polyadenylation signals, human type II collagen polyadenylation signals, and SV40 polyadenylation signals.

[0164] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any antibody can be introduced into suitable host cells for antibody production. The host cells can be cultured under suitable conditions for expressing the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered from the cultured cells (e.g., cells or culture supernatant) via conventional methods (e.g., affinity purification). If necessary, the antibody polypeptide chain can be incubated under suitable conditions for a suitable period of time to allow antibody production.

[0165] In some embodiments, the methods described herein for preparing antibodies involve a recombinant expression vector encoding both the heavy and light chains of the anti-TfR1 antibody as described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains that form the antibody, the polypeptide chains of which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow antibody formation.

[0166] In one example, two recombinant expression vectors are provided: one encoding a heavy chain of an anti-TfR1 antibody, and the other encoding a light chain of an anti-TfR1 antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each expression vector can be introduced into a suitable host cell individually. Positive transformants can be selected and cultured under suitable conditions that allow for antibody polypeptide chain expression. When both expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow for antibody formation. When the two expression vectors are introduced into different host cells, each expression vector can be recovered from the respective host cell or culture medium. The two polypeptide chains can then be incubated under suitable conditions for antibody formation.

[0167] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. For example, some antibodies can be separated by affinity chromatography using a protein A or protein G-conjugated matrix.

[0168] Any nucleic acid encoding the heavy chain, light chain, or both of the anti-TfR1 antibody as described herein, a vector containing such nucleic acid (e.g., an expression vector), and a host cell containing the vector are all within the scope of this disclosure. Methods for producing anti-TfR1 antibodies via expressing such nucleic acids in host cells and harvesting the resulting antibodies from the host cells and / or culture supernatant are also within the scope of this disclosure.

[0169] II. Anti-TfR1 antibody conjugate In some aspects, any anti-TfR1 antibody disclosed herein can be linked to one or more payloads to form an anti-TfR1 antibody conjugate. In some embodiments, the anti-TfR1 antibody in any conjugate provided herein can be in Fab form. Exemplary anti-TfR1 antibodies (e.g., Fab antibodies) are provided in Table 5 below, any of which can be used to prepare the conjugates disclosed herein. In some specific examples, the anti-TfR1 antibody is derived from Fab0061 (e.g., Fab0061 or a functional variant thereof). In other specific examples, the anti-TfR1 antibody is derived from Fab0070 (e.g., Fab0070 or a functional variant thereof).

[0170] Any anti-TfR1 antibody conjugate provided herein, such as a TfR1 Fab conjugate payload such as interfering RNA, can deliver the payload (e.g., the interfering RNA molecule) into the central nervous system (CNS) via the patient's brain endothelial cells. Without being bound by theory, the conjugate can bind to the transferrin receptor (TfR1) on the surface of brain endothelial cells. The payload, such as the interfering RNA, can be attached by any acceptable means for linking the anti-TfR1 antibody (e.g., Fab) to the payload (e.g., the interfering RNA), such that the payload (e.g., the interfering RNA) can be transferred across the cell membrane in a pharmaceutically active form. In some embodiments, chemical conjugation techniques can be used to link TfR1-specific antibodies (e.g., Fab) to TfR1-specific antibodies. In addition to covalent bonding, the conjugate can also be formed using non-covalent bonds, such as those formed by bifunctional antibodies, ionic bonds, hydrogen bonds, hydrophobic interactions, etc.

[0171] The payload, such as an interfering RNA molecule, can be directly covalently linked to a TfR1-specific antibody, such as Fab. Alternatively, the payload, such as the interfering RNA, can be linked to the antibody via a spacer. In some instances, the spacer can be a glycine spacer (e.g., having 1, 2, 3, or 4 glycines). In some embodiments, the spacer can be a glycine spacer containing 2 or 3 glycines.

[0172] A. Oligonucleotide-based reagents, including RNAi reagents The payload in any conjugate can be any type of molecule with the desired biological activity. In some cases, the payload can be a therapeutic agent. Alternatively, the payload can be a diagnostic agent. In some embodiments, the payload can be an oligonucleotide-based reagent.

[0173] As used in this article, "oligonucleotide-based reagents" contain approximately 10-50 (For example, 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 1) 4, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 1 8 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32,28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 Nucleotides comprising 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50 nucleotides or nucleotide base pairs. In some embodiments, the oligonucleotide-based reagent has a nucleobase sequence that is at least partially complementary to the coding sequence in the target nucleic acid or target gene expressed in the cell. In some embodiments, upon delivery to a cell expressing the target gene, the oligonucleotide-based reagent is capable of inhibiting the expression of the underlying gene and is referred to herein as an "expression-inhibiting oligonucleotide-based reagent". Gene expression can be suppressed in vitro or in vivo.

[0174] "Oligonucleotide-based reagents" include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligonucleotide-based reagent is a single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO). In some embodiments, the oligonucleotide-based reagent is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide-based reagent is a double-stranded oligonucleotide that is an RNAi reagent.

[0175] In some embodiments, the oligonucleotide-based reagent may be an “RNAi reagent,” which, as defined herein, is a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. As used herein, RNAi reagents may function via RNA interference mechanisms (i.e., by inducing RNA interference through interaction with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC), or through any alternative mechanism or pathway. While it is considered that RNAi reagents function primarily through RNA interference mechanisms as used herein, the disclosed RNAi reagents are not bound to or limited by any particular pathway or mechanism of action. The RNAi reagents disclosed herein comprise sense and antisense strands and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi reagents described herein is at least partially complementary to the mRNA to be targeted. RNAi reagents may include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0176] Typically, an RNAi reagent may comprise at least a sense strand (also known as a guest strand) including a first sequence and an antisense strand (also known as a guide strand) including a second sequence. The sense and antisense strands of the RNAi reagent may each be 16 to 49 nucleotides in length. In some embodiments, the sense and antisense strands of the RNAi reagent are independently 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. The sense and antisense strands may be the same length or different lengths. The RNAi reagent includes an antisense strand sequence that is at least partially complementary to a sequence in the target gene, and upon delivery to cells expressing the target, the RNAi reagent can inhibit the expression of one or more target genes in vivo or in vitro.

[0177] All positions in a given compound do not need to be uniformly modified. Instead, more than one modification can be incorporated into a single oligonucleotide-based reagent, or even into a single nucleotide.

[0178] The sense and antisense strands of the RNAi reagent can be synthesized and / or modified by methods known in the art. Further disclosures relating to RNAi reagents may be found, for example, in modified disclosures, such as International Patent Application No. PCT / US2017 / 045446 (WO2018027106) granted to Arrowhead Pharmaceuticals, Inc., which is also incorporated herein by reference for the purposes and purposes described herein.

[0179] B. Oligonucleotide-based reagent modification Any oligonucleotide-based reagent, such as an RNAi reagent, may contain one or more modifications. Exemplary modifications may include any modification to sugars, nucleobases, nucleoside-to-nucleotide bonds (e.g., linkages to phosphate ester / phosphodiester bonds / phosphodiester backbones), and any combination thereof. Some exemplary modifications provided herein are described in detail below.

[0180] (a) Modified nucleotides In some embodiments, any oligonucleotide-based reagents provided herein, such as RNAi reagents, may comprise modified nucleotides and / or one or more non-phosphodiester bonds. As used herein, “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxynucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides in the oligonucleotide-based reagents, such as RNAi reagents, may be modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, debased nucleotides, 2′-modified nucleotides, 3′ to 3′-bonded (reverse) nucleotides, nucleotides containing non-natural bases, bridged nucleotides, peptide nucleic acids, 2′,3′-open-ring nucleotide mimics (unlocked nucleobase analogs, locked nucleotides, 3′-O-methoxy (2′-nucleoside-linked) nucleotides, 2′-F-arabinonucleotides, 5′-Me, 2'-Fluoronucleotides, morpholinonucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. 2′-Modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring) include, but are not limited to, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, 2′-methoxyethyl (2′-O-2-methoxyethyl) nucleotides, 2′-amino nucleotides, and 2′-alkyl nucleotides. All positions in a given compound need not be uniformly modified. Instead, more than one modification can be incorporated into a single target RNAi reagent, or even into a single nucleotide. The sense and antisense strands of the target RNAi reagent can be synthesized and / or modified using methods known in the art. Modification at one nucleotide does not depend on modification at another nucleotide.

[0181] Furthermore, one or more nucleotides in oligonucleotide-based reagents, such as RNAi reagents, can be linked via non-standard bonding or a backbone (i.e., modified internucleotide bonding or a modified backbone). Modified internucleotide bonding can be phosphate-free covalent internucleotide bonding. Modified internucleotide bonds or skeletons include, but are not limited to, 5′-thiophosphate groups, chiral thiophosphates, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, alkylphosphonates (e.g., methylphosphonates or 3′-alkylenephosphonates), chiral phosphonates, hypophosphonates, aminophosphates (e.g., 3′-aminoaminophosphates, aminoalkylaminophosphates or thionophosphoramidates), thioalkylphosphonates, thioalkyl phosphate triesters, morpholino bonds, borane phosphates with normal 3′-5′ bonds, analogs of borane phosphates with 2′-5′ linkages, or borane phosphates with reverse polarity, wherein adjacent nucleoside unit pairs are 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′ linked.

[0182] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of 2-aminoadenine, adenine, and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- Thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazoguanine, 7-deazoadenine, 3-deazoguanine and 3-deazoadenine.

[0183] In some embodiments, all or substantially all nucleotides of the RNAi reagent are modified nucleotides. An RNAi reagent, as used herein, in which substantially all nucleotides are modified nucleotides, is an RNAi reagent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are unmodified ribonucleotides in both the sense and antisense strands. A sense strand, as used herein, in which substantially all nucleotides are modified nucleotides, is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the sense strand. An antisense strand, as used herein, in which substantially all nucleotides are modified nucleotides, is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the sense strand. In some embodiments, one or more nucleotides of the RNAi reagent are unmodified ribonucleotides.

[0184] (b) Modified internucleotide bonds In some embodiments, one or more nucleotides of the RNAi reagent are linked by non-standard bonds or a backbone (i.e., modified internucleotide bonds or a modified backbone). Modified internucleotide bonds or backbones include, but are not limited to, thiophosphate groups (represented herein as lowercase "s"), chiral thiophosphates, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, alkylphosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, hypophosphonates, aminophosphates (e.g., 3'-aminoaminophosphates, aminoalkylaminophosphates, or thioaminophosphates), thioalkylphosphonates, thioalkylphosphate triesters, morpholino bonds, borane phosphates with normal 3'-5' bonds, analogs of 2'-5' linked borane phosphates, or borane phosphates with reverse polarity, wherein adjacent nucleoside unit pairs are 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′ linked. In some embodiments, the modified internucleotide bonds or backbones lack a phosphorus atom. Modified internucleotide bonds lacking a phosphorus atom include, but are not limited to, bonds between short-chain alkyl or cycloalkyl sugars, bonds between mixed heteroatoms and alkyl or cycloalkyl sugars, or bonds between one or more short-chain heteroatoms or heterocyclic sugars. In some embodiments, the modified internucleotide backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, olefin-containing backbones, aminosulfonate backbones, methyleneimino and methylenehydrazine backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0185] In some embodiments, the sense strand of the RNAi reagent may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds, and the antisense strand of the RNAi reagent may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds, or both the sense and antisense strands may independently contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds. In some embodiments, the sense strand of the RNAi reagent may contain 1, 2, 3, or 4 phosphate-thioester bonds, and the antisense strand of the RNAi reagent may contain 1, 2, 3, or 4 phosphate-thioester bonds, or both the sense and antisense strands may independently contain 1, 2, 3, or 4 phosphate-thioester bonds.

[0186] In some embodiments, the sense strand of the RNAi reagent contains at least two phosphate-thioester nucleoside bonds. In some embodiments, the at least two phosphate-thioester nucleoside bonds are located between nucleotides at positions 1-3 starting from the 3' end of the sense strand. In some embodiments, one phosphate-thioester nucleoside bond is located at the 5' end of the sense strand, and the other phosphate-thioester bond is located at the 3' end of the sense strand. In some embodiments, two phosphate-thioester nucleoside bonds are located at the 5' end of the sense strand, and the other phosphate-thioester bond is located at the 3' end of the sense strand. In some embodiments, the sense strand does not include any phosphate-thioester nucleoside bonds between nucleotides, but contains one, two, or three phosphate-thioester bonds between terminal nucleotides at both the 5' and 3' ends and optionally present reverse debasement residues at the end cap. In some embodiments, the targeting ligand is linked to the sense strand via phosphate-thioester bonds.

[0187] In some embodiments, the antisense strand of the RNAi reagent contains four phosphate-thioester nucleoside bonds. In some embodiments, the four phosphate-thioester nucleoside bonds are between nucleotides at positions 1-3 starting from the 5' end of the antisense strand, and between nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 starting from the 5' end. In some embodiments, three phosphate-thioester nucleoside bonds are located between positions 1-4 starting from the 5' end of the antisense strand, and a fourth phosphate-thioester nucleoside bond is located between positions 20-21 starting from the 5' end of the antisense strand. In some embodiments, the RNAi reagent contains at least three or four phosphate-thioester nucleoside bonds in the antisense strand.

[0188] In some embodiments, the RNAi reagent contains one or more modified nucleotides and one or more modified nucleosides. In some embodiments, a 2′-modified nucleoside is combined with the modified nucleoside linker.

[0189] Exemplary modifications of oligonucleotide-based reagents, such as RNAi reagents, are provided in Tables 8 and 9 below, each within the scope of this disclosure.

[0190] C. CNS gene targets based on oligonucleotide reagents The oligonucleotide-based reagents described herein, such as RNAi reagents, may have targeted complementary antisense sequences for mRNAs encoding proteins such as amyloid precursor protein (APP; UniProt: P05067), ataxia-1, ataxia-2 ​​(ATXN2; UniProt: Q99700), ataxia-3 (ATXN3; UniProt: P54252), ataxia-7, androgen receptor (AR; UniProt: P10275), superoxide dismutase [Cu-Zn] (SOD1; UniProt: P00441), huntingtin (HTT; UniProt: P42858), chromosome 9 open reading frame 72 (C9ORF72; UniProt: Q96LT7), leucine-rich serine / threonine protein kinase 2 (LRRK2; Q5S007), and complement C3. (C3; UniProt: P01024), microtubule-associated protein tau (MAPT; UniProt: P10636), α-2A adrenergic receptor (ADRA2A; UniProt: P08913), sodium channel protein type 9 subunit α (SCN9A; UniProt: Q15858), apolipoprotein E (APOE; UniProt: P02649), α-synuclein (SNCA; UniProt: P37840), possible G protein-coupled receptor 75 (GPR75; UniProt: O95800), and RNA-binding protein FUS (FUS: UniProt: P35637), GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME and DYRK1A. Knockdown of these proteins is expected to be effective in treating or preventing CNS conditions including: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau proteinosis, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinosis, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

[0191] In some implementations, the oligonucleotide-based reagents that target androgen receptor (AR) mRNA are selected from those shown in Tables 8 and 9.

[0192] The ability of interfering RNA to knock down endogenous target gene expression levels in cells such as HeLa can be evaluated in vitro as follows. HeLa cells were plated in standard growth medium (e.g., DMEM supplemented with 10% fetal bovine serum) 24 hours prior to transfection. Transfection was performed using, for example, Dharmafect 1 (Dharmacon, Lafayette, Colo.) at interfering RNA concentrations ranging from 0.1 nM to 100 nM, according to the manufacturer's instructions. SiCONTROL™ Non-TargetingsiRNA #1 and siCONTROL™ Cyclophilin B siRNA (Dharmacon) were used as negative and positive controls, respectively. 24 hours post-transfection, target mRNA and cyclophilin B mRNA (PPIB, NM_000942) levels were evaluated by qPCR using, for example, the TAQMAN® Gene Expression Assay (Applied Biosystems, Foster City, Calif.), which preferably overlaps with the target site. When transfection efficiency was 100%, the positive control siRNA gave near-complete knockdown of cyclin B mRNA. Therefore, target mRNA knockdown was corrected for transfection efficiency by referencing cyclin B mRNA levels in cells transfected with cyclin B siRNA. Target protein levels can be evaluated, for example, by Western blotting, approximately 72 hours post-transfection (actual time depends on protein turnover). Standard techniques for isolating RNA and / or proteins from cultured cells are well known to those skilled in the art. To reduce the probability of nonspecific off-target effects, the lowest possible concentration of interfering RNA that produces the desired knockdown level of target gene expression is used. Cultured human endothelial cells can be used to assess the ability of interfering RNA to knock down endogenous target gene levels.

[0193] D. Preparation of anti-TfR1 antibody conjugates The anti-TfR1 antibody conjugates provided herein can be prepared using conventional techniques (e.g., chemical conjugation techniques) or methods disclosed herein (e.g., see Example 6 below). In some cases, oligonucleotide-based reagents, such as RNAi reagents, can be conjugated to chemical linkers, such as L20-p shown in Example 6, via a standard amide reaction. Modified oligonucleotide-based reagents can be conjugated to anti-TfR1 antibodies (e.g., in Fab form) via conventional chemical reactions.

[0194] In some embodiments, the anti-TfR1 antibody may be modified (capped) with a chemical entity, such as CP-113 or N-ethylmaleimide, before or after conjugation with an oligonucleotide-based reagent (see Example 6 below). In some cases, the antibody-oligonucleotide conjugates provided herein may contain chemical linkers and / or caps.

[0195] III. Pharmaceutical Composition Any anti-TfR1 antibody or anti-TfR1 antibody conjugate can be formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable loads. Depending on the payload conjugated to the antibody, such pharmaceutical compositions can be used for therapeutic or diagnostic purposes, for example, in the treatment methods disclosed herein.

[0196] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are substances other than the active pharmaceutical ingredient (API, therapeutic product) that are intentionally included in a drug delivery system. Excipients do not exert or are not intended to exert therapeutic effects at the intended dosage. Excipients may be used to: a) facilitate the processing of the drug delivery system during manufacturing; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other property of the overall safety and effectiveness of API delivery during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0197] Excipients include, but are not limited to: absorption enhancers, anti-adhesion agents, defoamers, antioxidants, adhesives, buffers, carriers, coating agents, pigments, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, flow aids, wetting agents, lubricants, oils, polymers, preservatives, brine, salt, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, stretching agents, mediators, waterproofing agents, and wetting agents.

[0198] The pharmaceutical compositions described herein may contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional components are pharmaceutically active materials. Pharmaceutically active materials include, but are not limited to: antipruritics, astringents, local anesthetics or anti-inflammatory drugs (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers and / or vaccines.

[0199] Pharmaceutical compositions may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts for altering osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other agents with known therapeutic benefits.

[0200] Depending on whether local or systemic treatment is desired and the area to be treated, the pharmaceutical composition can be administered in a variety of ways. Administration can be performed in any manner commonly known in the art, including, but not limited to, local (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or blowing in of powder or aerosol, including via nebulizer, intratracheal, or intranasal), epidermal, transdermal, oral, or parenteral administration. Parenteral administration includes, but is not limited to, intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal (e.g., via an implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered via subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, sugar-coated tablets, hard gelatin capsules or soft gelatin capsules, solutions, emulsions, or suspensions. Administration can also be performed rectally, for example, by suppository; locally or transdermally, for example, by ointment, cream, gel, or solution; or parenterally, for example, by injectable solutions.

[0201] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (when water-soluble) or dispersions, as well as sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor® EL (BASF, Parsippany, NJ), or phosphate-buffered saline. It should be stable under the conditions of manufacture and storage and should be preservative against microbial contamination such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0202] A sterile injectable solution can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above as required into a suitable solvent, followed by filtration and sterilization. Generally, a dispersion is prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0203] Formulations suitable for intra-articular application may be in the form of sterile aqueous formulations of any ligand described herein, which may be in microcrystalline form, such as in the form of an aqueous microcrystalline suspension. Liposome formulations or biodegradable polymer systems may also be used to deliver any ligand described herein for both intra-articular and ocular application.

[0204] The active compound can be prepared together with a carrier that protects the compound from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0205] Pharmaceutical compositions may contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, “pharmacologically effective amount,” “therapeuticly effective amount,” or simply “effective amount” refers to the amount of the active pharmaceutical ingredient that produces a pharmacological, therapeutic, or preventative result.

[0206] The present invention also aims to produce pharmaceutical agents containing anti-TfR1 antibodies or antibody fragments, whether conjugated or unconjugated to oligonucleotide-based compounds, and similarly, methods for manufacturing such pharmaceutical agents, comprising preparing one or more anti-TfR1 antibodies or antibody fragments disclosed herein, and one or more other substances having known therapeutic benefits, if necessary, into a pharmaceutically acceptable form.

[0207] The anti-TfR1 antibodies and antibody fragments disclosed herein, as well as pharmaceutical compositions comprising anti-TfR1 antibodies and antibody fragments, can be packaged into or included in kits, containers, packaging, or dispensers. The anti-TfR1 antibodies and antibody fragments, as well as pharmaceutical compositions comprising said anti-TfR1 antibodies and antibody fragments, can be packaged into pre-filled syringes or vials.

[0208] The pharmaceutical compositions provided herein may encompass more than one TfR1 antibody (e.g., Fab) conjugated to the same oligonucleotide-based reagent (e.g., interfering RNA), such as a cocktail of different antibodies (e.g., Fab) that react with TfR1, or more than one oligonucleotide-based reagent, such as interfering RNA, that targets different locations on a single mRNA.

[0209] The pharmaceutical composition is a formulation comprising, by weight, up to 99% of interfering RNA or its antibody conjugate mixed with a physiologically acceptable carrier medium, including, for example, water, buffer, saline, glycine, hyaluronic acid, mannitol, etc. The pH of the formulation is from about pH 4.0 to about pH 9.0, or from about pH 4.5 to about pH 7.4. Methods for preparing nanoparticles and their use in delivering pharmaceutical formulations are described in U.S. Patent No. 6,632,671, the entire disclosure of which is incorporated herein by reference. Methods for preparing nanoparticle-Fab conjugates and their use in delivering pharmaceutical formulations are described in U.S. Patent No. 6,372,250, the entire disclosure of which is incorporated herein by reference.

[0210] IV. Treatment Methods In some aspects, this disclosure provides a method for attenuating the expression of target mRNA in the CNS of a patient, comprising: (a) providing an anti-TfR1 antibody (e.g., Fab) conjugated oligonucleotide-based reagent, such as interfering RNA; and (b) administering the anti-TfR1 antibody conjugation to the CNS of a patient, wherein the oligonucleotide-based reagent, such as interfering RNA molecule, attenuates the expression of target mRNA in the CNS.

[0211] In some embodiments, this disclosure provides a method for preventing or treating CNS symptoms in a patient, the method comprising administering an anti-TfR1 antibody conjugate (e.g., an anti-TfR1 Fab conjugated to interfering RNA) to the patient. The antibody portion binds to TfR1, and the oligonucleotide-based reagent, such as interfering RNA, is transported into the CNS via the patient's brain endothelial cells.

[0212] Exemplary CNS conditions are selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, and tau disease. CNS brain regions for systemic targeting may include the cerebellum, cortex, striatum, tuberous sclerosis complex (TCS), or thoracic spinal cord.

[0213] In some implementations, the patient suffers from a CNS condition associated with endothelial cells, astrocytes, pericytes, microglia, neurons or oligodendrocytes, or another cell type of the CNS.

[0214] The methods disclosed herein can be used to attenuate the expression of specific genes in the CNS of a patient using RNA interference. In some embodiments, the TfR1-specific Fab-interfering RNA conjugate contains an interfering RNA molecule that targets genes associated with CNS conditions. Examples of mRNA target genes designed to target the interfering RNA of the present invention include genes associated with CNS conditions.

[0215] In some embodiments, methods for treating CNS diseases involve targeting specific cell types in the CNS. In some embodiments, TfR1 Fab-interfering RNA conjugates or TfR1 Fab-conjugated nanoparticles are delivered to the CNS via brain endothelial cells.

[0216] Animal models were used to test the activity of systemically delivered interfering RNA molecules. Examples of this application demonstrate the successful and efficient in vivo delivery of interfering RNA molecules to the CNS. For example, as described in Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr; 29(4): 341-5. doi: 10.1038 / nbt.1807. e.g., 20 March 2011. PMID: 21423189, targeted RNA interference can be tested in mice.

[0217] In other embodiments, the method of delivering interfering RNA molecules includes administering to a patient an antibody conjugated to nanoparticles, wherein the interfering RNA molecule is encapsulated within the nanoparticles, and the nanoparticles are linked to a TfR1 antibody, which transports the antisense oligonucleotide molecule into the patient's CNS via brain endothelial cell transport. Other embodiments of the invention provide methods for preventing or treating CNS conditions, the methods comprising delivering antisense oligonucleotide molecules into a patient's CNS via brain endothelial cells using Fab-conjugated nanoparticles.

[0218] TfR1 antibody (e.g., Fab) conjugated oligonucleotide-based reagents (e.g., interfering RNA) and nanoparticles containing such reagents can be administered via intravenous injection, oral administration, intramuscular injection, intranasal injection, intraperitoneal injection, intrathecal injection, inhalation, transdermal administration, sublingual administration, or transmucosal administration. The form and concentration (e.g., capsules, tablets, solutions, emulsions) of the TfR1 antibody (e.g., Fab) conjugated oligonucleotide-based reagent (e.g., interfering RNA) and nanoparticles containing such reagents will depend at least in part on the route of administration.

[0219] In some implementations, treatment of CNS conditions with interfering RNA molecules is achieved by directly administering a TfR1 antibody (e.g., Fab)-conjugated oligonucleotide-based reagent (e.g., interfering RNA) and nanoparticles containing such reagents covering the interfering RNA composition to the CNS. Therapeutic treatment of patients with interfering RNA targeting the target mRNA is expected to be more beneficial than small molecule therapies by increasing the duration of action, thereby allowing for less frequent dosing and greater patient compliance, and by increasing target specificity, thereby reducing side effects.

[0220] For example, the therapeutically effective dose of the formulation may depend on factors such as: the patient's age, race, and sex; the severity of the disease; the target gene transcript / protein turnover rate; antibody (e.g., Fab) affinity; the number of oligonucleotide-based reagents (e.g., interfering RNA molecules) per antibody (e.g., Fab); the potency of the oligonucleotide-based reagents (e.g., interfering RNA); and the stability of the oligonucleotide-based reagents (e.g., interfering RNA). In one embodiment, the interfering RNA is delivered at a therapeutic dose to improve the progression of the target gene-related disease.

[0221] In some embodiments, the present invention provides specific pharmaceutical compositions for the prevention or treatment of CNS conditions in patients, comprising, in a CNS-acceptable carrier and in a therapeutically effective amount of the interfering RNA-TfR1 Fab conjugate or nanoparticle-TfR1 Fab conjugate of the present invention. A therapeutically effective amount of the interfering RNA used in the compositions of the present invention results in an extracellular concentration of about 100 pM to 1 μM, or about 1 nM to 100 nM, or about 5 nM to about 50 nM, or about 25 nM at the surface of target cells. The dose required to achieve this local concentration will vary depending on a variety of factors, including in particular the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cells or tissue, and whether delivery is via intracranial local or systemic administration. For example, the composition may be delivered to the CNS via intravenous injection every two weeks, once a month, or for longer periods, depending on the judgment of a skilled clinician. The interfering RNA-TfR1 Fab conjugate and nanoparticle-TfR1 Fab conjugate of the present invention are administered as solutions, suspensions, or emulsions. TfR1 Fab-conjugated interfering RNA can be delivered in solutions, suspensions, or in biodegradable or non-biodegradable delivery devices.

[0222] V. Reagent Kit In some embodiments, this disclosure also provides a kit comprising reagents for attenuating mRNA expression in cells, as cited herein. The kit contains the necessary components for the production of oligonucleotide-based reagent-antibody conjugates (e.g., interfering RNA-Fab conjugates) and / or interfering RNA-Fab conjugates (e.g., interfering RNA molecules and Fab and necessary materials for ligation). The kit may also contain positive and negative control siRNA or shRNA expression vectors (e.g., non-targeted control siRNA or siRNA targeting unrelated mRNAs). The kit may also contain reagents for evaluating the knockdown of the intended target gene (e.g., primers and probes for quantitative PCR to detect the target mRNA, and / or antibodies against the corresponding protein for Western blotting). Alternatively, the kit may contain an siRNA sequence or shRNA sequence, along with instructions and materials necessary for generating siRNA by in vitro transcription or constructing an shRNA expression vector.

[0223] Further, a drug combination in the form of a kit is provided, comprising a carrier tool and a first container tool packaged together, the carrier tool being adapted to contain the container tool in a tightly constrained manner, the first container tool comprising a TfR1 antibody-conjugated interfering RNA composition. Where desired, such kits may further include one or more of a variety of conventional drug kit components, such as containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. The kit may also include printed instructions as inserts or labels indicating the quantity of components to be administered, instructions on administration, and / or instructions for mixing components.

[0224] The references cited in this article are incorporated by way of reference to the extent that they provide exemplary procedures or other details that complement the content set forth herein.

[0225] Based on this disclosure, those skilled in the art will understand that obvious modifications can be made to the embodiments disclosed herein without departing from the spirit and scope of the invention. In view of this disclosure, all embodiments disclosed herein can be prepared and performed without excessive experimentation. The full scope of the invention is set forth in the disclosure and its equivalent embodiments. This specification should not be construed as unduly narrowing the full scope of protection enjoyed by the invention.

[0226] General Technology Unless otherwise stated, practice of this disclosure will employ conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well described in the art. Such techniques are well illustrated in the literature, for example... Molecular Cloning: A Laboratory Manual, Second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, edited 1984); Methods in Molecular Biology Humana Press; Cell Biology: A Laboratory Notebook(JE Cellis, ed., 1989) Academic Press; Animal Cell Culture (RIFreshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, ed., 1993–8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, ed., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); Current Protocols in Immunology (JEColigan et al., editors, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., editors, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, editors, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, editors Harwood Academic Publishers, 1995);. DNA Cloning: A practical Approach Volumes I and II (edited by DN Glover, 1985); Nucleic Acid Hybridization ( Edited by BDHames & S.J. Higgins (1985); Transcription and Translation ( BD Hames & S.J. Higgins, editors (1984); Animal Cell Culture RI Freshney, editor (1986); Immobilized Cells and Enzymes IRL Press, (1986); and B. Perbal, A practical guide to Molecular Cloning 1984); FM Ausubel et al. (editors).

[0227] Without further elaboration, it is believed that those skilled in the art will be able to utilize the invention to its fullest extent based on the foregoing description. Therefore, the specific embodiments described below should be construed as exemplary only and do not limit the remainder of this disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subjects described herein. Example

[0228] Example 1. Humanization of TfR1 Fab Heavy chain subgroup III (humIII) and light chain ƙ Subgroup I (hum κ The human shared sequence (Carter P, Presta et al. Proc. Natl. Acad. Sci. USA 89, 4285-4289 (1992); Presta LG, et al. J. Immunol 151, 2623-2632 (1993); Kabat, EA, et al. Sequences of Proteins of Immunological Interest. 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) was used for the humanization of the anti-hTfR1 antibody. To generate the template pAY2 for future CDR exchange, humIII and hum ƙThe I gene is inserted into a phagemid designed to display human Fab on the surface of an M13 bacterial phage. Under the control of the phoA promoter, two open reading frames (OPFs) encode two peptide chains, one for each chain. The first ORF encodes the light chain, and the second ORF encodes the heavy chain fused to the C-terminal domain of the M13 minor capsid protein P3. Both peptide chains are guided for secretion via an N-terminal stII signal sequence.

[0229] single-stranded DNA (ssDNA) template for pAY2 Then, pAY2 was transformed into chemicompetent CJ236 cells (uracil deglycosidase deficient) using a micropulse electroporator (Bio-Rad). Single colonies were inoculated into 1 ml of 2YT starting medium containing 100 µg / ml ampicillin and 10 µg / ml chloramphenicol, and the resulting cultures were incubated at 37°C with shaking for 6 hours. M13KO7 helper phage (~10 g / ml) was then added. 10 The mixture was prepared by shaking at 37°C for 10 minutes and then transferring 300 ml of the mixture to 30 ml of 2YT containing 100 µg / ml ampicillin and 0.25 µg / ml uridine. After 18 hours of growth at 37°C, the phage was purified and ssDNA containing uracil was isolated using the EZNA® M13 DNA Mini Kit (Omega Biotek Inc).

[0230] Example 2. Six CDRs were incorporated into humIII for VH and humI for VL to generate humanized hmab1a. Kunkel mutagenesis was employed (Kunkel TA Proc Natl Acad Sci USA 82, 488-92 (1985); Sidhu S. S et al. Methods Enzymol 328, 333-63 (2000)) to construct the CDR exchange form of the humanized anti-hTfR1 antibody hmab1a. Six primers were designed to exchange the six CDRs of the mab0039 antibody into pAY2. The primers were individually phosphorylated with T4 polynucleotide kinase (NEB) at 37°C for 1 hour, then annealed at 90°C to a uracil-containing ssDNA template for 1 minute, followed by 3 minutes at 50°C and on ice. The oligonucleotides were extended with T7 DNA polymerase and ligated with T4 DNA ligase at 37°C for 1.5 hours to form covalently closed circular DNA. DNA was desalted and affinity purified using the Qiagen QIAquick DNA Purification Kit, and then transformed into XL-1 blue cells (a strain containing uracil glycosidase) via heat shock transformation. Small-scale DNA was purified using the Qiagen miniprep kit and sequenced to confirm the sequence. The plasmid hmab1a was then used to prepare uracil-containing ssDNA for humanized library construction.

[0231] Humanized Library (Construction of Library AY1) Based on published work (Baca M, et al. J. Biol. Chem. 272, 10678-10684 (1997)) and other therapeutic antibody sequences, the library was designed to include mouse and common human amino acid compositions at the following sites: VL: M4 (MTG), F71 (TWC); VH: A24 (RYC), V37 (RTC), F67 (NYC), I69 (HTT), R71 (SKT), D73 (RAW), K75 (ARM), N76 (ARC), L78 (KYG), A93 (DYG), R94 (ARG). The degenerate codons used for each site are shown in parentheses. M = A or C, W = A or T, R = A or G, Y = C or T, N = A, C, G or T, H = A, C or T, K = G or T, S = G or C, D = A, G or T. Two primers for VL and three primers for VH were designed to introduce these degenerate codons at desired sites via Kunkel mutagenesis. Primer phosphorylation and Kunkel mutagenesis were performed as described (Sidhu S. S et al. Methods Enzymol 328, 333-63 (2000)). The resulting covalently closed circular DNA was electroporated into electrocompetent SS320 cells to prepare a hmab1 humanized Fab library, as described (Sidhu S. S et al. Methods Enzymol 328, 333-63 (2000)). The titrated apparent diversity was 6.9 x 10⁸, greater than the designed theoretical diversity of 4.7 x 10⁶.

[0232] Selection of humanized hmab1 clones The selection process was similar to that previously described (Ye JD, et al., Proc Natl Acad Sci USA 105, 82-87 (2008)). The biotinylated ECD domain of hTfR1 (hTfR1-B) was used as the antigen. In the first round, 0.5 nmol of hTfR1-B was immobilized on magnetic beads (Promega) and incubated for 15 min with 10¹²–13 cfu of phage in 1 ml of PT (1 x PBS with 0.05% Tween 20), the PT being supplemented with 0.5% BSA and 0.2 mg / ml streptoacidin. The solution was then removed, and the beads were washed twice with PT and amplified for selection in subsequent rounds. In subsequent rounds, the purified phage pools were first incubated with streptoacidin beads for 15 min, and the supernatant was used for subsequent selection on a KingFisher magnetic particle processor (Thermo Fisher). Phages (10¹⁰–1¹ CFU) were incubated with decreasing concentrations of hTfR1-B (20–0.1 nM) for 15 minutes. Streptomycin beads were then added to the solution for 15 minutes to allow for capture of hTfR1-B along with the bound phages. The beads were washed five times with PT and resuspended in 100 µl of PT. After each round of selection, the recovered phages were amplified as described (Sidhu S. S et al. Methods Enzymol 328, 333–63 (2000)).

[0233] Screening using phage ELISA with low antigen concentrations (PESLA) After four rounds of selection, individual clones were analyzed by phage ELISA (Ye JD, et al., Proc Natl Acad Sci US A105, 82–87 (2008)). The procedure was modified to screen for high-affinity binding agents in a high-throughput manner. Ninety-six or more individual colonies were picked from fresh LB / Amp plates and inoculated into 300 μL of 2YT medium (containing 100 μg / mL ampicillin and 10¹⁰ PFU / mL M13KO7 helper phage) in 96-well deep-well plates and incubated overnight at 37 °C with shaking at 300 rpm. The deep-well plates were then centrifuged at 3500 rpm and 4 °C for 15 minutes to allow cell pellet formation. The supernatant was diluted 3-fold with phage dilution buffer (PBS, 0.5% BSA, and 0.05% Tween 20) to prepare phage solutions. 96-well Maxisorp plates were coated overnight at 4 °C with 100 μL of 0.1–0.2 μg / mL hTfR1 in 100 mM sodium bicarbonate coating buffer (pH 9.6). The coating solution was removed, and the Maxisorp plates were blocked for 1 hour with 200 μL / well of PBS containing 1% (w / v) BSA. After removing the blocking solution, the Maxisorp plates were washed four times with PBS (PT) with 0.05% (v / v) Tween 20 and incubated at room temperature with 100 μL / well of phage solution for 1 hour. After washing six times with PT buffer, the Maxisorp plates were incubated at room temperature for 30 minutes with 100 μL / well of anti-M13 / horseradish peroxidase conjugate (diluted 3000× in phage dilution buffer). After washing six times with PT buffer, Maxisorp plates were incubated with 100 μL / well of Ultra TMB-ELISA substrate for 5–10 minutes, quenched with 100 μL / well of 1 M phosphate, and read spectrophotometrically at 450 nm using a microplate reader. Higher signals were associated with more tightly bound clones due to the limiting effect of low antigen concentration. These tighter bound clones were micro-prepared and sequenced to identify unique clones.

[0234] Characterization of unique clones using competitive phage ELISA Twenty-four unique clones were identified from library AY1. Competitive phage ELISA was used to characterize the binding affinity of these clones. MaxiSorp ELISA plates were coated with hTfR1 overnight at 4°C and blocked with BSA at RT for 1 hour. Serial dilutions of hTfR1 were incubated with subsaturated concentrations of phage at RT for 1 hour, then added to blocked and washed ELISA plates. After 15 minutes of incubation and washing, the anti-M13 antibody / HRP conjugate was added and incubated for 30 minutes, followed by TMB development for 5–10 minutes and quenching with 1 M phosphate. Binding signals were analyzed using a plate reader. Mab1G showed the best binding affinity for hTfR1 (0.78 nM). Example 3. General method for affinity maturation of mAb1G The Construction of mab1G Affinity Mature Library (Library AY2) A uracil-containing single-stranded DNA template for mab1G was generated using a procedure similar to that described in Sidhu S. S et al., Methods Enzymol 328, 333–63 (2000). All six CDRs in the library were screened simultaneously. A single-amino acid CDR walking strategy was employed (Yang WP, et al., J. Mol. Biol. 254, 392–403 (1995)). Randomized positions included 24–25 and 27–34 in CDR-L1, 50–56 in CDR-L2, 90–97 in CDR-L3, 27–35 in CDR-H1, 50 and 52–58 in CDR-H2, and 95–102 in CDR-H3. Each position was individually randomized at a given CDR using the degenerate codon NNS to encode all 20 amino acids. Randomization was incorporated into the mab1G ssDNA template via Kunkel mutagenesis (Kunkel TA ProcNatl Acad Sci USA 82, 488–92 (1985); Sidhu S. et al. Methods Enzymol 328, 333–63 (2000)). Primer phosphorylation and Kunkel mutagenesis were performed as described (Sidhu S. et al. Methods Enzymol 328, 333–63 (2000)). The resulting covalently closed circular DNA was electroporated into electrocompetent SS320 cells to prepare the AY2 library, as described (Sidhu S. et al. Methods Enzymol 328, 333–63 (2000)). The titrated apparent diversity was 1.1 x 10¹⁰.

[0235] Selection of Affinity Maturation Antibodies Choose something similar to what has been previously described (Ye JD, et al.) Proc Natl Acad Sci USA 105 ， 82–87 (2008) were performed. The biotinylated ECD domain of hTfR1 (hTfR1-B) was used as the antigen. In each round except the first, the purified phage pool was first incubated with streptoacidin beads for 15 minutes, and the supernatant was used for subsequent selection on a KingFisher magnetic particle processor (Thermo Fisher). Phage (10 10-11 CFU was incubated with hTfR1-B (20, 0.3, 0.05, and 0.05 nM for rounds 1–4, respectively) for 15 min–1 h. Streptomycin magnetic beads were then added to the solution for 1.5–15 min to allow hTfR1-B to be captured along with the bound phage. In the third round, separate selection was performed in addition to the routine selection. After capture of the antigen / antibody complex on the beads, the beads were washed with PBS (PT) supplemented with 0.05% Tween 20, and >1000-fold competitive non-biotinylated hTfR1 was incubated with the beads at RT for 2.5 h. The beads were then washed five times with PT and resuspended in 100 µl PT. After each round of selection, the recovered phages were amplified as described (Sidhu S. S et al. Methods Enzymol 328, 333–63 (2000)).

[0236] Example 4. Combining beneficial mutations from different CDRs PESLA was applied to the third-round output (without hTfR1 competition) and the fourth-round output (with hTfR1 competition in the third round). Unique mutations were identified by sequencing. These mutations were combined using two different methods. In method one, individual clones with combined mutations were prepared by Kunkel mutagenesis. In the second method, a mini-library was generated by combining all identified mutations, and selection was performed for hTfR1-B. These methods collectively yielded 15 Fab clones (Fab0060-Fab0074).

[0237] Table 1-2 shows the CDR1 and CDR2 variants of VL and VH with combined beneficial mutations from Fab 0060-0074. CDR3 is the same sequence in 15 Fab clones VL (QHFWGTPLT; SEQ ID NO: 13) and VH (GTRAYHY; SEQ ID NO: 24).

[0238] Table 1. Complementarity Determining Regions (CDRs) of Light Chains Table 2. Complementarity Determining Regions (CDRs) of Heavy Chains Table 3 shows exemplary light chain framework regions used in the exemplary anti-TfR1 antibody provided herein. Exemplary CL sequences for use in the Fab light chain are also provided in Table 3.

[0239] Table 3. Exemplary Light Chain Framework Regions and Constant Regions Table 4 shows exemplary heavy chain framework regions used in the exemplary anti-TfR1 antibodies provided herein. Table 4 also provides exemplary CH1 sequences for use in the Fab heavy chain.

[0240] Table 4. Exemplary Heavy Chain Frame Region and Constant Region Fragments Table 5 below lists the VH and VL sequences of the exemplary anti-TfR1 antibodies provided herein, as well as their heavy and light chain sequences in Fab form.

[0241] Table 5. Sequences of exemplary anti-TfR1 Fab antibodies Example 5. Affinity binding parameters of TfR1 antibody determined by competitive binding. cTfR BLI dynamics Samples were analyzed via biolayer interferometry (BLI) to determine the kinetics against cynomolgus transferrin receptor 1 (cTfR1). In short, the Octet RH16 (Sartorius) was used for BLI measurements using a streptavidin biosensor (Sartorius p / n 18-5019) to irreversibly immobilize biotinylated cTfR1 (Acro Bio p / n TFR-C8249). Samples were serially diluted to 150–5 nM depending on the expected affinity, and five concentrations within this range were used for analysis. Sample dilutions were prepared internally and consisted of 10 mM PBS pH 7.4 (150 mM NaCl), 0.01% BSA, 0.002% Tween 20, and 0.01% kathon. 96-well plates were used for analysis, filled with the desired sample solutions and a row of 10 µg / mL cTfR1 for loading the streptavidin sensor. For each sample, one well was reserved for a reference sensor control (assessment of non-specific binding of cTfR1 to the surface at the highest sample concentration) and a sample reference (buffered in only the sample loaded with cTfR1). One well was also reserved for a second replicate of one of the five concentrations to assess sensor and method variability. Eight channels were used for the highest sensitivity provided by the Octet RH16. For each assay performed, the procedure was as follows: a sensor check for baseline assessment of the sensor alone in buffer was performed for 30 seconds, followed by 60 seconds for cTfR1 loading; then a baseline check for 90 seconds to verify the stability of cTfR1 immobilized to the sensor surface, followed by 180 seconds for sample binding, and finally 300 seconds for sample dissociation. An oscillation rate of 1000 was used for all steps. Sample results were analyzed in Octet Analysis software.

[0242] hTfR BLI dynamics Samples were analyzed via biolayer interferometry (BLI) to determine the kinetics against human transferrin receptor 1 (hTfR1). In short, an Octet RH16 (Sartorius) was used for BLI measurements using a streptavidin biosensor (Sartorius p / n 18-5019) to irreversibly immobilize biotinylated hTfR1 (Acro Bio p / nTFR-H82E5). Samples were serially diluted to 150–5 nM depending on the expected affinity, and five concentrations within this range were used for analysis. Sample dilutions were prepared internally and consisted of 10 mM PBS pH 7.4 (150 mM NaCl), 0.01% BSA, 0.002% Tween 20, and 0.01% kathon. 96-well plates were used for analysis, filled with the desired sample solutions and a row of 10 µg / mL hTfR1 for loading the streptavidin sensor. For each sample, one well was reserved for a reference sensor control (assessment of non-specific binding of hTfR1 to the surface at the highest sample concentration) and a sample reference (buffered in only the sample loaded with hTfR1). One well was also reserved for a second replicate of one of the five concentrations to assess sensor and method variability. Eight channels were used for the highest sensitivity provided by the Octet RH16. For each assay performed, the procedure was as follows: a sensor check for baseline assessment of the sensor alone in buffer was performed for 30 seconds, followed by 60 seconds for cTfR1 loading; then a baseline check for 90 seconds to verify the stability of cTfR1 immobilized to the sensor surface, followed by 180 seconds for sample binding, and finally 300 seconds for sample dissociation. An oscillation rate of 1000 was used for all steps. Sample results were analyzed in Octet Analysis software.

[0243] cTfR1 SPR dynamics Samples were analyzed via surface plasmon resonance (SPR) to determine the kinetics against cynomolgus transferrin receptor 1 (cTfR1). Briefly, a Biacore X100 (Cytiva) was used for SPR measurements using a CM5 chip (carboxymethylated dextran surface, Cytiva p / n BR100399), where antihistamine antibodies were irreversibly immobilized onto the surfaces of two flow cells for reversible capture of his-tagged cTfR1 (Acro Biosystems p / n TFR-C524a). The antihistamine antibodies were irreversibly immobilized via amine coupling using the Cytiva Amine Coupling Kit (p / n BR100050) and His Capture Kit (p / n28995056). Samples were serially diluted to 150–5 nM depending on the expected affinity, and five concentrations within this range were used for analysis. Sample diluents were prepared internally and consisted of 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20. Cytiva caps and vials compatible with the Biacore X100 were used for sample analysis. Each analytical cycle consisted of a capture step, followed by a sample or blank injection, and a final regeneration step that removed any sample and cTfR1 from the sensor surface. The capture step was performed at 5 µL / min with only a 24-second contact time on the active flow cell, followed by a jet wash with 0.1% Tween 20. Generally, 2 µg / mL hTfR1 was a sufficient concentration of appropriate hTfR1 to be observed for consistent capture between cycles. The sample or buffer was injected at 30 µL / min with a 180-second contact time and a 180-second dissociation time, followed by a jet wash with 0.1% Tween 20. Regeneration was performed using a 10 mM glycine solution (pH 1.5, provided in the His Capture Kit) at a rate of 30 µL / min with a 60-second contact time. Cycles were performed for each sample concentration analyzed, along with one cycle repeated for each concentration for variability assessment, and 2–3 buffer cycles. Results were analyzed in Biacore Evaluation Software. The buffer cycles were averaged and subtracted from the sample injections before kinetic fitting. For the analyzed samples, either a monovalent fit or a kinetic fit was specified. The accuracy of the fit was evaluated, and data points were excluded where applicable.

[0244] hTfR1 SPR dynamics Samples were analyzed via surface plasmon resonance (SPR) to determine the kinetics against human transferrin receptor 1 (hTfR1). In short, a Biacore X100 (Cytiva) was used for SPR measurements using a CM5 chip (carboxymethylated dextran surface, Cytiva p / n BR100399), where antihistamine antibodies were irreversibly immobilized onto the surfaces of two flow cells for reversible capture of his-tagged hTfR1 (Acro Biosystems p / n CD1-H5243). The antihistamine antibodies were irreversibly immobilized via amine coupling using the Cytiva Amine Coupling Kit (p / n BR100050) and His Capture Kit (p / n28995056). Samples were serially diluted to 150–5 nM depending on the expected affinity, and five concentrations within this range were used for analysis. Sample diluents were prepared internally and consisted of 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20. Cytiva caps and vials compatible with the Biacore X100 were used for sample analysis. Each analytical cycle consisted of a capture step, followed by a sample or blank injection, and a final regeneration step that removed any sample and hTfR1 from the sensor surface. The capture step was performed at 5 µL / min with only a 24-second contact time on the active flow cell, followed by a jet wash with 0.1% Tween 20. Generally, 2 µg / mL hTfR1 was a sufficient concentration of appropriate hTfR1 to be observed for consistent capture between cycles. The sample or buffer was injected at 30 µL / min with a 180-second contact time and a 180-second dissociation time, followed by a jet wash with 0.1% Tween 20. Regeneration was performed using a 10 mM glycine solution at pH 1.5 (provided in the His Capture Kit) at a rate of 30 µL / min with a 60-second contact time. Cycles were performed for each sample concentration analyzed, including one cycle repeated for each concentration for variability assessment, and 2–3 buffer cycles. Results were analyzed using Biacore Evaluation Software. The buffer cycles were averaged and subtracted from the sample injections before kinetic fitting. For the analyzed samples, either a monovalent fit or a kinetic fit was specified. The accuracy of the fit was evaluated, and data points were excluded where applicable. Figure 4 The binding affinity of the selected TfR1Fab, in KdD, is shown for Fab0002. Fab0002 has the structure shown below: Tables 6 and 7 show the binding parameters as measured by competitive ELISA, SPR, and BLI using siRNA conjugates.

[0245] Table 6. Human transferrin receptor 1 (hTfR1) binding parameters as measured by competitive ELISA, SPR, and BLI. Table 7. Cynomolgus monkey transferrin receptor 1 (cTfR1) binding parameters as measured by competitive ELISA, SPR, and BLI. Example 6. Synthesis of RNAi reagents and TfR1-specific conjugates The double strands of the RNAi reagent disclosed herein were synthesized according to the following procedure: A. Synthesis .

[0246] The sense and antisense strands of the RNAi reagent were synthesized using a solid-phase phosphoramidite technique employed in oligonucleotide synthesis. Depending on scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was performed on a solid support made of controlled-porosity glass (CPG, 500 Å or 600 Å, from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the 2'-O-methylphosphoramidite used comprised the following: (5'-O-dimethoxytriphenylmethyl-N... 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-O-dimethoxy-triphenylmethyl-N 4 -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphamide, (5'-O-dimethoxytriphenylmethyl-N 2-(isobutyryl)-2'-O-methylguanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide and 5'-O-dimethoxytriphenylmethyl-2'-O-methyluridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2'-Deoxy-2'-fluorophosphamide carries the same protecting group as 2'-O-methylRNAphosphamide (amidite). 5'-Dimethoxytriphenylmethyl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from Glen Research (Virginia). Reverse debasing (3'-O-dimethoxytriphenylmethyl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from ChemGenes. (Wilmington, MA, USA). The following UNA phosphoramids were used: 5'-(4,4'-dimethoxytriphenylmethyl)-N6-(benzoyl)-2',3'-open-ring-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-2',3'-open-ring-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-2',3'-open-ring-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-(4,4'-dimethoxytriphenylmethyl)-N-isobutyryl-2 ',3'-open-ring-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide and 5'-(4,4'-dimethoxytriphenylmethyl)-2',3'-open-ring-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide. The TFA-linked phosphoramide was also commercially available (ThermoFisher). Cyclopropylphosphonate phosphoramide was synthesized according to International Patent Application Publication No. WO2017 / 214112 (see also Altenhofer et al., Chem. Communications (Royal Soc. Chem.), 57(55): 6808-6811 (July 2021)).

[0247] B. Cleavage and deprotection of oligomers bound to support agents.

[0248] After solid-phase synthesis was complete, the dried solid support was treated with a 1:1 volume solution of 40% by weight aqueous methylamine and 28% to 31% ammonium hydroxide solution (Aldrich) at 30°C for 1.5 hours. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0249] C. Conjugation of RNAi reagents with Fab and capping with CP-1113.

[0250] The RNAi reagent containing free amines described in this article, after cleavage from the solid phase, is reacted with L20-p using standard amide reaction chemistry. Conjugation. Freshly prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) in PBS (5-20 equivalents, 70 mM) was added to a solution of Fab in PBS (0.2 μmol, 1.0-10.0 mg / mL in PBS). The reaction was allowed to proceed overnight at room temperature in the dark. The next day, TCEP was removed by loading the reaction mixture onto a PD-10 desalting column equilibrated with PBS and eluted with PBS. The concentration of Fab in the eluent was determined using the theoretical absorbance at 280 nm. A solution of the L20-modified sense chain in sodium phosphate buffer was prepared, and the concentration was determined using the theoretical absorbance at 260 nm. The L20-modified sense chain (1-1.3 equivalents, 0.5-2.5 mM) was added to the desalted Fab solution, and the reaction was carried out by inverted mixing. Analysis by SEC Method 1 and AIEX Method 1 showed the mixture of starting Fab, DAR1, and DAR2. After 1 hour, CP-1113-p: The solution in DMSO was added to the reaction mixture (3 equivalents, 36 mM). After 1 hour, the solution of L-cysteine ​​in PBS was added to the reaction mixture (6–10 equivalents, 165 mM). Finally, the conjugate was annealed by adding antisense (1.2–1.5 equivalents, 0.5–2.5 mM). The conjugate was purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was pumped onto the column and eluted with a gradient of 10–40% buffer B. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AEX Method 1, and Nanodrop 260 / 280 readings. The DAR1 fraction was combined, and PBS was replaced with PD-10 desalting column buffer. The purified conjugate was analyzed by SEC method 1 and eluted as a monomer peak with a retention time of 13.2 minutes.

[0251] D. Conjugation of RNAi reagents with Fab and capping with NEM.

[0252] The RNAi reagent containing free amines described in this article, after cleavage from the solid phase, is reacted with L20-p using standard amide reaction chemistry. Conjugation. A freshly prepared solution of TCEP-HCl in PBS (5–20 equivalents, 70 mM) was added to a solution of Fab in PBS (10 mg, 0.2 μmol, 1.0–10.0 mg / mL in PBS). The reaction was allowed to proceed overnight at room temperature in the dark. The next day, TCEP was removed by loading the reaction mixture onto a PD-10 desalting column equilibrated with 20 mM tris pH 8 and eluted with 20 mM tris pH 8. The concentration of Fab in the eluent was determined using the theoretical absorbance at 280 nm. A solution of the L20-modified sense chain in sodium phosphate buffer was prepared, and its concentration was determined using the theoretical absorbance at 260 nm. The L20-modified sense chain (1–1.3 equivalents, 0.5–2.5 mM) was added to the desalted Fab solution, and the reaction was carried out by inverted mixing. Analysis by SEC Method 1 and AIEX Method 1 showed the mixture of starting Fab, DAR1, and DAR2. One hour later, a solution of N-ethylmaleimide (NEM) in 20 mM tris pH 8 was added to the reaction mixture (12 equivalents, 160 mM). One hour later, the conjugate was annealed by adding antisense (1.2–1.5 equivalents, 0.5–2.5 mM). The conjugate was purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10–40% buffer B. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AEX Method 1, and UV-Vis 260 / 280 measurements. The DAR1 fraction was combined, and PBS was replaced with PD-10 column buffer. The purified conjugate was analyzed by SEC method 1 and eluted as a monomer peak with a retention time of 13.2 minutes.

[0253] SEC Method 1 AIEX Method 1 The RNAi reagent containing free amines described in this article is first coupled with L1026-p using standard solid-phase synthesis techniques: Concatenation is performed to generate a semantic chain modified by L-1026: (NH2-C6)s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb) (i.e., CS915332, SEQ ID NO.: 151). After cleavage from the solid phase, the RNAi reagent described herein is synthesized according to the following procedure, as exemplified by the synthesis of AC006448: A freshly prepared solution of TCEP-HCl in PBS (5 equivalents, 70 mM, 42 uL) was added to a solution of Fab0070 (28 mg, 0.59 μmol, 5.55 mg / mL in PBS). The reaction was inverted and mixed at ambient temperature for 15 minutes, then kept overnight at 5°C without stirring. The next day, TCEP was removed by loading the reaction mixture onto two PD-10 desalting columns (Cytiva), which were equilibrated with 20 mM tris 50 mM NaCl pH 7.6 (alternatively, 20 mM tris pH 8 or PBS buffer) and eluted with the same buffer. The concentration of Fab in the eluent was determined using the theoretical absorbance at 280 nm. A solution of L-1026-modified sense strand (CS915332) was prepared in 10 mM sodium phosphate buffer pH 6.0–6.5, and its concentration was determined using the theoretical absorbance at 260 nm. L-1026-modified CS915332 (1.15 equivalents, 2.75 mM, 240 μL) was added to the desalted Fab solution, and the reaction was carried out by inverted mixing at ambient temperature. Analysis by SEC Method 1 and AIEX Method 1 showed a mixture of starting Fab0070, DAR1 product, and DAR2 product. After 30 minutes, an L-cysteine ​​solution in 20 mM tris 50 mM NaCl pH 7.6 (alternatively, some L-1026 conjugates have been prepared in 20 mM tris pH 8 or PBS buffer) was added to the reaction mixture (10 equivalents, 165 mM, 36 μL). After 30 minutes, the conjugate was annealed by adding antisense chain (CA003820) (1.3 equivalents, 1.45 mM in water, 529 μL). The conjugates were purified using an AKTA Pure FPLC system equipped with 20 mMtris pH 8 (buffer A), 20 mMtris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10–40% buffer B. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AIEX Method 1, and UV-Vis 260 / 280 measurements. The DAR1 fraction was combined, and two PD-10 column buffers were replaced with PBS. The purified conjugates were analyzed by SEC Method 1 and eluted as monomeric peaks with a retention time of 7.2 min.

[0254] SEC Method 1 AIEX Method 1 E. Conjugation of RNAi reagents with Fab.

[0255] Synthesis of Fab0070-L-1288 Add freshly prepared TCEP-HCl solution (5 equivalents) to a Fab0070 solution in PBS (82 mg, 8.2 mg / mL). Incubate the reaction mixture overnight at 5°C. The next day, using four PD-10 columns equilibrated with the same buffer, buffer-exchange the reaction mixture to 20 mM Tris 50 mM NaCl pH 7.6. Determine the concentration using Nanodrop and dilute the solution to 2 mg / mL with 20 mM Tris 50 mM NaCl pH 7.6. Dilute the solution with DMSO (1.95 mL). Prepare an L-1288 solution (2 mg / mL in DMSO) and load it over 15 minutes (1.6 mL, 3.19 mg, 3 equivalents). Quench the reaction with cysteine ​​(10 equivalents) after 10 minutes. Remove insoluble material by centrifugation. The supernatant was collected, filtered, diluted 1:1 with PBS, and concentrated / desalted to a volume of 8 mL using a Pierce 10K MWCO centrifuge column. Finally, the solution was buffer-exchanged by loading it onto a HiPrep 26 / 10 desalting column and eluting with PBS. The final yield was approximately 80 mg.

[0256] L-1289 bioconjugation Lyophilized CS915332 (86 mg, 11.6 μmol) was dissolved in DMF (1.58 mL) and water (215 μL) and sonicated. Triethylamine (9.7 μL, 6 equivalents) and a solution of L-1289 in DMF (50 mg / mL, 344 μL, 3 equivalents) were added to the solution. The reaction was monitored by LCMS. After 1 hour, additional triethylamine (4 equivalents) and L-1289 solution (2 equivalents) were added. Complete conversion was observed by LCMS after 20 minutes. The reaction mixture was acidified with phosphoric acid (200 mg / mL in water, 5 equivalents). The crude solution was added dropwise to a mixture of acetonitrile (43 mL) and PBS (1.7 mL). The precipitate was collected by centrifugation, and the supernatant was discarded. The precipitate was dissolved in water (1.5 mL) and added to acetonitrile (43 mL). The precipitate was collected by centrifugation and dissolved in PBS (5 mL). The yield was 88 mg (98%).

[0257] The concatenation of semantic chains modified by Fab0070-L-1288 and L-1307 Add the L1307-modified sense chain solution to the Fab0070-L-1288 solution in PBS (12.5 mg, 6.97 mg / mL, 1.79 mL, 1 equivalent): L-1307s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb) (i.e., CS009529, SEQ ID NO.: 152) in PBS (6.18 mg, 17.9 mg / mL, 345 uL, 3 equivalents). The combined solutions were inverted and mixed for 10 minutes, then stored at 5°C without stirring. The next day, the reaction was heated to room temperature and annealed with the antisense chain: cPrpusUfsgauuAfgcagGfuCfaAfaagsusg (i.e., AC003820, SEQ ID NO.: 153), 6.23 mg, 10.4 mg / mL, 599 uL, 3.3 equivalents. The conjugates were purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was diluted to 40 mL with MPA, loaded onto the column, and eluted with a gradient of 10–40% buffer B. Fractions containing DAR1 were combined and exchanged for PBS using two PD-10 column buffers. The conjugates were analyzed by SEC Method 1 and found to be 99% pure with a retention time of 7.2 min. This procedure was also followed to generate the Fab-L-1288-L-1289-RNAi conjugates disclosed herein.

[0258] SEC Method 1 F. Synthesis of antibody-siRNA linkers The following provides the synthesis of various Fab connectors used throughout this application.

[0259] Synthesis of 2,3,5,6-tetrafluorophenyl 16-((3,5-bis(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)amino)-16-oxo-4,7,10,13-tetraoxazhexadecanoate (i.e., L-1026-p) Compound 6 (2.35 g, 7.31 mmol; according to Sarbisheh et al.) Bioconjugate ChemistryPrepared in 202031 (12), 2789-2806) 、 EDC-HCl (2.38 g, 12.43 mmol) and K-Oxyma (2.50 g, 13.9 mmol) were used as a solid mixture and slurried in DMF (190 mL) at ambient temperature under N2. Compound 7 (1.192 g, 5.48 mmol) was added as a solution (10 mL) in DMF. After 5 minutes, triethylamine (4.5 mL, 32.2 mmol) was added dropwise at ambient temperature. The reaction mixture was heated at 50°C for 2 days. The reaction mixture was concentrated under reduced pressure to a red oil, which was slurried in DCM (250 mL) and washed with a saturated aqueous sodium bicarbonate solution (200 mL). The layers were separated, and the aqueous layer was further extracted with DCM (100 mL). The combined organic phases were washed with water (200 mL) and brine (200 mL). The organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by normal-phase SiO2 chromatography using a gradient of ethyl acetate in DCM (0-100%). Yield of compound 8: 1.77 g (49%), partially contaminated with compound 6. Calculated mw for compound 8: 653.77 g / mol, measured m / z (ESI, positive ion mode): 654.83.

[0260] Compound 8 (1.77 g, 2.71 mmol) was dissolved in TFA:DCM [1:1] (18 mL) and stirred at ambient temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and then co-evaporated with toluene (3 x 30 mL). The residue was purified by normal-phase SiO2 chromatography using a gradient (0-7%) of DCM containing 0.1% formic acid and methanol. Yield of compound 9: 1.30 g (80%). Calculated mw for compound 9: 597.66 g / mol, measured m / z (ESI, positive ion mode): 598.79.

[0261] Compound 9 (1.30 g, 2.18 mmol) was dissolved in DCM (50 mL) and cooled to 0°C. By dissolving 10.38 g... m -CPBA (77 wt%) was dissolved in 80 mL of DCM and dried over sodium sulfate until clear to prepare 100 mg / mL. m -CPBA solution. At 0°C, 58 mL of the solution of compound 9 was added dropwise. m -CPBA (5.85 g, 26.1 mmol)。 The reaction mixture was heated to ambient temperature and allowed to stand overnight. The reaction mixture was concentrated, slurried in 0.1% formic acid (50 mL) in DCM, and filtered. The filtrate was purified by normal-phase SiO2 chromatography using a gradient (0-10%) of DCM containing 0.1% formic acid and methanol. Yield of compound 10: 1.03 g (72%). Calculated mw for compound 10: 661.65 g / mol, measured m / z (ESI, positive ion mode): 662.65.

[0262] At 0°C, EDC (0.448 g, 2.34 mmol) was added to a solution of compound 10 (1.03 g, 1.56 mmol) in DCM:ACN [4:1] (15 mL), followed by a solution of TFP (0.310 g, 1.87 mmol) in DCM:ACN [4:1] (5 mL). After 5 minutes, the reaction mixture was heated to ambient temperature. After 1.5 hours, the reaction mixture was concentrated to dryness. The crude product was purified by preparative reversed-phase HPLC (Phenomenex Gemini C18 50 mm x 250 mm, 10 μm) using a water / acetonitrile gradient containing 0.1% TFA. The fraction containing the product was concentrated under reduced pressure. Yield of L-1026-p: 1.10 g (87%). The calculated mw for compound L-1026-p is 809.71 g / mol, and the measured m / z (ESI, positive ion mode) is 810.62. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 2.64 (t, 2H), 3.00 (t, 2H), 3.49 (m, 12H), 3.74 (m, 10H), 7.92 (m, 1H), 8.34 (t, 1H), 8.68 (d, 2H), 10.67(s, 1H).

[0263] Synthesis of L20-p At room temperature, methyl iodine (4.20 mL, 67.50 mmol) was added to a suspension of compound 1 (5.00 g, 22.50 mmol) and Cs₂CO₃ (25.66 g, 78.75 mmol) in anhydrous DMF (80 mL). The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was quenched with water (200 mL) and extracted with EtOAc (3 x 100 mL). The organic phases were combined and washed with water and brine. The organic layer was dried over anhydrous Na₂SO₄ and concentrated. Compound 2 was given as a pale yellow solid, 5.41 g, 96%. Compound 2 was used directly without further purification. LC-MS: [M+H] calculated value 251.05, found value 251.18.

[0264] At room temperature, LiOH (2.59 g, 108.08 mmol) was added to a solution of compound 2 (5.41 g, 21.62 mmol) in THF / H₂O (50 mL / 50 mL). The reaction mixture was stirred at room temperature for 1 hour. After removing THF under vacuum, the pH was adjusted to ~2 by [C]HCl. Then, EtOAc (3 x 60 mL) was used for extraction. The organic layers were combined, washed with brine, dried over anhydrous Na₂SO₄, and concentrated. Compound 3 was given as a grayish-white solid, 5 g, 98%. Compound 3 was used directly without further purification. LC-MS: calculated [M+H] 237.03, found 237.26.

[0265] At room temperature, EDC (7.07 g, 36.90 mmol), DMAP (0.30 g, 2.46 mmol), and compound 4 (6.13 g, 36.90 mmol) were added to a solution of compound 3 (5.81 g, 24.60 mmol) in THF / DMF (80 mL / 20 mL). The reaction mixture was stirred overnight at room temperature. After solvent removal under vacuum... ， The residue was loaded onto a 120 g column and compound 5 was eluted with hexane containing 0–50% EtOAc. Compound 5 was given as a white solid, 9.36 g, 99%. LC-MS: calculated [M+H] 385.03, found 385.46.

[0266] At 0 °C, 70% m-CPBA (5.14 g, 27.79 mmol) was added to a solution of compound 5 (2.29 g, 5.96 mmol) in DCM (110 mL). The reaction mixture was stirred at room temperature for 6 hours. Then, 1.8 g of m-CPBA was added at room temperature. The reaction mixture was stirred overnight at room temperature. After filtration, the solvent was removed under vacuum. The residue was recrystallized twice from DCM / EtOAc (50 mL / 50 mL). Compound L20-p was given as white needle-like crystals, 1.93 g, 78%. LC-MS: calculated [M+H] 417, measured 417.

[0267] Example 7. After delivery of a TfR1 Fab-conjugated siRNA antisense targeting androgen receptor (AR) mRNA, Relative expression of mRNA The siRNA duplexes are shown as sense and antisense in Tables 8 and 9, respectively. The antisense strand is complementary to the AR mRNA. The sense siRNA strand is conjugated to the Fab shown in Table 8 below.

[0268] Table 8. siRNA sense sequences used to target AR mRNA.

[0269] *Fab0044 is identical to Fab0002, except that the valine residue at position 205 of the light chain has been mutated to cysteine. **Fab0046 is identical to Fab0002, except that the alanine at position 117 of the heavy chain has been mutated to cysteine.** Symbol explanation: (a) represents 2′-O-methyladenosine, (c) represents 2′-O-methylcytidine, (g) represents 2′-O-methylguanosine, and (u) represents 2′-O-methyluridine; (Af) represents 2′-fluoroadenosine, (Cf) represents 2′-fluorocytidine, (Gf) represents 2′-fluoroguanosine, and (Uf) represents 2′-fluorouridine; (s) represents phosphate thioester bond; (invAb) and (invAb)s represent reverse debasing nucleotides ( The structures are shown in Table 13 below; cPrpus represents 5′-cyclopropylphosphonate-2′-O-methyluridine-3′-thiophosphate (structures are shown in Table 13 below); (NH-C6)s structure is shown in Table 13 below; L20 structure is shown in Table 13 below; [CP-1113] indicates that Fab has been capped using CP-1113p according to the procedure of Example 6C; [NEM] indicates that Fab has been capped using N-ethylmaleimide according to the procedure of Example 6D.

[0270] Table 9. siRNA antisense sequences for targeting AR mRNA Symbol explanation: (a) represents 2′-O-methyladenosine, (c) represents 2′-O-methylcytidine, (g) represents 2′-O-methylguanosine, and (u) represents 2′-O-methyluridine; (Af) represents 2′-fluoroadenosine, (Cf) represents 2′-fluorocytidine, (Gf) represents 2′-fluoroguanosine, and (Uf) represents 2′-fluorouridine; (s) represents thiophosphate bond; cPrpus represents 5′-cyclopropylphosphonate-2′-O-methyluridine-3′-thiophosphate (structures are in Table 13 below).

[0271] Table 10 shows the structures of cPrpus, (invAb)s, (invAb), L20, and NH-C6 as defined in the symbol descriptions of the meaningful and antisense sequences in Tables 8 and 9 above. Example 8. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice According to Table 11 below, on day 1 of the study, Tg B-hTfr1 mice were administered intravenously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-7) at a dose of 250 μL / 25g, and were administered the same formulation again on day 2. Table 11: Drug administration groups in mice of Example 8.

[0272] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 1 The relative AR mRNA knockdown was shown for each group in the four analyzed tissues.

[0273] Example 9. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice According to Table 12 below, on day 1 of the study, Tg B-hTfr1 mice were administered intravenously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-10) at a dose of 250 μL / 25g, and were administered the same formulation again on day 2. Table 12: Drug administration groups in mice of Example 9.

[0274] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 2 The relative AR mRNA knockdown was shown for each group in the four analyzed tissues.

[0275] Example 10. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice According to Table 13 below, on day 1 of the study, Tg B-hTfr1 mice were administered intravenously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-6) at a dose of 250 μL / 25g, and were administered the same formulation again on day 2. Table 13: Drug administration groups in mice of Example 10.

[0276] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 3 The relative AR mRNA knockdown was shown for each group in the four analyzed tissues.

[0277] Example 11. The effect of different antibody conjugate groups on the selected brain regions compared to the AC003313 (Fab0002) control group AR mRNA gene expression in vivo In vivo AR mRNA gene knockdown of the Fab-doublet was measured in selected brain regions. AR mRNA gene expression in each group was normalized for the AC003313 (Fab0002 conjugated siRNA) group. Figure 4 A graph showing relative expression values ​​is displayed. The Fab0070-siRNA duplex group appears to have the lowest relative expression (highest gene knockdown).

[0278] Example 12. In vivo knockdown of AR mRNA gene expression in cynomolgus monkeys According to Table 17 below, on day 0 of the study, cynomolgus monkeys were administered subcutaneously with PBS or a compound formulation containing 3 mg / kg of anti-TfR1 Fab-siRNA conjugate at a concentration of 1.5 mg / mL. On day 7 of the study, groups 2, 3, and 4 were administered the same formulation as on day 0. All animals were administered at a volume of 2 mL / kg.

[0279] Table 14: Dosing groups in non-human primates in Example 12.

[0280] Three monkeys (n=3) were administered the drug in groups 1 (control), 3, and 4 (trigger treatment), and two monkeys (n=2) were administered the drug in group 2. On day 28 of the study, animals from all groups were euthanized, and tissues from each animal were collected including: frontal cortex, temporal cortex, caudate nucleus, cerebellum (cortex), putamen, motor cortex, medulla oblongata, pons, hippocampus, thalamus, hypothalamus, midbrain, substantia nigra, dorsal cervical root ganglion (DRG), thoracic DRG, lumbar DRG, cervical spinal cord, lumbar spinal cord, thoracic spinal cord, visual cortex, gastrocnemius muscle, triceps surae muscle, left atrium, right atrium, left ventricle, and right ventricle. Samples were analyzed by qPCR for AR mRNA knockdown. The mean results for each group relative to group 1 are shown in Table 15 below: Table 15. Relative expression of AR mRNA in various tissues by qPCR analysis for each dosing group in Example 12.

[0281] As shown in Table 15 above, a persistent decrease in AR mRNA expression was observed in multiple tissues in non-human primates treated with anti-TfR1 antibody-siRNA conjugates. Except for the thoracic and lumbar DRGs, groups 3 (Fab0061-siRNA conjugate) and 4 (Fab0070-siRNA conjugate) outperformed group 2 (Fab002-siRNA conjugate) in almost every tissue analyzed.

[0282] Table 15 also shows that Fab0061 and Fab0070 are capable of delivering siRNA to skeletal muscle tissues such as the gastrocnemius and triceps muscles. Delivery to cardiac tissue is also possible using Fab0061 and Fab0070, where deep knockdown of AR mRNA was demonstrated in each segment of the collected heart.

[0283] Example 13. In vivo knockdown of MAPT in cynomolgus monkeys As shown in Table 15 above, a persistent decrease in AR mRNA expression was observed in multiple tissues in non-human primates treated with anti-TfR1 antibody-siRNA conjugates. Except for the thoracic and lumbar DRGs, groups 3 (Fab0061-siRNA conjugate) and 4 (Fab0070-siRNA conjugate) outperformed group 2 (Fab002-siRNA conjugate) in almost every tissue analyzed.

[0284] According to Table 16 below, on day 0 of the study, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound preparation containing AC007414 at a concentration of 1.5 mg / mL and a volume of 2 mL / kg, 3 mg / kg, 7.5 mg / mL and a volume of 2 mL / kg, or 30 mg / kg of AC007414 at a concentration of 7.5 mg / mL and a volume of 4 mL / kg. Table 16: Drug administration groups in non-human primates in Example 13.

[0285] Four monkeys (n=4) in each group were administered the drug. Subcutaneous injections were given on days 0, 7, and 14. On day 29 of the study, animals from each group were euthanized, and brain and spinal cord tissues were collected from each animal. Samples were analyzed for MAPT mRNA knockdown by qPCR. Samples were analyzed for protein knockdown by JESS. The mean mRNA knockdown in the frontal cortex, hippocampus, and thoracic spinal cord for each group relative to group 1 is shown in Table 17 below: Table 17. Relative expression of MAPT mRNA in various tissues by qPCR analysis for each dosing group in Example 13. Compared to Group 1, the mean protein knockdown in the frontal cortex, hippocampus, and thoracic spinal cord of each group was shown in Figure 5 middle.

[0286] As shown in Table 17 and Figure 5As can be seen, when injected subcutaneously, the MAPT RNAi reagent, which binds to anti-transferrin Fab070, achieved dose-dependent and deep knockdown in CNS tissues. The results confirm that treatment of MAPT-related diseases and symptoms can be achieved through subcutaneous administration of the RNAi reagent.

[0287] It should be understood that the foregoing disclosure highlights certain specific embodiments of the invention, and all equivalent modifications or alternatives are within the spirit and scope of the invention as set forth in the appended claims.

[0288] Example 14. In vivo administration of HTT RNAi reagent in cynomolgus monkeys.

[0289] In vivo evaluation of HTT RNAi reagent containing anti-Tfr Fab in cynomolgus monkeys 。 On days 1, 8, and 15, four (n=4) male cynomolgus monkeys in each test group were administered HTT RNAi reagent at doses of 1.5 mg / ml, 2.0 ml / kg, or 3.0 mg / kg (adjusted for individual animal weight) in PBS, or via PBS. Each dose was administered subcutaneously (SC) and based on the most recent body weight of each animal. Dosing was performed according to Table 18 below.

[0290] Table 18. Administration of drugs to cynomolgus monkeys in Example 14.

[0291] The test animals were non-human primates, male cynomolgus fascicularis, not first-time test subjects, aged 3–5 years. Prior to administration, the test animals were acclimatized to laboratory conditions for at least 14 days according to facility and acclimatization standard operating procedures. RNAi reagent test samples were administered subcutaneously (SC) via syringe and needle into the mid-scapular region.

[0292] The animal was shaved before administration. The day 1 dose was delivered to the left scapular region, the day 8 dose to the right scapular region, and the day 15 dose to the left scapular region. Each dose was administered using a syringe with a 23-25 ​​gauge needle.

[0293] Individual weights of the test animals were recorded once 7 days before treatment, then weekly throughout the study, and once before necropsy.

[0294] On day -7, ~0.5 mL of cerebrospinal fluid (CSF) was collected from all groups. On day 43 (the day of autopsy), ~2.0 mL of CSF was collected from all groups. For the CSF collection procedure, all test animals were anesthetized by intravenous (IV) administration of ketamine (10 mg / kg, IV) and dexmedetomidine (0.02 mg / kg, IM, IM) and placed in a lateral decubitus position. The skin covering the insertion site was shaved off and wiped several times with individual chlorhexidine swabs. The animal's head was gently bent until the chin almost touched the chest, but the airway was not obstructed. A professional palpated the occipital protuberance and the atlantoaxial (C1) wing. The needle was inserted vertically above the atlantoaxial vertebra, through the skin, into the subarachnoid space. If bone was encountered, the needle was reoriented forward or backward until the designated entry point was found. Once proper placement was achieved, CSF would flow freely through the needle and be collected in a cryotube. The needle was removed, and direct pressure was applied to the puncture site for at least two minutes. Once the procedure is complete, administer carbofen 2–4 mg / kg to the animals every 12 hours via SC for 1 day. If necessary, administer atemexazole (0.225 mg / kg, IM).

[0295] On day 43, the cynomolgus monkeys were euthanized. The following CNS tissues were collected from the test animals: left and right cerebral hemispheres, spinal cord, and dorsal root ganglia (DRG). Other tissues besides those listed above may also be collected, and if such additional tissues are collected, their biological data are similarly presented below. The collected tissues were analyzed for biological parameters.

[0296] The cHTT mRNA transcription level was quantified from collected tissues by qPCR, with cPPIB used as an endogenous control gene, and normalized for group 1 cynomolgus monkeys administered PBS. cHTT expression data are shown in Table 19 below.

[0297] Table 19. cHTT expression in the CNS tissues of cynomolgus monkeys in Example 14.

[0298] In the temporal cortex, frontal cortex, motor cortex, caudate nucleus, and hippocampus, groups 2–5 showed a reduction in HTT transcripts up to at least day 43 post-dose. In the putamen, groups 2, 4, and 5 showed a reduction in HTT transcripts up to at least day 43 post-dose. Most notably, the most significant reduction in HTT transcripts was observed in the hippocampus, with three doses of 3.0 mg / kg AC007867 showing ~69% HTT transcript inhibition (0.306), and three doses of 3.0 mg / kg AC007865 showing ~70% HTT transcript inhibition (0.302).

[0299] HTT protein expression was analyzed in cynomolgus monkey tissues using the Jess protein assay, normalized for group 1 cynomolgus monkeys administered with PBS. HTT protein was quantified using an anti-huntingtin protein antibody (Sigma-Aldrich®, catalog MAB2166). HTT protein expression data are shown in Table 20 below.

[0300] Table 20. HTT protein expression in the CNS tissue of cynomolgus monkeys in Example 14.

[0301] In the temporal cortex, frontal cortex, motor cortex, caudate nucleus, putamen, hippocampus, and cerebellum, groups 2–5 showed a decrease in HTT protein up to at least day 43 post-dose. In the liver, groups 2, 3, and 5 showed a decrease in HTT protein up to at least day 43 post-dose. Most notably, the most significant decrease in HTT protein was observed in the motor cortex, with three doses of 3.0 mg / kg AC007867 showing ~86% HTT protein inhibition (0.143), and three doses of 3.0 mg / kg AC007865 showing ~85% HTT protein inhibition (0.146).

[0302] Example 15. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0303] The AR RNAi reagent was evaluated in vivo in mice. On days 1 and 2, five (n=5) female B-hTFR1 mice were administered saline or the AR RNAi reagent (prepared in saline at 0.75 or 1.5 mg / kg body weight) via subcutaneous (SC) injection at a total dose volume of 250 μL / 25 g. Dosage was performed according to Table 21 below.

[0304] Table 21. Drug administration groups in mice of Example 15.

[0305] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0306] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding portion having a modified sequence as shown in the double-stranded structure herein.

[0307] Mice were euthanized on day 15. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 22 below.

[0308] Table 22. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 15.

[0309] In the thoracic spinal cord, right cerebellum, right cortex, and right striatum, groups 2–12 showed a decrease in mAR up to at least day 15. Dose responses were observed in AC004791, AC004792, and AC004798 in the thoracic spinal cord, right cerebellum, right cortex, and right striatum.

[0310] Example 16. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0311] The ARRNAi reagent was evaluated in vivo in mice. On days 1 and 2, five (n=5) female B-hTFR1 mice were administered saline or the ARRNAi reagent (prepared in saline at a dose concentration of 1.5 mg / kg body weight) via subcutaneous (SC) injection. Dosage was performed according to Table 23 below.

[0312] Table 23. Drug administration groups for mice in Example 16.

[0313] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0314] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding portion having a modified sequence as shown in the double-stranded structure herein.

[0315] Mice were euthanized on day 15. CNS and muscle tissue were harvested from the mice and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 24 below.

[0316] Table 24. Relative expression of mAR in mouse CNS and muscle tissue in Example 16.

[0317] In the cerebellum, cortex, triceps, gastrocnemius, thoracic spinal cord, and right striatum, groups 2 and 3 showed a reduction in mAR up to at least day 15. Most notably, both doses of 1.5 mg / kg AC011951 achieved ~84% inhibition (0.157) in the cerebellum.

[0318] Example 17. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0319] The AR RNAi reagent was evaluated in vivo in mice. Five (n=5) female B-hTFR1 mice were administered saline or the AR RNAi reagent (prepared in saline at doses of 0.75, 1.5, 3.0, and 12.0 mg / kg body weight) via subcutaneous (SC) injection on days 1, 1 and 2, or 1 and 2 and 3 and 4. Dosing was performed according to Table 25 below.

[0320] Table 25. Drug administration groups in mice of Example 17.

[0321] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0322] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding portion having a modified sequence as shown in the double-stranded structure herein.

[0323] Mice were euthanized on day 15. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 26 below.

[0324] Table 26. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 17.

[0325] In the thoracic spinal cord, cerebellum, cortex, striatum, and midbrain, groups 2–10 showed a reduction in mAR up to at least day 15. Most notably, four doses of 3.0 mg / kg AC006372 achieved ~85% inhibition (0.153) in the cerebellum. Dose responses were observed with single, two, and four doses of AC006372 in the thoracic spinal cord and cerebellum. Dose responses were observed with single and two doses of AC006372 in the cortex. Dose responses were observed with single and four doses of AC006372 in the striatum. Dose responses were observed with two and four doses of AC006372 in the midbrain.

[0326] Example 18. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0327] The AR RNAi reagent was evaluated in vivo in mice. On days 1 and 2, five (n=5) female B-hTFR1 mice were administered saline or the AR RNAi reagent (prepared in saline at 1.5 mg / kg body weight) via subcutaneous (SC) injection at a total dose volume of 250 μL / 25 g. Dosage was performed according to Table 27 below.

[0328] Table 27. Drug administration groups for mice in Example 18.

[0329] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0330] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding portion having a modified sequence as shown in the double-stranded structure herein.

[0331] Mice were euthanized on day 15. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 28 below.

[0332] Table 28. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 18.

[0333] In the thoracic spinal cord, cerebellum, cortex, and striatum, groups 2–7 showed a decrease in mAR up to at least day 15.

[0334] Example 19. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0335] In vivo evaluation of AR RNAi reagents in mice 。 On days 1, 2, and 3, four (n=4) female B-hTFR1 mice were administered saline or AR RNAi reagent (prepared in saline at 3.0 mg / kg body weight) via subcutaneous (SC) injection at a total volume of 250 μL / 25 g. Dosing was performed according to Table 29 below.

[0336] Table 29. Drug administration groups in mice of Example 19.

[0337] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0338] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding portion having a modified sequence as shown in the double-stranded structure herein.

[0339] Mice were euthanized at days 4, 10, 17, 31, 45, 59, 80, 100, or 122, according to Table 58 above. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested from the mice and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene; groups 3–7 were normalized to group 1 mice treated with saline, and groups 9–11 were normalized to group 8 mice treated with saline. mAR expression data are shown in Tables 30 and 31 below.

[0340] Table 30. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 19.

[0341] In the thoracic spinal cord, cerebellum, left cortex, striatum, and brainstem, groups 3–7 showed a reduction in mAR up to at least day 59. Most notably, at day 59, all three doses of 3.0 mg / kg AC006448 achieved ~85% inhibition (0.147) in the cerebellum.

[0342] Table 31. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 19.

[0343] In the thoracic spinal cord, cerebellum, and striatum, groups 9–11 showed a reduction in mAR up to at least day 122. Most notably, at day 122, all three doses of 3.0 mg / kg AC006448 achieved ~39% inhibition (0.612) in the striatum.

[0344] Other implementation plans All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature that performs the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of an equivalent or similar feature in a general series.

[0345] Based on the above description, those skilled in the art can readily determine the essential characteristics of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also included within the claims.

[0346] Equivalent scheme Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and such variations and / or modifications are each considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to determine, many equivalent embodiments of the specific inventive embodiments described herein using no more than conventional experimentation. Therefore, it should be understood that the foregoing embodiments are presented only as examples, and that inventive embodiments may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. The inventive embodiments of this disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, if such features, systems, articles, materials, kits, and / or methods are not inconsistent with each other, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of this disclosure.

[0347] As defined and used herein, all definitions should be understood to take precedence over dictionary definitions, definitions in incorporated documents by reference, and / or the general meaning of the defined terms.

[0348] All references, patents, and patent applications disclosed herein are incorporated by reference to their respective subjects, and in some cases, this may cover the entire document.

[0349] Unless there is an explicit indication to the contrary, the indefinite articles “a” and “an” as used herein in the specification and claims shall be understood to mean “at least one / a type”.

[0350] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or both” of the elements so connected, i.e., elements that coexist in some cases and exist separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., “one or more” of the elements so connected. Other elements may optionally be present in addition to those explicitly identified by the “and / or” clause, whether related to or unrelated to those explicitly identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” reference to “A and / or B” may, in one embodiment, refer only to A (optionally including elements other than B); in another embodiment, refer only to B (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so on.

[0351] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when items are listed separately, “or” or “and / or” should be interpreted as inclusive, that is, including at least one of many elements or a list of elements, but also including more than one, and optionally additional unlisted items. Only terms that explicitly indicate the opposite, such as “only one” or “exactly one”, or “consisting of” as used in the claims, refer to including exactly one of many elements or a list of elements. In general, when preceded by an exclusive term, such as “any,” “one of,” “only one,” or “exactly one,” the term “or” as used herein should be interpreted only as indicating an exclusive alternative (i.e., “one or the other, but not both”). When used in the claims, “consisting substantially of” should have its ordinary meaning as used in the field of patent law.

[0352] As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or equivalently "at least one of A and / or B") in one embodiment may refer to at least one (optionally including more than one) A, without B (and optionally including elements other than B); in another embodiment, it may refer to at least one (optionally including more than one) B, without A (and optionally including elements other than A); in yet another embodiment, it may refer to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0353] It should also be understood that, unless there is an explicit instruction to the contrary, in any method for which protection is sought herein and which includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described herein.

Claims

1. An antibody that binds to the transferrin receptor (TfR1), said antibody comprising a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL); The VH includes: The heavy chain complementarity determination region 1 (HC CDR1) is shown as GX1TFX2SYWX3H, where X1 is F or Y; X2 is K, N or T; and X3 is M or V. The heavy chain complementarity determination region 2 (HC CDR2) is shown as EINPTNGRX4NYIEKFKS (SEQ ID NO: 23), where X4 is F, S, T, or V; and The heavy chain complementarity determination region 3 (HC CDR3) shown as GTRAYHY (SEQ ID NO: 24) is displayed; and The VL mentioned therein includes: The light chain complementarity determination region 1 (LC CDR1) is shown as RASDX5LYX6NLA (SEQ ID NO: 5), where X5 is G, K or N, and X6 is R or S; The light chain complementarity determination region 2 (LC CDR2) is shown as DAX7X8LAS, where X7 is F, K, R, or T; and X8 is K, L, N, or R; and The light chain complementarity determination region 3 (LC CDR3) is shown as QHFWGTPLT (SEQ ID NO: 13).

2. The antibody according to claim 1, wherein X1 is F or Y; X2 is T; X3 is M; X4 is T or V; X5 is G or K; X6 is S; X7 is T; and X8 is L or K.

3. The antibody according to claim 1, wherein the antibody comprises HC CDR1, HC CDR2 and HC CDR2 of any one of Fab0070, Fab0061, Fab0060, Fab0062, Fab0063, Fab0064, Fab0065, Fab0066, Fab0067, Fab0068, Fab0069, Fab0071, Fab0072, Fab0073, Fab0074, Fab0165, Fab0166, Fab0167 or Fab0168 as shown in Table 2. CDR3; and / or wherein the antibody comprises LC CDR1, LC CDR2 and LC CDR3 of any one of Fab0070, Fab0061, Fab0060, Fab0062, Fab0063, Fab0064, Fab0065, Fab0066, Fab0067, Fab0068, Fab0069, Fab0071, Fab0072, Fab0073, Fab0074, Fab0165, Fab0166, Fab0167 or Fab0168 shown in Table 1.

4. The antibody according to claim 3, wherein the antibody comprises: (a) HC CDR1, HC CDR2, and HC CDR3 as SEQ ID NO: 16, 19, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 as SEQ ID NO: 4, 11, and 13, respectively; or (b) HC CDR1, HC CDR2 and HC CDR3 shown as SEQ ID NO: 14, 20 and 24 respectively, and LC CDR1, LC CDR2 and LC CDR3 shown as SEQ ID NO: 2, 7 and 13 respectively.

5. The antibody according to any one of claims 1-4, wherein the antibody is a humanized antibody; optionally, wherein the antibody comprises the light chain framework region shown in Table 3 and / or the heavy chain framework region shown in Table 4.

6. The antibody of claim 5, wherein the antibody comprises any one of the VH sequences shown in Table 5; and / or wherein the antibody comprises any one of the VL sequences shown in Table 5.

7. The antibody according to any one of claims 1-6, wherein the antibody is a full-length antibody, its antigen-binding fragment, or a single-chain variable region fragment (scFv) antibody.

8. The antibody according to any one of claims 1-7, wherein the antibody is a Fab molecule; wherein the heavy chain comprises VH and CH1 heavy chain constant region fragments; and wherein the light chain comprises VL and light chain constant region (CL).

9. The antibody according to claim 8, wherein CH1 comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 34); and / or The CL contains the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29).

10. The antibody according to claim 8 or 9, wherein the VH comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTLVTVSS (SEQ ID NO: 59), and / or wherein the VL comprises the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 60).

11. The antibody according to claim 8 or 9, wherein: (a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 61, and the light chain comprises the amino acid sequence of SEQ ID NO: 62; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO: 45, and the light chain contains the amino acid sequence of SEQ ID NO:

46.

12. The antibody according to any one of claims 1-11, wherein the antibody comprises an Fc fragment, optionally wherein the antibody is a Fab-Fc molecule.

13. A conjugate comprising an antibody (anti-TfR1 antibody) that binds to the transferrin receptor (TfR1) and an oligonucleotide-based reagent, wherein the anti-TfR1 antibody is set forth in any one of claims 1-12.

14. The conjugate according to claim 13, wherein the oligonucleotide-based reagent is directly conjugated to the anti-TfR1 antibody via a covalent or non-covalent bond.

15. The conjugate of claim 13, wherein the oligonucleotide-based reagent is conjugated to the anti-TfR1 antibody via a linker, the linker optionally being a chemical linker, peptide linker, polymer linker, or nucleic acid-binding polypeptide.

16. The conjugate according to claim 13, wherein the linker is a peptide linker, which is a glycine spacer having 1-4 glycine residues.

17. The conjugate according to any one of claims 13-16, wherein the oligonucleotide-based reagent comprises one or more chemically modified nucleotides.

18. The conjugate of claim 17, wherein the nucleotide is chemically modified by: substitution with alkyl, alkoxy, amino, denitrogenated, halogenated, hydroxyl and / or thiol groups; sugar modification; one or more non-nucleotides; one or more deoxyribonucleotides; one or more non-phosphodiester bonds; or combinations thereof.

19. The conjugate according to claim 18, wherein: The sugar modification includes a 2′ OH group replaced by a 2′ amino group, a 2′ O-methyl group, a 2′ fluorine group, a 2′ methoxyethyl group, or a 2′-O, 4′-C methylene bridge; The one or more non-nucleotides comprise xanthine, hypoxanthine, azapurine, methionine, 7-deadenosine, or O- and N-modified nucleotides; and / or The one or more non-phosphodiester bonds involve the oxygen in the phosphate group being replaced by one or more nitrogen or sulfur atoms.

20. The conjugate according to any one of claims 13-19, wherein the oligonucleotide-based reagent is an RNAi reagent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double strand.

21. The conjugate of claim 20, wherein the RNAi reagent attenuates the expression of target genes selected from the following: APP, ataxia-1, ataxia-2, ataxia-3, ataxia-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich serine / threonine protein kinase 2, complement C3, microtubule-associated protein tau, α-2A adrenergic receptor, sodium channel protein type 9 subunit α, apolipoprotein E, α-synuclein, possible G protein-coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, and DYRK1A.

22. The conjugate according to claim 21, wherein the target gene is selected from APP, ATXN2, ATXN3, AR, SOD1, HTT, C9ORF72, LRRK2, C3, MAPT, ADRA2A, SCN9A, APOE, SNCA, GPR75 and FUS.

23. The conjugate according to any one of claims 20-22, wherein the sense chain is conjugated to an anti-TfR1 antibody, wherein the anti-TfR1 antibody is a Fab molecule.

24. The conjugate according to any one of claims 13-23, wherein the anti-TfR1 antibody is a Fab molecule; wherein the oligonucleotide-based reagent is conjugated with a linker having the following formula: ， in Represents the attachment point to the Fab molecule, and This represents the attachment point of the oligonucleotide-based reagent moiety in the linker and conjugate.

25. The conjugate according to any one of claims 13-23, wherein the anti-TfR1 antibody is a Fab molecule comprising a cap portion; wherein the oligonucleotide-based reagent is conjugated to a linker, and wherein the oligonucleotide-based reagent is conjugated to the Fab molecule via a linker.

26. The conjugate according to claim 25, wherein the cap portion has the following formula: (CP-1113); and / or said connector has The structure in which Represents the attachment point to the Fab molecule, and This represents the attachment point of the oligonucleotide-based reagent moiety in the linker and conjugate.

27. The conjugate according to claim 25 or claim 26, wherein the oligonucleotide-based reagent is an RNAi reagent comprising a sense strand and an antisense strand forming a double strand; and wherein the sense strand is conjugated to a linker.

28. A pharmaceutical composition comprising an anti-TfR1 antibody or a conjugate thereof and a pharmaceutically acceptable carrier, wherein the anti-TfR1 antibody is set forth in any one of claims 1-12.

29. The pharmaceutical composition of claim 27, comprising a conjugate of an anti-TfR1 antibody, wherein the conjugate is set forth in any one of claims 13-27.

30. A method for attenuating the expression of a target gene in the brain of a subject, comprising administering to the subject in need an effective amount of a conjugate or a pharmaceutical composition comprising such a conjugate, The conjugate comprises an anti-TfR1 antibody conjugated with an oligonucleotide-based reagent. The anti-TfR1 antibody described herein is set forth in any one of claims 1-12; and The oligonucleotide-based reagents described therein attenuate the expression of the target gene.

31. The method of claim 30, wherein the conjugate is set forth in any one of claims 12-27.

32. The method of claim 30 or claim 31, wherein the subject is a person who has or is suspected of having a central nervous system (CNS) disease.

33. The method according to claim 32, wherein the CNS disease is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau protein disease, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 protein disease, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

34. The method according to any one of claims 30-33, wherein the conjugate or pharmaceutical composition comprising such conjugate is administered to the subject by intravenous injection, subcutaneous injection, intramuscular injection, intraparenchymal injection or intrathecal injection.

35. A method for alleviating CNS disease, comprising administering to a subject in need an effective amount of the conjugate set forth in any one of claims 12-26 or a pharmaceutical composition comprising such conjugate; The conjugates described herein contain oligonucleotide-based reagents that attenuate the expression of target genes selected from: APP, ataxia-1, ataxia-2, ataxia-3, ataxia-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich serine / threonine protein kinase 2, complement C3, microtubule-associated protein tau, α-2A adrenergic receptor, sodium channel protein type 9 subunit α, apolipoprotein E, α-synuclein, possible G protein-coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, and DYRK1A; and The subjects mentioned therein had or were suspected of having a CNS disease selected from the following: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tau proteinosis, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prions, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinosis, traumatic brain injury, Down syndrome, neuropathic pain, hereditary erythromelalgia, paroxysmal extreme pain disorder, and hereditary small fiber neuropathy.

36. A method for attenuating the expression of a target gene in the skeletal muscle of a subject, comprising administering to a subject in need an effective amount of a conjugate or a pharmaceutical composition comprising such a conjugate, The conjugate comprises an anti-TfR1 antibody conjugated with an oligonucleotide-based reagent. The anti-TfR1 antibody described herein is set forth in any one of claims 1-12; and The oligonucleotide-based reagents described therein attenuate the expression of the target gene.

37. The method of claim 36, wherein the target gene is selected from DUX4, DM1, ACVR2A, ACVR2B, MSTN, and GYS1.

38. The method of claim 36 or 37, wherein the subject is a person who has or is suspected of having a skeletal muscle disease.

39. The method of claim 38, wherein the skeletal muscle disease is selected from facioscapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy and myotonic dystrophy type 1, spinal muscular atrophy, or metabolic myopathy including but not limited to Pompe disease, or neuromuscular junction diseases including but not limited to myasthenia gravis (MG), or metabolic syndrome.

40. A nucleic acid or nucleic acid group comprising nucleotide sequences encoding the heavy and light chains of the anti-TfR1 antibody set forth in any one of claims 1-12.

41. The nucleic acid or nucleic acid group according to claim 40, wherein it is a vector or vector group.

42. The nucleic acid or nucleic acid group according to claim 41, wherein the vector or vector group is an expression vector or expression vector group.

43. A host cell comprising the nucleic acid or nucleic acid group set forth in any one of claims 40-42.

44. A method for producing an anti-TfR1 antibody, comprising: (iii) Culture the host cells according to claim 43 under conditions that allow for the expression of anti-TfR1 antibodies; and (iv) Harvest the anti-TfR1 antibodies produced as a result.

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