Anti-transferrin receptor antibody, its conjugate, and its uses

Abstract

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

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] The present application claims the benefit of priority of U.S. provisional application serial number 63 / 624,417 filed January 24, 2024; U.S. provisional application serial number 63 / 573,151 filed April 2, 2024; U.S. provisional application serial number 63 / 662,243 filed June 20, 2024; and U.S. provisional application serial number 63 / 718,062 filed November 8, 2024, the full contents of each of which are incorporated herein by reference.

[0003] Field of invention

[0004] The present invention relates to a composition comprising an anti-transferrin receptor (TfR1) antibody conjugated to an oligonucleotide-based agonist, and a related method for treating various diseases in the CNS, skeletal muscle, heart, and other tissues using said conjugate or said composition.

[0005] Sequence list

[0006] The present application contains a sequence list submitted in XML format (compliant with standard ST26), the full text of which is incorporated herein by reference. The XML sequence list file is named 30728-WO_SeqListing.xml, was created on January 22, 2025, and is 906 kb in size. Background Technology

[0007] RNA interference (RNAi) is a process that silences gene expression by degrading RNA. One type of RNAi molecule is a short (i.e., <30 nucleotides) double-stranded RNA ("dsRNA") molecule present in cells (Fire et al., 1998, Nature 391:806-811). These short dsRNA molecules, called "short interfering RNA" or "siRNA," cause the disruption of messenger RNA ("mRNA") that shares sequence homology with siRNA (Elbashir et al., 2001, Genes Dev, 15:188-200). One strand of siRNA is believed to be incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Using this siRNA strand, RISC identifies mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then either cleaves these target mRNAs or inhibits their translation. siRNA is clearly recycled in a manner very similar to multiplexerases, and a single siRNA molecule can induce the cleavage of approximately 1,000 mRNA molecules. siRNA-mediated RNAi degradation of mRNA is a safe and effective technique for suppressing the expression of target genes.

[0008] RNAi offers a highly interesting approach for treating and / or preventing diseases. Compared to various traditional therapeutic approaches, some of the key benefits of RNAi include the fact that it can reduce or eliminate off-target effects by targeting very specific genes involved in disease processes with high specificity; that RNAi is a normal cellular process that causes highly specific RNA degradation; and that interfering RNA can be modified so as not to trigger a host immune response.

[0009] Various interfering RNA delivery methods are being tested and developed for in vivo use. For example, siRNA can be delivered "naked" in saline solutions; complexed with polyvalent cations, cationic lipids / lipid transfection reagents, or cationic peptides; delivered as a component of defined molecular conjugates (e.g., cholesterol-modified siRNA, TAT-DRBD / siRNA complex); as a component of liposomes; and as a component of nanoparticles. These approaches have demonstrated varying degrees of success. However, for RNAi to achieve therapeutic success through gene expression knockdown, it must be delivered to the affected cell type. Therefore, there is a need for novel and improved methods to target RNAi molecules for in vivo delivery to achieve and enhance the therapeutic potential of RNAi.

[0010] The present disclosure provides novel anti-transferrin receptor (TfR1) antibodies that exhibit high binding affinity and / or high expression yield for the target antigen TfR1. The TfR1 antibodies provided herein are suitable for conjugation to oligonucleotide-based agonists, such as small interfering RNA, wherein the antibodies bind to TfR1 to facilitate delivery across the blood-brain barrier (BBB) ​​as well as to desired cell types, including but not limited to cells in the CNS, skeletal muscle, and heart. Accordingly, the TfR1 antibody-conjugated interfering RNA of the present disclosure can be used to deliver interfering RNA molecules to the central nervous system (CNS) of a patient. The present disclosure also provides a therapeutic method using a TfR1 antibody conjugated to interfering RNA to deliver interfering RNA molecules in vivo.

[0011] Accordingly, one aspect of the present disclosure features an antibody that binds to a transferrin receptor (TfR1), said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0012] VH comprises (a) heavy chain complementarity determining region 1 (HC CDR1) presented as GX1TFX2SYWX3H (where X1 is F or Y; X2 is K, N, or T; and X3 is M or V); (b) heavy chain complementarity determining region 2 (HC CDR2) presented as EINPTNGRX4NYIEKFKS (SEQ ID NO: 23) (where X4 is F, S, T, or V); and (c) heavy chain complementarity determining region 3 (HC CDR3) presented as GTRAYHY (SEQ ID NO: 24). VL comprises (a) light chain complementarity determining region 1 (LC CDR1) presented as RASDX5LYX6NLA (SEQ ID NO: 5) (where X5 is G, K, or N; and X6 is R or S); (b) light chain complementarity determining region 2 (LC CDR2) presented as DAX7X8LAS (where X7 is F, K, R, or T; X8 is K, L, N, or R); and (c) light chain complementarity determining region 3 (LC CDR3) presented as QHFWGTPLT (sequence identification number: 13).

[0013] In some embodiments, 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.

[0014] In some examples, the antibodies include HC CDR1, HC CDR2, and HC CDR3 as presented in Table 2. Alternatively or additionally, the antibodies include LC CDR1, LC CDR2, and LC CDR3 as presented in Table 1.

[0015] In some examples, the antibodies include HC CDR1, HC CDR2, and HC CDR3 presented as sequence identification numbers: 16, 19, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 presented as sequence identification numbers: 4, 11, and 13, respectively (i.e., HC and LC CDRs identical to clone Fab0070).

[0016] In another example, the antibodies include HC CDR1, HC CDR2, and HC CDR3 presented as sequence identification numbers: 14, 20, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 presented as sequence identification numbers: 2, 7, and 13, respectively (i.e., HC and LC CDRs identical to clone Fab0061).

[0017] In some embodiments, the anti-TfR1 antibody disclosed herein is a humanized antibody. Such humanized antibodies may comprise a light chain framework region as presented in Table 3. Alternatively or additionally, the antibody comprises a heavy chain framework region as presented in Table 4. In some examples, the anti-TfR1 antibody provided herein may comprise VH as presented in Table 5 and / or; the antibody comprises VL as presented in Table 5.

[0018] In some cases, the anti-TfR1 antibody provided herein is a full-length antibody, its antigen-binding fragment, or a single-chain variable fragment (scFv) antibody.

[0019] In a specific example, the antibody Fab molecule comprises the following: (a) a heavy chain comprising VH (e.g., any of those provided in Table 5) and a CH1 heavy chain constant region fragment; and (b) a light chain comprising VL (e.g., any of those provided in Table 5) and a light chain constant region (CL). In some cases, CH1 may be that of a human IgG heavy chain comprising, for example, the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (Sequence ID: 34). Alternatively or additionally, CL may be of a human light chain invariant region (e.g., k or l chain) containing, for example, the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (sequence identification number: 29).

[0020] In one specific example, the anti-Tf1R antibody provided herein may comprise a heavy chain comprising (e.g., composed thereof) the amino acid sequence of SEQ ID NO: 61 and a light chain comprising (e.g., composed thereof) the amino acid sequence of SEQ ID NO: 62 (e.g., clone Fab0070). 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., clone Fab0061).

[0021] In some cases, the anti-Tf1R antibody provided herein may contain an Fc fragment. For example, the anti-Tf1R antibody may be a Fab-Fc molecule comprising a VH-CH1 chain, a VL-CL chain, and an Fc fragment attached to the VH-CH1 chain or the VL-CL chain. One example (xAb000293) is provided in Table 5.

[0022] In another aspect, a conjugate comprising an antibody (anti-TfR1 antibody) that binds to the transferrin receptor (TfR1) and an oligonucleotide-based agonist is provided herein. In some cases, the anti-TfR1 antibody is any of those provided herein (e.g., those listed in Tables 1, 2 and 5, e.g., clones Fab0061 and Fab0070, or their functional equivalents which may include heavy and light chain CDRs identical to those of Fab0061 or Fab0070).

[0023] In some embodiments, oligonucleotide-based agonists may be directly conjugated to an anti-TfR1 antibody via covalent or non-covalent bonds (e.g., ionic bonds, hydrogen bonds, or hydrophobic interactions). In some cases, oligonucleotide-based agonists may be conjugated to the antibody via, for example, peptides, polymers, or nucleic acid-binding proteins. In some examples, oligonucleotide-based agonists may be conjugated to an anti-TfR1 antibody via a linker. Examples include, but are not limited to, chemical linkers, peptide linkers, polymer linkers, or nucleic acid-binding polypeptides. In one example, the linker may be a peptide linker, for example, a glycine spacer of 1 to 4 glycine residues.

[0024] An oligonucleotide-based agent in any of the conjugates disclosed herein may comprise one or more chemically modified nucleotides. In some embodiments, the nucleotides may be chemically modified by substitution with alkyl, alkoxy, amino, deaza, halo, hydroxyl, and / or thiol groups; sugar modification; one or more non-nucleotides; one or more deoxyribonucleotides; one or more non-phosphodiester linkages; or a combination thereof. In some examples, the oligonucleotide-based agent 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 crosslink. In some examples, the oligonucleotide-based agent may comprise one or more non-nucleotides, which may comprise xanthine, hypoxanthine, azapurine, methylthioadenine, 7-deaza-adenosine, or O- and N-modified nucleotides. Alternatively or additionally, the oligonucleotide-based agent may comprise one or more non-phosphodiester linkages, which may comprise one or more nitrogen or sulfur substitutions for oxygen in the phosphate group.

[0025] In some examples, the oligonucleotide-based agonist in any of the conjugates provided herein may be an RNAi agonist 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.

[0026] In some cases, oligonucleotide-based agents, such as RNAi agents, 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 repeat serine / threonine-protein kinase 2, complement C3, microtubule-associating protein tau, alpha-2A adrenergic receptor, sodium channel protein type 9 subunit alpha, apolipoprotein E, alpha-synuclein, putative 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.

[0027] In some cases, the anti-TfR1 antibody is a Fab molecule, which is conjugated to the cap moiety. Oligonucleotide-based agonists are conjugated to the linker. Oligonucleotide-based agonists are conjugated to the Fab molecule via the linker. In some examples, the cap moiety has the following structure:

[0028] Alternatively or additionally, the linker has the following structure:

[0029] In a specific example, an oligonucleotide-based agonist is an RNAi agonist comprising a sense strand and an antisense strand that form a double strand. The sense strand can be conjugated to a linker.

[0030] In another aspect, the present disclosure provides a pharmaceutical composition comprising an anti-TfR1 antibody or a conjugate thereof (e.g., any of those provided herein) and a pharmaceutically acceptable carrier. The anti-TfR1 antibody may be any of those provided herein, e.g., those listed in Tables 1, 2 and 5, e.g., clones Fab0061 and Fab0070, or a functional equivalent thereof that may comprise heavy chain and light chain CDRs identical to those of Fab0061 or Fab0070.

[0031] Additionally, a method for attenuating the expression of a target gene in the brain of a subject is provided herein, said method comprising administering an effective amount of a conjugate or a pharmaceutical composition containing the same to a subject requiring attenuation of the expression of a target gene in the brain. The conjugate comprises an anti-TfR1 antibody conjugated to an oligonucleotide-based agonist. The anti-TfR1 antibody may be any of those provided herein, for example, those listed in Tables 1, 2, and 5, for example, clones Fab0061 and Fab0070, or a functional equivalent thereof that may comprise heavy and light chain CDRs identical to those of Fab0061 or Fab0070. The oligonucleotide-based agonist (e.g., an RNAi agonist) attenuates the expression of the target gene of interest, for example, that provided herein. Any of the conjugates provided herein may be used in the method disclosed herein.

[0032] In some embodiments, the subject is a human patient who has or is 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, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy.

[0033] In some embodiments, the conjugate or the pharmaceutical composition containing it is administered to a subject by intravenous, subcutaneous, intramuscular, parenchymal, or intrathecal injection.

[0034] Additionally, the present invention provides a method for alleviating a CNS disease or disorder, a skeletal muscle disease, a heart disease or disorder, or a disease in other tissues, comprising administering an effective amount of any of the conjugates disclosed herein or a pharmaceutical composition containing such a conjugate to a subject who requires alleviation of a CNS disease or disorder, a skeletal muscle disease, a heart disease or disorder, or a disease in other tissues. The conjugate comprises an anti-Tf1R antibody as disclosed herein (e.g., those listed in Tables 1, 2, and 5, e.g., clones Fab0061 and Fab0070, or a functional equivalent thereof that may comprise heavy and light chain CDRs identical to those of Fab0061 or Fab0070) and target genes of interest, e.g., APP, ataxin-1, ataxin-2, ataxin-3, ataxin-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich repeat serine / threonine-protein kinase 2, complement C3, microtubule-associating protein tau, alpha-2A adrenergic receptor, sodium channel protein type 9 subunit alpha, apolipoprotein E, alpha-synuclein, putative G-protein coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, Includes oligonucleotide-based agonists (e.g., RNAi agonists) that attenuate the expression of MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, DYRK1A, DUX4, DM1, ACVR2A, ACVR2B, MSTN, and GYS1.The subject is a CNS disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, hereditary small fiber neuropathy, or muscular dystrophy or atrophy, e.g., to a non-limiting extent, facioscapulohumeral dystrophy (FSHD), Duchenne muscular dystrophy, and myotonic dystrophy type 1, spinal muscular atrophy, or metabolic myopathy, e.g., to a non-limiting extent, Pompe disease, or It may be a human patient who has or is suspected of having a neuromuscular junction disease, such as, to a limited extent, myasthenia gravis (MG), or metabolic syndrome.

[0035] Additionally, any of the conjugates provided herein for use in treating diseases such as those disclosed herein, or pharmaceutical compositions containing such conjugates, or the use of conjugates / pharmaceutical compositions for manufacturing medicines for use in treating diseases is within the scope of the present disclosure.

[0036] Additionally, the present disclosure provides a nucleic acid or set of nucleic acids comprising a nucleotide sequence encoding the heavy and light chains of the anti-TfR1 antibody disclosed herein, for example those listed in Tables 1, 2, and 5, for example, clones Fab0061 and Fab0070, or a functional equivalent thereof that may include heavy and light chain CDRs identical to those of Fab0061 or Fab0070. In some cases, the nucleic acid or set of nucleic acids is a vector or set of vectors. In some examples, the vector or set of vectors is an expression vector or set of expression vectors. Additionally, a host cell comprising the nucleic acid or set of nucleic acids disclosed herein is provided herein.

[0037] Additionally, the present disclosure features a method for producing an anti-TfR1 antibody, comprising: (i) culturing a host cell having coding nucleic acid(s) for any of the anti-TfR1 antibodies provided herein (e.g., those listed in Tables 1, 2 and 5, e.g., clones Fab0061 and Fab0070, or functional equivalents thereof that may include heavy and light chain CDRs identical to those of Fab0061 or Fab0070) under conditions allowing the expression of the anti-TfR1 antibody; and (ii) collecting the anti-TfR1 antibody produced thereby.

[0038] Additional embodiments of the present disclosure are provided below.

[0039] Embodiment 1. An antibody that binds to a transferrin receptor (TfR1), comprising a heavy chain including a heavy chain variable region (VH) and a light chain including a light chain variable region (VL);

[0040] Here, VH is:

[0041] Heavy chain complementarity determination region 1 (HC CDR1) presented as GX1TFX2SYWX3H, where X1 is F or Y; X2 is K, N, or T; and X3 is M or V;

[0042] Heavy chain complementarity determining region 2 (HC CDR2) presented as EINPTNGRX4NYIEKFKS (sequence identification number: 23), where X4 is F, S, T, or V; and

[0043] It includes heavy chain complementarity determining region 3 (HC CDR3) presented as GTRAYHY (sequence identification number: 24);

[0044] Here, VL is:

[0045] Light chain complementarity determining region 1 (LC CDR1) presented as RASDX5LYX6NLA (sequence identification number: 5), where X5 is G, K, or N, and X6 is R or S;

[0046] Light chain complementarity determination region 2 (LC CDR2) presented as DAX7X8LAS, where X7 is F, K, R, or T; X8 is K, L, N, or R; and

[0047] Light chain complementarity determining region 3 (LC CDR3) presented as QHFWGTPLT (Sequence ID: 13)

[0048] An antibody that contains

[0049] Embodiment 2. The antibody of Embodiment 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.

[0050] Embodiment 3. In Embodiment 1, comprising any one of HC CDR1, HC CDR2, and HC CDR3 as presented in Table 2, such as Fab0070, Fab0061, Fab0060, Fab0062, Fab0063, Fab0064, Fab0065, Fab0066, Fab0067, Fab0068, Fab0069, Fab0071, Fab0072, Fab0073, Fab0074, Fab0165, Fab0166, Fab0167, or Fab0168; An antibody comprising 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 presented in Table 1.

[0051] Embodiment 4. In Embodiment 3,

[0052] (a) HC CDR1, HC CDR2, and HC CDR3 presented as sequence identification numbers: 16, 19, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 presented as sequence identification numbers: 4, 11, and 13, respectively; or

[0053] (b) HC CDR1, HC CDR2, and HC CDR3 presented as sequence identification numbers: 14, 20, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 presented as sequence identification numbers: 2, 7, and 13, respectively

[0054] Antibody containing

[0055] Embodiment 5. Any one of Embodiments 1-4, wherein the antibody is a humanized antibody; optionally comprising a light chain framework region presented in Table 3 and / or a heavy chain framework region presented in Table 4.

[0056] Embodiment 6. An antibody according to Embodiment 5, comprising any one of the VH sequences presented in Table 5 and / or any one of the VL sequences presented in Table 5.

[0057] Embodiment 7. An antibody that is a full-length antibody, its antigen-binding fragment, or a single-chain variable fragment (scFv) antibody in any one of Embodiments 1-6.

[0058] Embodiment 8. An antibody in any one of Embodiments 1-7, wherein the Fab molecule; the heavy chain comprises VH and a CH1 heavy chain constant region fragment; and the light chain comprises VL and a light chain constant region (CL).

[0059] Embodiment 9. In Embodiment 8, CH1 comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (Sequence Identification No.: 34);

[0060] An antibody in which CL contains the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence identification number: 29).

[0061] Embodiment 10. An antibody according to Embodiment 8 or 9, wherein VH comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTLVTVSS (Sequence Identification No.: 59) and / or VL comprises the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (Sequence Identification No.: 60).

[0062] Embodiment 11. In Embodiment 8 or 9,

[0063] (a) the heavy chain comprises the amino acid sequence of sequence identification number: 61, and the light chain comprises the amino acid sequence of sequence identification number: 62; or

[0064] (b) An antibody in which the heavy chain comprises the amino acid sequence of sequence identification number: 45 and the light chain comprises the amino acid sequence of sequence identification number: 46.

[0065] Embodiment 12. An antibody comprising an Fc fragment in any one of Embodiments 1-11, optionally a Fab-Fc molecule.

[0066] Embodiment 13. A conjugate comprising an antibody that binds to a transferrin receptor (TfR1) (anti-TfR1 antibody) and an oligonucleotide-based agonist, wherein the anti-TfR1 antibody is the one presented in any one of Embodiments 1-12.

[0067] Embodiment 14. A conjugate in which an oligonucleotide-based agent is directly conjugated to an anti-TfR1 antibody via a covalent or non-covalent bond in Embodiment 13.

[0068] Embodiment 15. A conjugate in which an oligonucleotide-based agent is conjugated to an anti-TfR1 antibody through a linker that is optionally a chemical linker, a peptide linker, a polymer linker, or a nucleic acid-binding polypeptide linker in Embodiment 13.

[0069] Embodiment 16. The conjugate of Embodiment 13, wherein the linker is a peptide linker that is a glycine spacer of 1 to 4 glycine residues.

[0070] Embodiment 17. A conjugate in any one of Embodiments 13-16, wherein the oligonucleotide-based agent comprises one or more chemically modified nucleotides.

[0071] Embodiment 18. The conjugate of Embodiment 17, wherein the nucleotide is chemically modified by substitution with an alkyl, alkoxy, amino, deaza, halo, hydroxyl, and / or thiol group; sugar modification, one or more non-nucleotides; one or more deoxyribonucleotides; one or more non-phosphodiester linkages; or a combination thereof.

[0072] Embodiment 19. In Embodiment 18,

[0073] The sugar modification comprises in which the 2' OH group is replaced by a 2' amino group, a 2' O-methyl group, a 2' fluoro group, a 2' methoxyethyl group, or a 2'-O, 4'-C methylene crosslink;

[0074] One or more non-nucleotides comprise xanthine, hypoxanthine, azapurine, methylthioadenine, 7-deaza-adenosine, or O- and N-modified nucleotides;

[0075] A conjugate in which one or more non-phosphodiester links comprise one or more nitrogen or sulfur substitutions for oxygen in the phosphate group.

[0076] Embodiment 20. A conjugate of RNAi agonists comprising a sense strand and an antisense strand forming a double strand, wherein the oligonucleotide-based agonist is a conjugate in any one of Embodiments 13-19.

[0077] Embodiment 21. In Embodiment 20, the RNAi agonist comprises APP, ataxin-1, ataxin-2, ataxin-3, ataxin-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich repeat serine / threonine-protein kinase 2, complement C3, microtubule-associating protein tau, alpha-2A adrenergic receptor, sodium channel protein type 9 subunit alpha, apolipoprotein E, alpha-synuclein, putative 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. A conjugate that weakens the expression of a target gene selected from the group.

[0078] Embodiment 22. A conjugate in which the target gene in Embodiment 21 is selected from the group consisting of APP, ATXN2, ATXN3, AR, SOD1, HTT, C9ORF72, LRRK2, C3, MAPT, ADRA2A, SCN9A, APOE, SNCA, GPR75, and FUS.

[0079] Embodiment 23. In any one of Embodiments 20-22, the sense strand is conjugated to an anti-TfR1 antibody, wherein the anti-TfR1 antibody is a conjugate in which the Fab molecule.

[0080] Embodiment 24. In any one of Embodiments 13-23, the anti-TfR1 antibody is a Fab molecule; wherein the oligonucleotide-based agent is conjugated to a linker of the following chemical formula:

[0081] ,

[0082] Here represents the attachment site for the Fab molecule, and A conjugate that represents the attachment site of the linker to the oligonucleotide-based agonist portion of the conjugate.

[0083] Embodiment 25. In any one of Embodiments 13-23, the anti-TfR1 antibody is a Fab molecule containing a cap moiety; wherein the oligonucleotide-based agonist is conjugated to a linker, and the oligonucleotide-based agonist is conjugated to the Fab molecule through the linker.

[0084] Embodiment 26. In Embodiment 25, the cap moiety has the following chemical formula and / or:

[0085] ; The linker has the following structure

[0086] , here represents the attachment site for the Fab molecule, and A conjugate that represents the attachment site of the linker to the oligonucleotide-based agonist portion of the conjugate.

[0087] Embodiment 27. The oligonucleotide-based agent of Embodiment 25 or Embodiment 26 is an RNAi agent comprising a sense strand and an antisense strand forming a double strand; wherein the sense strand is a conjugate joined to a linker.

[0088] Embodiment 28. A pharmaceutical composition comprising an anti-TfR1 antibody or a conjugate thereof and a pharmaceutically acceptable carrier, wherein the anti-TfR1 antibody is the one presented in any one of Embodiments 1-12.

[0089] Embodiment 29. A pharmaceutical composition according to Embodiment 27, comprising a conjugate of an anti-TfR1 antibody, wherein the conjugate is as presented in any one of Embodiments 13-27.

[0090] Embodiment 30. A method for weakening the expression of a target gene in the brain of a subject, comprising administering an effective amount of a conjugate or a pharmaceutical composition containing the same to a subject who requires weakening of the expression of a target gene in the brain, and

[0091] Here, the conjugate comprises an anti-TfR1 antibody conjugated to an oligonucleotide-based agonist, and

[0092] Herein, the anti-TfR1 antibody is presented in any one of embodiments 1-12;

[0093] Here, the method involves an oligonucleotide-based agonist attenuating the expression of a target gene.

[0094] Embodiment 31. A method in which, in Embodiment 30, the composite is as presented in any one of Embodiments 12-27.

[0095] Embodiment 32. The method of Embodiment 30 or Embodiment 31, wherein the subject is a human patient who has or is suspected of having a central nervous system (CNS) disease.

[0096] Embodiment 33. The method of Embodiment 32, wherein the CNS disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy.

[0097] Embodiment 34. A method in which, in any one of Embodiments 30-33, the conjugate or the pharmaceutical composition containing the same is administered to a subject by intravenous, subcutaneous, intramuscular, parenchymal, or intrathecal injection.

[0098] Embodiment 35. A method for alleviating CNS disease, comprising administering an effective amount of the conjugate presented in any one of Embodiments 12-26 or a pharmaceutical composition containing the same to a subject requiring alleviation of CNS disease, and

[0099] Here, the conjugate is an oligonucleotide-based that attenuates the expression of target genes selected from the group consisting of APP, ataxin-1, ataxin-2, ataxin-3, ataxin-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich repeat serine / threonine-protein kinase 2, complement C3, microtubule-associating protein tau, alpha-2A adrenergic receptor, sodium channel protein type 9 subunit alpha, apolipoprotein E, alpha-synuclein, putative 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 Includes an agent;

[0100] A method in which the subject has or is suspected of having a CNS disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy.

[0101] Embodiment 36. A method for weakening the expression of a target gene in the skeletal muscle of a subject, comprising administering an effective amount of a conjugate or a pharmaceutical composition containing the same to a subject who requires weakening of the expression of a target gene in the skeletal muscle, and

[0102] Here, the conjugate comprises an anti-TfR1 antibody conjugated to an oligonucleotide-based agonist, and

[0103] Herein, the anti-TfR1 antibody is presented in any one of embodiments 1-12;

[0104] Here, the method involves an oligonucleotide-based agonist attenuating the expression of a target gene.

[0105] Embodiment 37. The method of Embodiment 36, wherein the target gene is selected from the group consisting of DUX4, DM1, ACVR2A, ACVR2B, MSTN, and GYS1.

[0106] Embodiment 38. The method of Embodiment 36 or 37, wherein the subject is a human patient who has or is suspected of having a skeletal muscle disease.

[0107] Embodiment 39. The method of Embodiment 38, wherein the skeletal muscle disease is selected from the group consisting of facial-scapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy, and myotonic dystrophy of type 1, spinal muscular atrophy, or metabolic myopathy, e.g., Pompe disease, to the contrary, or neuromuscular junction disease, e.g., myasthenia gravis (MG), to the contrary, or metabolic syndrome.

[0108] Embodiment 40. A nucleic acid or set of nucleic acids comprising a nucleotide sequence encoding the heavy chain and light chain of an anti-TfR1 antibody presented in any one of Embodiments 1-12.

[0109] Embodiment 41. In Embodiment 40, a nucleic acid or nucleic acid set that is a vector or vector set.

[0110] Embodiment 42. In Embodiment 41, the nucleic acid or nucleic acid set in which the vector or vector set is an expression vector or expression vector set.

[0111] Embodiment 43. A host cell comprising the nucleic acid or set of nucleic acids presented in any one of Embodiments 40-42.

[0112] Embodiment 44. A method for producing an anti-TfR1 antibody,

[0113] (i) a step of culturing the host cells of Embodiment 43 under conditions allowing the expression of an anti-TfR1 antibody; and

[0114] (ii) A step of collecting the anti-TfR1 antibody produced accordingly.

[0115] A method including

[0116] Specific preferred embodiments of the present invention will become apparent from the following more detailed description of specific preferred embodiments and the claims. Brief explanation of the drawing

[0117] These and other features, aspects, and advantages of the present disclosure can be better understood by reading the following detailed description with reference to the accompanying drawings. Figure 1 shows the relative androgen receptor (AR) mRNA expression knockdown compared to control siRNA after delivery of selected TfR1-targeting Fab conjugated to siRNA having an antisense strand complementary to the androgen receptor mRNA in different brain regions. Figure 2 shows the relative androgen receptor mRNA expression knockdown compared to control siRNA after delivery of selected TfR1-targeting Fab conjugated to siRNA having an antisense strand complementary to androgen receptor mRNA in different brain regions. Figure 3 shows the relative androgen receptor mRNA expression knockdown compared to control siRNA after delivery of selected TfR1-targeting Fab conjugated to siRNA having an antisense strand complementary to androgen receptor mRNA in different brain regions. Figure 4 shows the binding affinity of the selected TfR1-targeted Fab normalized for Fab0002, K D It shows as. Figure 5 shows the knockdown of MAPT protein in various tissues according to the description of Example 13. Specific details for implementing the invention

[0118] The details set forth herein are merely for illustrative purposes and for the exemplary discussion of preferred embodiments of the invention, and are presented to provide what is considered to be the most useful and easily understood description of the principles and concepts of various embodiments of the invention. In this regard, no attempt has been made to describe the structural details of the invention in greater detail than is necessary for a fundamental understanding of the invention, and the description, together with the drawings and / or embodiments, makes clear to those skilled in the art how various forms of the invention can be practically implemented.

[0119] definition

[0120] The following definitions and descriptions are intended and intended to be dominant in any future constructions, unless otherwise clearly and unambiguously modified in the following examples, or where the application of the meaning would render any construction meaningless or essentially meaningless. Where the construction of a term would render it meaningless or essentially meaningless, the definitions should be taken from the literature [Webster's Dictionary, 3rd sup. rd Edition] or dictionaries known to those skilled in the art, such as the literature [Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004)].

[0121] All percentages used herein are weight percentages unless otherwise noted.

[0122] Unless otherwise indicated, singular terms used herein are interpreted to mean "one," "at least one," or "one or more." Unless otherwise required by the context, singular terms used herein will include the plural, and plural terms will include the singular.

[0123] As used herein, the term "receptor" is intended to encompass the entire receptor or its ligand-binding portion. These portions of the receptor include regions sufficient for specific binding of a ligand to occur, particularly regions that can be recognized by an antibody or antibody fragment.

[0124] The antibody (used interchangeably in plural form) is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody,” e.g., anti-TfR1 antibody, encompasses intact (e.g., full-length) polyclonal or monoclonal antibodies, as well as their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-strand antibodies (scFv), fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., VH monobody), multispecific antibodies (e.g., bispecific antibodies), and any other modified forms of immunoglobulin molecules containing an antigen recognition site of required specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies, e.g., anti-TfR1 antibody, include antibodies of any class, e.g., IgD, IgE, IgG, IgA, or IgM (or their subclasses), and the antibody does not need to be of any specific class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of their heavy chain constant domain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isoforms), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional shapes of the different classes of immunoglobulins are widely known.

[0125] The term “Fab” as used herein refers to a fragment antigen-binding (Fab) region that binds to the TfR1 antigen and includes six complementarity determining regions (CDRs), where three CDRs are on the variable light chain (VL) and three CDRs are on the variable heavy chain (VH).

[0126] The term "ligand" is defined as any molecule or atom that binds to a recipient molecule, for example, a protein, nucleotide or fragment, a binding domain or its complement.

[0127] As used herein, the term "protein" includes peptides, polypeptides, consensus molecules, fusion proteins, purified naturally occurring proteins, artificially synthesized proteins, recombinant proteins, antibodies, antibody fragments, and analogs, derivatives, or combinations thereof.

[0128] Additionally, the term "antigen-binding fragment" (e.g., Fab) is intended to encompass fully humanized and chimeric antibody fragments, bifunctional antibody fragments, etc., which include portions from more than one species.

[0129] The term "HA2 peptide" refers to a peptide containing 20 amino acids at the N-terminus of the influenza virus hemagglutinin protein.

[0130] The term “conjugate” is defined as comprising two or more molecules; or two or more entities coupled together. Preferably, the two molecules or entities are conjugated by non-specific or specific protein-protein interactions, by covalent bonds, by non-covalent bonds, or by coordination chemistry. In the context of the invention, the first molecule may be an interfering RNA molecule, while the second molecule may be a ligand for a receptor on a target cell as defined herein. In some embodiments, the term conjugate comprises an oligonucleotide-based agonist, e.g., TfR1 Fab coupled to siRNA, which may also comprise a linker and / or peptide or protein for the release of the oligonucleotide-based agonist from the endosome as described herein.

[0131] As used herein, the terms “interfering RNA” and “interfering RNA molecule” refer to any RNA or RNA-like molecule that interacts with RISC and can participate in RISC-mediated changes in gene expression.

[0132] As used herein, the term "siRNA" refers to double-stranded interfering RNA unless otherwise indicated.

[0133] As used herein, the term "therapeutic effective dose" refers to an amount of interfering RNA or a pharmaceutical composition containing interfering RNA that is determined to produce a therapeutic response in mammals. Such therapeutic effective dose is readily identified by a person skilled in the art and using methods as described herein.

[0134] As used herein, the phrase “attenuating expression” in relation to a gene or mRNA means administering or expressing an amount of interfering RNA (e.g., siRNA) that reduces the translation of target mRNA into protein through mRNA cleavage or direct inhibition of translation.

[0135] As used herein, the terms “inhibit,” “silence,” and “attenuate” refer to the expression of a target mRNA or a measurable reduction of the expression of a target mRNA or a corresponding protein in the absence of the interfering RNA of the present invention or compared with the corresponding protein.

[0136] The term "knockdown" refers to the expression of a target mRNA or a reduction in the corresponding protein.

[0137] As used herein, the terms "target sequence" and "target mRNA" refer to a portion of mRNA or mRNA sequence that can be recognized by the interfering RNA used in the method of the present invention, wherein the interfering RNA can silence gene expression as discussed herein.

[0138] As used herein, the term “CNS disorder” includes conditions or disorders associated with the central nervous system (CNS), including but not limited to the patient’s brain and spinal cord. In some embodiments, CNS disorder 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, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy.

[0139] As used herein, the term “skeletal muscle disorder” includes conditions or disorders associated with skeletal muscle tissue, including but not limited to FSHD, myotonic dystrophy of type 1, and Duchenne muscular dystrophy.

[0140] As used herein, the term "patient" means a human or other mammal that has or is at risk of having a CNS disorder.

[0141] As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the substitution is made. Variants may be prepared according to methods of modifying polypeptide sequences known to those skilled in the art, as identified in references compiling such methods, such as, for example, [Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York]. Conservative amino acid substitutions include substitutions made from amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0142] The "percent identity" of two amino acid sequences is determined using the algorithm from [Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990] and a modified algorithm as in [Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993]. These algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) from [Altschul, et al., J. Mol. Biol. 215:403-10, 1990]. BLAST protein search 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. If a gap exists between the two sequences, a gapd BLAST is performed [Altschul et al., Nucleic Acids Res. It can be used as described in [25(17):3389-3402, 1997]. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0143] The transferrin receptor (TfR1) is an intrinsic membrane glycoprotein that mediates iron uptake by individual cells. There is a correlation between the number of receptors on the cell surface and cell proliferation, with the highest number of receptors present 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) suggest that the TfR1-binding domain of Tf is located within the C-lobe of human Tf, residues 334-679. Residues 365-401 (particularly 381-401), 415-433, and 457-470 appear to be particularly important for receptor binding.

[0144] Targeting the TfR1 receptor is a promising approach to facilitate the delivery of drugs to the brain across the blood-brain barrier (BBB). An anti-TfR1 antibody having a high binding affinity for TfR1 and other excellent characteristics also provided herein, such as high expression yield, is provided herein. This anti-TfR1 antibody is reported herein to successfully facilitate the delivery of an exemplary siRNA molecule conjugated thereto into brain cells, thereby attenuating the expression of the gene targeted by the siRNA. Thus, the anti-TfR1 antibody provided herein can be used to achieve efficient drug delivery to the brain, thereby addressing the greatest challenge associated with developing therapeutic agents for treating central nervous system (CNS) diseases.

[0145] I. Anti-TfR1 Fab and Anti-TfR1 Antibodies

[0146] The present disclosure provides an antibody that binds to TfR1, e.g., human TfR1. In some embodiments, the anti-TfR1 antibody disclosed herein may bind to TfR1 expressed on a cell surface, e.g., on brain endothelial cells. Thus, the antibody disclosed herein may be used to facilitate the delivery of a therapeutic or diagnostic agent conjugated to the antibody into the brain. In some cases, the anti-TfR1 antibody provided herein may also be used to detect the presence of the TfR1 receptor in vitro or in vivo. As used herein, the term "anti-TfR1 antibody" refers to any antibody capable of binding to a TfR1 polypeptide (e.g., a TfR1 polypeptide expressed on a cell surface, e.g., on brain endothelial cells), which may be from a suitable source, e.g., human or non-human mammal (e.g., mouse, rat, rabbit, primate, e.g., monkey).

[0147] A typical antibody molecule typically has a variable heavy chain region (V) associated with antigen binding. H ) and light chain variable region (V L Includes ). V H and V L The region may be further subdivided into a hypervariable region, also known as the "complementarity determination region" ("CDR"), which is interspersed with more conserved regions, known as the "framework region" ("FR"). Each V H and V LIt typically consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The scope of the framework region and CDRs can be accurately identified using methodologies known in the relevant art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or contact definition, all of which are widely known in the relevant art. For example, the literature [Kabat, EA, 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. See Al-lazikani et al., (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004)]. Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0148] The anti-TfR1 antibody described herein may be a full-length antibody containing two heavy chains and two light chains, each containing 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 included within the term "antigen-binding fragment" of a full-length antibody include: (i) a Fab fragment which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment which is a divalent fragment consisting of two Fab fragments connected by a disulfide crosslink at a hinge region; (iii) a Fd fragment consisting of VH and CH1 domains; (iv) a Fv fragment consisting of VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR) possessing functionality. Additionally, the two domains of the Fv fragment, VL and VH, are encoded by individual genes, but they can be linked by a synthetic linker using a recombination method so that the VL and VH regions pair up to form a monovalent molecule known as single-stranded Fv (scFv), which can then be prepared as a single protein chain. For example, see the literature [Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883].

[0149] In some embodiments, the anti-TfR1 antibody provided herein is a TfR1 fragment antigen-binding region (Fab) that specifically binds to TfR1. In some examples, the TfR1-specific Fab binds to TfR1 on brain endothelial cells. In some embodiments, the TfR1-specific Fab can specifically recognize and bind to one or more epitopes on TfR1.

[0150] The anti-TfR1 antibodies disclosed herein, such as the TfR1-specific Fab, can competitively bind to TfR1 and transferrin (Tf), a physiological TfR1 ligand. When Fab binds to TfR1 on the cell surface, the delivery of Fab and attached interfering RNA enters the cell via constitutive clathrin-mediated endocytosis.

[0151] The antibodies described herein may be of a suitable origin, e.g., murine, rat, or human. These antibodies are non-naturally occurring antibodies, that is, they would not be produced in animals without human action (e.g., immunizing these animals with the target antigen or fragments thereof, or isolating them from an antibody library). Any of the antibodies described herein, e.g., anti-TfR1 antibodies, may be monoclonal or polyclonal. "Monoclonal antibody" refers to a homologous antibody population, and "polyclonal antibody" refers to a heterologous antibody population. These two terms do not limit the source of the antibodies or the method of their preparation.

[0152] In some embodiments, the anti-TfR1 antibody is a human antibody, which may be isolated from a human antibody library or generated in transgenic mice. For example, a fully human antibody may be obtained by using commercially available mice engineered to express a specific human immunoglobulin protein. Transgenic animals designed to generate a more desirable (e.g., fully human antibody) or more robust immune response may also be used for humanization or the generation of human antibodies. Examples of such technologies are Xenomouse™ from Amgen, Inc. (Fremont, California) and HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, New Jersey). In another alternative, the antibody may be recombinantly produced by phage display or yeast technology. For example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and see the literature [Winter et al., (1994) Annu. Rev. Immunol. 12:433-455]. Alternatively, human antibodies and antibody fragments can be produced in vitro from an immunoglobulin variable (V) domain gene repertoire from a non-immunized donor using antibody library display techniques known in the art, such as phage, yeast display, mammalian cell display, or mRNA display techniques.

[0153] In another embodiment, the anti-TfR1 antibody may be a humanized antibody. A humanized antibody refers to a form of a non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or its antigen-binding fragment containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, one or more Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Additionally, the humanized antibody may include residues that are not found in the CDR or framework sequences introduced in the recipient antibody, but are included to further refine and optimize antibody performance. In some cases, the humanized antibody may substantially comprise at least one, typically two, variable domains, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulin, and all or substantially all FR regions are of the human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least one portion of the immunoglobulin constant region or domain (Fc), typically of 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 (1, 2, 3, 4, 5, or 6) modified relative to the original antibody, which are also referred to as one or more CDRs derived from one or more CDRs from the original antibody. The humanized antibody may also involve affinity maturation. Methods for constructing humanized antibodies are also widely known in the art. For example, the literature [Queen et al., Proc. Natl. Acad. Sci.See USA, 86:10029-10033 (1989)].

[0154] In some embodiments, the anti-TfR1 antibody described herein specifically binds to a corresponding target antigen (here TfR1) or its epitope. An antibody that "specifically binds" to an antigen or epitope is a term widely understood in the art. A molecule is referred to as exhibiting "specific binding" when it reacts with a specific target antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than with an alternative target. An antibody "specifically binds" to a target antigen or epitope when it binds with greater affinity, binding strength, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (TfR1) or its antigen epitope is an antibody that binds to this target antigen with greater affinity, binding strength, more readily, and / or for a longer duration than it binds to other antigens or other epitopes of the same antigen. Under these definitions, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it may include it). In some examples, an antibody that "specifically binds" to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen (i.e., only baseline binding activity may be detected by conventional methods).

[0155] In some embodiments, the anti-TfR1 antibody as described herein has a suitable binding affinity for a target antigen (e.g., TfR1) or its antigenic epitope. As used herein, "binding affinity" refers to the apparent association constant or K ARefers to. K A is the dissociation constant (K D It is the reciprocal of ). The anti-TfR1 antibody described herein has a binding affinity (K) of at least 2 nM, 1 nM, 0.5 nM, 0.1 nM, or less for TfR1. D It can have ). Increased binding affinity is reduced K D It corresponds to. The higher affinity binding of the antibody to the first antigen compared to the second antigen is K for binding to the second antigen. A (or numerical K D Higher K for binding to the first antigen than ) A (or a smaller value K) D It can be represented by ). In such cases, the antibody has specificity for the first antigen (e.g., the first protein in the first conformation or its mimic) compared to the second antigen (e.g., the same first protein in the second conformation or its mimic; or the second protein). The difference in binding affinity (e.g., specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 90, 100, 500, 1000, 10,000, or 10 5 It may be a multiple. In some embodiments, any of the anti-TfR1 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or its antigen epitope.

[0156] 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). An exemplary condition for evaluating binding affinity is in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). Using these techniques, the concentration of the bound binding protein can be measured as a function of the target protein concentration. The concentration of the bound binding protein ([Bound]) is generally related to the concentration of the free target protein ([Free]) by the following equation: [Bound] = [Free] / (Kd + [Free]).

[0157] However, K A It is not always necessary to determine precisely, and this is sometimes determined using methods such as ELISA or FACS analysis, K A This is because it is sufficient to obtain quantitative measurements of affinity, which can be used for comparisons such as determining whether a higher affinity is, for example, 2 times higher, since it is proportional to, obtain qualitative measurements of affinity, or obtain estimates of affinity by activity in, for example, functional tests, for example in vitro or in vivo tests.

[0158] A. Exemplary anti-TfR1 antibody

[0159] In some embodiments, the anti-TfR1 antibodies provided herein are humanized antibodies derived from mouse monoclonal antibody clones. Briefly, to produce parental humanized antibodies, the heavy and light chain complementarity determining domains of the mouse parent are grafted onto the human consensus VH and VL frameworks. A humanization library was generated using Kunkel mutagenesis to introduce mutations at specific framework region sites, CDR-FR junction sites were constructed, and clones exhibiting high binding affinity for human TfR1 were screened. mab1G derived from the screening exhibited the highest binding affinity and was therefore selected for further affinity maturation. Refer to the following examples. An exemplary anti-TfR1 antibody derived from affinity maturation containing mutations at specific CDRs compared to the mab1G parental antibody exhibited further enhanced binding affinity. Specific clones, e.g., Fab0061 and Fab0070, exhibited superior rodent and sino-cross-activity compared to the mouse parent antibody (Fab version). Additionally, specific clones, e.g., Fab0061 and Fab0070, showed an approximately 4-fold increase in expression yield compared to the mouse parent antibody in Fab format.

[0160] Accordingly, the anti-TfR1 antibody provided herein (e.g., in Fab format) may comprise a heavy chain comprising three complementarity determining regions (CDRs), a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3, and a light chain comprising a light chain variable region (VL). In some cases, the anti-TfR1 antibody contains a heavy chain CDR3 presented as GTRAYHY (Sequence Identification No.: 24) and a light chain CDR3 presented as QHFWGTPLT (Sequence Identification No.: 13). The anti-TfR1 antibody may further comprise a heavy chain CDR1 having the consensus 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 the consensus motif of EINPTNGRX4NYIEKFKS (Sequence Identification No.: 23) (where X4 is F, S, T, or V). Alternatively or additionally, the anti-TfR1 antibody may comprise a light chain CDR1 having the consensus motif of RASDX5LYX6NLA (Sequence Identification No.: 5) (where X5 is G, K, or N; and X6 is R or S); and may further include a light chain CDR2 having the consensus motif of DAX7X8LAS (where X7 is F, K, R, or T; and X8 is K, L, N, or R). In some examples, in the consensus motif 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. In some embodiments, in the consensus motif X1 is F; X2 is T; X3 is M; and X4 is T. In some embodiments, in the consensus motif X5 is K; X6 is S; X7 is T; and X8 is L. In some embodiments, in the consensus motif 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.

[0161] In some embodiments, X1 is F. In some embodiments, X1 is Y.

[0162] In some embodiments, X2 is K. In some embodiments, X2 is N. In some embodiments, X2 is T.

[0163] In some embodiments, X3 is M. In some embodiments, X3 is V.

[0164] In some embodiments, X4 is F. In some embodiments, X4 is S. In some embodiments, X4 is T. In some embodiments, X4 is V.

[0165] In some embodiments, X5 is G. In some embodiments, X5 is K. In some embodiments, X5 is N.

[0166] In some embodiments, X6 is R. In some embodiments, X6 is S.

[0167] In some embodiments, X7 is F. In some embodiments, X7 is K. In some embodiments, X7 is R. In some embodiments, X7 is T.

[0168] In some embodiments, X8 is K. In some embodiments, X8 is L. In some embodiments, X8 is N. In some embodiments, X8 is R.

[0169] Exemplary combinations of light chain CDRs of the anti-TfR1 antibody provided herein are provided in Table 1 below. Exemplary combinations of heavy chain CDRs of the anti-TfR1 antibody provided herein are provided in Table 2 below. In some cases, the anti-TfR1 antibody may include one or more of the heavy and light chain framework regions provided in Tables 3 and 4 below.

[0170] In some embodiments, the anti-TfR1 antibody described herein binds to an epitope of the same TfR1 polypeptide as any of the exemplary antibodies described herein (e.g., those disclosed in Table 5, e.g., clone Fab0061 or Fab0070), or competes with the exemplary antibody for binding to the TfR1 antigen. In some examples, the exemplary antibody is Fab0061. In other examples, the exemplary antibody is Fab0070. “Epitope” refers to a site on the target antigen that is recognized and bound by the antibody. The site may consist entirely of amino acid components, entirely of chemical modifications of amino acids of the protein (e.g., glycosyl moiety), or a combination thereof. Overlapping epitopes include at least one common amino acid residue. Epitopes may be linear, which is typically 6 to 15 amino acids long. Alternatively, epitopes may be stereomorphic. The epitope to which the antibody binds may be determined by commercial technology, for example, an epitope mapping method (e.g., see description below). An antibody binding to the same epitope as the exemplary antibody described herein may bind to an epitope that is exactly the same as or substantially overlapping with the exemplary antibody (e.g., containing fewer than three non-overlapping amino acid residues, fewer than two non-overlapping amino acid residues, or only one non-overlapping amino acid residue). Whether two antibodies compete with each other for binding to a cognate antigen may be determined by a competition test widely known in the art.

[0171] In some examples, the anti-TfR1 antibody is the same V as the exemplary antibodies described herein (e.g., those provided in Table 5, e.g., Fab0061 or Fab0070). H and / or V L Includes CDR. Same V H and / or V LTwo antibodies having a CDR mean that their CDR is identical when determined by the same approach (e.g., the Kavat approach, the Cotia approach, the AbM approach, the contact approach, or the IMGT approach as known in the relevant art). For example, see bioinf.org.uk / abs / . These anti-TfR1 antibodies have the same V compared to the exemplary antibodies described herein. H , identical V L , or you can have both.

[0172] Functional variants of any of the exemplary anti-TfR1 antibodies disclosed herein are also within the scope of this disclosure. These functional variants are substantially similar to the exemplary antibodies in both structural and functional aspects. The functional variants are substantially identical to the exemplary antibodies V H and V L It includes a CDR. For example, it may contain only eight or fewer amino acid residue variations (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) within the total CDR region of the antibody and binds to the same epitope of TfR1 with substantially similar affinity (e.g., K of the same digit). D (Having a value). 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. These anti-TfR1 antibodies are V of the exemplary antibody H V having a CDR amino acid residue mutation only in heavy chain CDR1 compared to H It may contain fragments. In some examples, the anti-TfR1 antibody is the same V as the exemplary antibody. L CDR3, and arbitrarily the same V L V having CDR1 or VL CDR2 L You can include additional short stories.

[0173] Alternatively or additionally, amino acid residue modifications may be conservative amino acid residue substitutions.

[0174] In some embodiments, the anti-TfR1 antibody is V of the exemplary antibody described herein. H It may include heavy chain CDRs having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, compared with the CDRs. Alternatively or additionally, the anti-TfR1 antibody is V of the exemplary antibody described herein. L It may include light chain CDRs that have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, with respect to the CDR. As used herein, "individually" means that one CDR of the antibody shares the indicated sequence identity with respect to the corresponding CDR of the exemplary antibody. "Collectively" means three V of the antibody H or V L The CDR combination corresponds to the three V of the exemplary antibody H or V L This means that it shares sequence identity indicated relative to the CDR combination.

[0175] In some embodiments, the anti-TfR1 antibody may comprise a VH that is at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to 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 VH that is at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to one of the exemplary antibodies provided in Table 5 (e.g., the VL of Fab0061 or Fab0070). Such variants may comprise heavy chain and light chain CDRs identical to the exemplary antibodies having variations in only one or more of the FR regions.

[0176] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. Alternatively or additionally, the light chain of the anti-TfR1 antibody may further comprise a CL which may be any light chain constant region (CL) known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. Antibody heavy chains and light chain constant regions are widely known in the art and are available, for example, at the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0177] In some cases, the anti-TfR1 antibody is a Fab format comprising a heavy chain containing any of the VH regions disclosed herein and a CH1 fragment, and a light chain containing any of the VL regions disclosed herein and a light chain constant region (LC). The CH1 fragment may be of any immunoglobulin (Ig) heavy chain constant region (e.g., human Ig heavy chain constant region). In some cases, CH1 may be of an IgG (e.g., IgG1, IgG2, or IgG4) heavy chain. The LC may be of human origin in some cases. It may be a kappa chain. Alternatively, it may be a lambda chain. Examples of the anti-TfR1 Fab antibodies disclosed herein are provided in Table 5.

[0178] In some embodiments, the anti-TfR1 antibody disclosed herein may be a single-strand antibody (scFv). The scFv antibody may comprise a VH fragment and a VL fragment that can be linked via a flexible peptide linker. In some cases, the scFv antibody may be oriented VH→VL (from the N-terminus to the C-terminus). In other cases, the scFv antibody may be oriented VL→VH (from the N-terminus to the C-terminus).

[0179] Any of the anti-TfR1 antibodies disclosed herein may further comprise an Fc fragment, for example, an Fc fragment from human immunoglobulin (e.g., a human IgG molecule). In some examples, these 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.

[0180] B. Preparation of Anti-TfR1 Antibody

[0181] Antibodies capable of binding to TfR1 as described herein may be prepared by any method known in the art, for example, through recombinant technology. For example, refer to the literature [Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York].

[0182] If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma cell line or isolated from an antibody library) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or proliferation. The sequence encoding the antibody of interest can be maintained within the vector in a host cell, and the host cell can then be expanded and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic engineering, for example, to humanize the antibody or to improve the antibody's affinity (affinity maturation) or other characteristics. For example, if the antibody is from a non-human source and is to be used in clinical trials and treatments in humans, the constant region can be engineered to be more similar to the human constant region to avoid an immune response. Alternatively, or additionally, it may be desirable to genetically engineer the antibody sequence to obtain greater affinity and / or specificity for the target antigen and greater efficacy in enhancing TfR1 activity. It will be obvious to a person skilled in the art that one or more polynucleotide changes can be made to an antibody, and that this can still maintain its binding specificity to a target antigen.

[0183] Alternatively, antibodies capable of binding to a target antigen (TfR1 molecule) as described herein may be isolated from a suitable antibody library through commercial practice. The antibody library may be used to identify proteins that bind to the target antigen (e.g., human TfR1, e.g., cell surface TfR1) through a commercial screening process. In the selection process, polypeptide components are probed with the target antigen or a fragment thereof, and if the polypeptide component binds to the target, the antibody library member is typically identified by retention on a support. The retained display library member is recovered from the support and analyzed. The analysis may include amplification and subsequent selection under similar or dissimilar conditions. For example, positive and negative selection may be alternated. The analysis may also include determining the amino acid sequence of the polypeptide component for detailed characterization and purifying the polypeptide component.

[0184] A number of commercial methods for identifying and isolating antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosome display, or mammalian display techniques, are known in the relevant art.

[0185] Genetically modified antibodies, such as humanized antibodies, chimeric antibodies, single-strand antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant technology. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be easily isolated and sequenced using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Once isolated, the DNA can be inserted into one or more expression vectors and then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in recombinant host cells. For example, see PCT publication number WO 87 / 04462. Subsequently, DNA may be modified, for example, by substituting coding sequences for human heavy and light chain constant domains instead of homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851) or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. In this way, genetically engineered antibodies having binding specificity to target antigens, such as "chimeric" or "hybrid" antibodies, may be produced.

[0186] Methods for constructing humanized antibodies are also widely known in the relevant art. For example, refer to the literature [Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989)]. In one example, variable regions of VH and VL of a parent non-human antibody are applied to three-dimensional molecular modeling analysis according to methods known in the relevant art. Next, framework amino acid residues predicted to be important for the formation of an accurate CDR structure are identified using the same molecular modeling analysis. Simultaneously, human VH and VL chains having amino acid sequences homologous to those of the parent non-human antibody are identified from an arbitrary antibody gene database using the parent VH and VL sequences as a search query. Subsequently, human VH and VL recipient genes are selected.

[0187] A CDR region within a selected human recipient gene can be replaced with a CDR region from a parent non-human antibody or a functional variant thereof. If necessary, a corresponding residue within the human recipient gene can be replaced using a residue within a framework region of the parent chain that is predicted to be important for interacting with the CDR region (see description above).

[0188] Single-strand antibodies can be produced via recombinant technology by linking a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the technology described for the production of single-strand antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce phage-display, yeast-display, mammalian cell-display, or mRNA-display scFv libraries, and scFv clones specific to TfR1 can be identified from the library according to commercial procedures. Positive clones can be applied to further screening to confirm that they enhance CD19 activity.

[0189] Antibodies obtained according to the methods described herein and known in the art may be characterized using methods widely known in the art. For example, one method is identifying the epitope to which the antigen binds, or “epitope mapping.” For example, there are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including the interpretation of the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described in Chapter 11 of the literature [Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999]. In additional examples, epitope mapping may be used to determine the sequence to which the antibody binds. The epitope may be a linear epitope, i.e., contained within a single stretch of amino acids, or a stereochemical epitope formed by three-dimensional interactions of amino acids, which may not necessarily be contained within a single stretch (a linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids in length) may be isolated or synthesized (e.g., recombinantly) and used for antibody binding assays. In another example, the epitope to which the antibody binds may be determined by systematic screening using an overlapping peptide derived from the target antigen sequence and determining binding by the antibody. According to gene fragment expression assays, an open reading frame encoding the target antigen is fragmented randomly or by specific gene construction, and the reactivity of the antibody to be tested with the expressed fragment of the antigen is determined. The gene fragment may be produced, for example, by PCR, and then transcribed and translated into a protein in vitro in the presence of radioactive amino acids.Subsequently, the binding of antibodies to radiolabeled antigen fragments is determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using a large library of random peptide sequences (phage library) displayed on the surface of phage particles.

[0190] Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In additional examples, antigen-binding domain mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required for epitope binding and / or sufficient / or necessary. For example, domain swapping experiments can be performed using mutants of the target antigen, where various fragments of TfR1 are replaced (swapped) with sequences from closely related but antigenically distinct proteins (e.g., another member of the tumor necrosis factor receptor family). By evaluating the binding of antibodies to mutant TfR1, the importance of specific antigen fragments for antibody binding can be assessed.

[0191] Alternatively, a competitive test can be performed using another antibody known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competitive testing is widely known to those skilled in the art.

[0192] In some examples, anti-TfR1 antibodies (e.g., Fab format) are produced by recombinant technology as exemplified below.

[0193] The nucleic acids encoding the heavy and light chains of the anti-TfR1 antibody as described herein can be cloned into a single expression vector, and each nucleotide sequence is operably linked to a suitable promoter. In one example, each nucleotide sequence encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter so that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0194] In some examples, 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. In cases where the two chains are expressed in different cells, each of them can be isolated from the host cells expressing them, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to enable antibody formation.

[0195] Generally, a nucleic acid sequence encoding one or all strands of an antibody can be cloned into a suitable expression vector operably linked to a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be brought into contact with a restriction enzyme under suitable conditions to generate complementary ends on each molecule that can pair with each other and be linked together by a ligase. Alternatively, a synthetic nucleic acid linker can be ligated to the end of the gene. These synthetic linkers contain a nucleic acid sequence corresponding to a specific restriction site within the vector. The choice of expression vector / promoter will depend on the type of host cell to be used to produce the antibody.

[0196] Various promoters, including but not limited to cytomegalovirus (CMV) intermediate early promoters, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, monkey virus 40 (SV40) early promoters, E. coli lac UV5 promoters, and herpes simplex tk virus promoters, may be used for the expression of the antibodies described herein.

[0197] Regulatory promoters may also be used. These regulatory promoters include using a lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-possessing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], and using a tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16, or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0198] A modifiable promoter containing a repressor along with an operon may be used. In one embodiment, the lac repressor from E. coli functions as a transcriptional regulator to regulate transcription from a lac operator-possessing mammalian cell promoter [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)], tetracycline repressor (tetR) can be combined with a transcription activator (VP 16) to produce a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), and combined with a tetO-retaining minimal promoter derived from a human cytomegalovirus (hCMV) major ultra-early promoter to produce a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. Tetracycline repressor (tetR) alone, rather than tetR-mammalian cell transcription factor fusion derivatives, can function as a potent trans-modulator for regulating gene expression in mammalian cells when the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One specific advantage of this tetracycline inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transcription factor or repressor fusion proteins, which can be toxic to cells in some cases, to achieve its modulated effects (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0199] Additionally, the vector may contain, for example, some or all of the following: a selection marker gene, e.g., a neomycin gene for the selection of stable or transient transfectors in mammalian cells; an enhancer / promoter sequence from the ultra-early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; an SV40 polyoma replication origin and ColE1 for appropriate episome replication; an internal ribosome binding site (IRES), a multi-purpose multiple cloning site; and T7 and SP6 RNA promoters for the in vitro transcription of sense and antisense RNAs. Suitable vectors and methods for producing vectors containing transgenes are widely known and available in the relevant art.

[0200] Examples of polyadenylation signals useful for carrying out the method described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

[0201] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies may be introduced into host cells suitable for producing antibodies. The host cells may be cultured under conditions suitable for the expression of the antibody or any of its polypeptide chains. Such antibody or its polypeptide chain may be recovered from the cultured cells (e.g., from the cells or culture supernatant) by conventional methods, e.g., affinity purification. If necessary, the polypeptide chain of the antibody may be incubated under suitable conditions for a suitable period to enable the production of the antibody.

[0202] In some embodiments, the method for producing the antibody described herein also involves a recombinant expression vector encoding both the heavy and light chains of the anti-TfR1 antibody as described herein. The recombinant expression vector may be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, e.g., calcium phosphate-mediated transfection. Positive transformant host cells may be selected and cultured under suitable conditions that enable the expression of the two polypeptide chains that form the antibody, and these may be recovered from the cells or from the culture medium. If necessary, the two chains recovered from the host cells may be incubated under suitable conditions that enable the formation of the antibody.

[0203] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-TfR1 antibody and the other encoding the light chain of an anti-TfR1 antibody. Both of the two recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, e.g., calcium phosphate-mediated transfection. Alternatively, each expression vector can be introduced into suitable host cells. Positive transformants can be selected and cultured under suitable conditions that enable the expression of the antibody polypeptide chain. If the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or from the culture medium. If necessary, the polypeptide chain can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that enable antibody formation. If the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or from the corresponding culture medium. Next, two polypeptide chains can be incubated under conditions suitable for antibody formation.

[0204] Using standard molecular biology techniques, a recombinant expression vector is prepared, host cells are transfected, transformants are selected, host cells are cultured, and antibodies are recovered from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a protein A or protein G coupled matrix.

[0205] Any of the nucleic acids encoding the heavy chain, light chain, or both of an anti-TfR1 antibody as described herein, a vector containing such a vector (e.g., an expression vector); and a host cell comprising the vector are within the scope of the present disclosure. Additionally, a method for producing an anti-TfR1 antibody by expressing the anti-TfR1 antibody in a host cell and collecting the antibody produced thereby from the host cell and / or culture supernatant is within the scope of the present disclosure.

[0206] II. Anti-TfR1 Antibody Conjugate

[0207] In some aspects, any of the anti-TfR1 antibodies disclosed herein may be connected to one or more payloads to form an anti-TfR1 antibody conjugate. In some embodiments, the anti-TfR1 antibody in any of the conjugates provided herein may be in the Fab format. Exemplary anti-TfR1 antibodies (e.g., Fab antibodies) are provided in Table 5 below, any of which may 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).

[0208] Any of the anti-TfR1 antibody conjugates provided herein, for example, a TfR1 Fab-conjugated payload, such as interfering RNA, can deliver the payload (e.g., an interfering RNA molecule) into the central nervous system (CNS) through the patient's brain endothelial cells. Without being bound by theory, the conjugate may bind to a transferrin receptor (TfR1) on the surface of the brain endothelial cells. The payload, such as interfering RNA, may be attached by any permissible means for linking the anti-TfR1 antibody (e.g., Fab) to the payload (e.g., interfering RNA) so that the payload (e.g., interfering RNA) can be delivered across the cell membrane in a pharmaceutically active form. In some embodiments, a TfR1-specific antibody (e.g., Fab) may be linked to the TfR1-specific antibody using chemical conjugation techniques. In addition to covalent bonds, the conjugate may be formed using non-covalent bonds, such as those formed with a difunctional antibody, ionic bonds, hydrogen bonds, hydrophobic interactions, etc.

[0209] A payload, such as an interfering RNA molecule, can be directly covalently linked to a TfR1-specific antibody, such as Fab. Alternatively, a payload, such as interfering RNA, can be linked to an antibody spacer. In some examples, the spacer may be a glycine spacer (e.g., having 1, 2, 3, or 4 glycines). In some embodiments, the spacer may be a glycine spacer containing 2 or 3 glycines.

[0210] A. Oligonucleotide-based agonists including RNAi agonists

[0211] The payload in any of the conjugates may be any type of molecule having the desired biological activity. In some cases, the payload may be a therapeutic agent. Alternatively, the payload may be a diagnostic agent. In some embodiments, the payload may be an oligonucleotide-based agonist.

[0212] The "oligonucleotide-based agents" used herein comprise about 10 to 50 (e.g., 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 14, 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, 18 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, to 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 to 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,It is a nucleotide acid containing 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 agent has a nucleobase sequence that is at least partially complementary to the coding sequence of the target nucleic acid or target gene expressed in the cell. In some embodiments, the oligonucleotide-based agent can inhibit the expression of the underlying gene upon delivery to a cell expressing the target gene and is referred to herein as an "expression-inhibiting oligonucleotide-based agent." Gene expression can be suppressed in vitro or in vivo.

[0213] "Oligonucleotide-based agents" 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 agent is a single-stranded oligonucleotide, e.g., an antisense oligonucleotide (ASO). In some embodiments, the oligonucleotide-based agent is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide-based agent is a double-stranded oligonucleotide that is an RNAi agent.

[0214] In some embodiments, the oligonucleotide-based agonist may be an “RNAi agonist” as defined herein, which is a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading the mRNA transcript of a target messenger RNA (mRNA) or inhibiting its translation in a sequence-specific manner. The RNAi agonist as used herein may act via an RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells) or by any alternative mechanism(s) or pathway(s). Although the RNAi agonist is considered to act primarily via an RNA interference mechanism as the term is used herein, the disclosed RNAi agonist is not bound to or limited to any specific pathway or mechanism of action. The RNAi agonists disclosed herein consist of a sense strand and an antisense strand 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 agonists described herein is at least partially complementary to the mRNA to be targeted. The RNAi agonists may comprise one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0215] Typically, an RNAi agonist may consist of at least a sense strand (also referred to as a passenger strand) containing a first sequence and an antisense strand (also referred to as a guide strand) containing a second sequence. The lengths of the RNAi agonist sense and antisense strands may each be 16 to 49 nucleotides long. In some embodiments, the sense and antisense strands of the RNAi agonist are independently 17 to 26 nucleotides long. In some embodiments, the sense and antisense strands are independently 19 to 26 nucleotides long. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides long. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides long. The sense and antisense strands may be the same length or different lengths. RNAi agonists include an antisense strand sequence that is at least partially complementary to a sequence within a target gene, and when delivered to a cell expressing the target, RNAi agonists can inhibit the expression of one or more target genes in vivo or in vitro.

[0216] Not all positions within a given compound need to be uniformly modified. On the contrary, more than one modification may be incorporated into a single oligonucleotide-based agent or even its single nucleotide.

[0217] RNAi agonist sense strands and antisense strands may be synthesized and / or modified by methods known in the art. For example, additional disclosures regarding RNAi agonists can be found, and disclosures regarding modifications can be found, for example, in International Patent Application No. PCT / US2017 / 045446 (WO2018027106) of Arrowhead Pharmaceuticals, Inc., which is also incorporated herein by reference for the objects and purposes referenced herein.

[0218] B. Modification of Oligonucleotide-Based Agents

[0219] Any of the oligonucleotide-based agents, such as RNAi agents, may contain one or more modifications. Exemplary modifications may include any modifications to the sugar, nucleobase, and nucleoside linkages (e.g., linkage phosphate / phosphodiester linkages / phosphodiester backbones), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

[0220] (a) Modified nucleotide

[0221] In some embodiments, any of the oligonucleotide-based agents provided herein, such as RNAi agents, may consist of modified nucleotides and / or one or more non-phosphodiester linkages. As used herein, "modified nucleotides" are nucleotides other than ribonucleotides (2'-hydroxyl nucleotides). 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 of the oligonucleotide-based agents, such as RNAi agents, may be modified nucleotides. The modified nucleotides used herein include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3'-to-3' linked (inverted) nucleotides, non-natural base-containing nucleotides, cross-linked nucleotides, peptide nucleotides, 2',3'-seconucleotide mimics (non-locking nucleobase analogs), locking nucleotides, 3'-O-methoxy (linked between 2' nucleosides) nucleotides, 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, morpholinonucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate-containing nucleotides and cyclopropyl phosphonate-containing nucleotides. 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a 5-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoronucleotides, 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions within a given compound to be uniformly modified. On the contrary, more than one modification may be incorporated into a single target RNAi agent or even into a single nucleotide thereof.The target RNAi agonist sense strand and antisense strand may be synthesized and / or modified by methods known in the art. A modification at one nucleotide is independent of a modification at another nucleotide.

[0222] In addition, one or more nucleotides of an oligonucleotide-based agonist, e.g., an RNAi agonist, may be connected by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages may be non-phosphate-containing covalent internucleoside linkages. The modified internucleoside linkage or backbone is a 5'-phosphorothioate group, chiral phosphorothioate, thiophosphate, phosphorodithioate, phosphotriester, aminoalkyl-phosphotriester, alkyl phosphonate (e.g., methyl phosphonate or 3'-alkylene phosphonate), chiral phosphonate, phosphinate, phosphoramidate (e.g., 3'-amino phosphoramidate, aminoalkyl phosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonate, thionoalkyl phosphotriester, morpholino linkage, boranophosphate having a normal 3'-5' linkage, a 2'-5' linked analog of boranophosphate, or adjacent pairs of nucleoside units linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. It includes, but is not limited to, boranophosphates having reverse polarity.

[0223] Modified nucleobases are synthetic and natural nucleobases, e.g., 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, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) derivatives of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothimine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothimine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine, 7-deazaguanine, 7-deazadenine, 3-deazaguanine, and It contains 3-deazadenine.

[0224] In some embodiments, all or substantially all nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all nucleotides present are modified nucleotides is an RNAi agent in which four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand are non-modified ribonucleotides. As used herein, a sense strand in which substantially all nucleotides present are modified nucleotides is a sense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand are non-modified ribonucleotides. As used herein, an antisense sense strand in which substantially all nucleotides present are modified nucleotides is an antisense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand are non-modified ribonucleotides. In some embodiments, one or more nucleotides of the RNAi agent are unmodified ribonucleotides.

[0225] (b) Modified nucleoside linkage

[0226] In some embodiments, one or more nucleotides of the RNAi agent are connected by non-standard linkages or backbones (i.e., modified nucleoside linkages or modified backbones). The modified internucleoside linkage or backbone is a phosphorothioate group (represented herein by a lowercase "s"), chiral phosphorothioate, thiophosphate, phosphodithioate, phosphotriester, aminoalkyl-phosphotriester, alkyl phosphonate (e.g., methyl phosphonate or 3'-alkylene phosphonate), chiral phosphonate, phosphinate, phosphoramidate (e.g., 3'-amino phosphoramidate, aminoalkyl phosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonate, thionoalkyl phosphotriester, morpholino linkage, boranophosphate having a normal 3'-5' linkage, a 2'-5' linked analogue of boranophosphate, or adjacent pairs of nucleoside units from 3'-5' to 5'-3' or from 2'-5' It includes, but is not limited to, boranophosphates having reverse polarity connected by a 5'-2'. In some embodiments, the modified nucleoside linkage or backbone lacks a phosphorus atom. The modified nucleoside linkage lacking a phosphorus atom includes, but is not limited to, a linkage between short-chain alkyl or cycloalkyl sugars, a linkage between a mixed heteroatom and an alkyl or cycloalkyl sugar, or a linkage between one or more short-chain heteroatoms or heterocyclic sugars. In some embodiments, the modified nucleoside backbone comprises, but is not limited to, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl and thioformacetyl backbone, a methylene formacetyl and thioformacetyl backbone, an alkene-containing backbone, a sulfamate backbone, a methyleneimino and methylenehydrazino backbone, a sulfonate and sulfonamide backbone, an amide backbone, and other backbones having mixed N, O, S and CH2 components.

[0227] In some embodiments, the sense strand of the RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate links, or the antisense strand of the RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate links, or both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate links. In some embodiments, the sense strand of the RNAi agent may contain 1, 2, 3, or 4 phosphorothioate links, or the antisense strand of the RNAi agent may contain 1, 2, 3, or 4 phosphorothioate links, or both the sense strand and the antisense strand may independently contain 1, 2, 3, or 4 phosphorothioate links.

[0228] In some embodiments, the RNAi agonist sense strand contains at least two phosphorothioate nucleoside linkages. In some embodiments, at least two phosphorothioate nucleoside linkages are located between nucleotides at positions 1–3 from the 3’ end of the sense strand. In some embodiments, one phosphorothioate nucleoside linkage is located at the 5’ end of the sense strand, and another phosphorothioate linkage is located at the 3’ end of the sense strand. In some embodiments, two phosphorothioate nucleoside linkages are located at the 5’ end of the sense strand, and another phosphorothioate linkage is located at the 3’ end of the sense strand. In some embodiments, the sense strand does not contain any phosphorothioate nucleoside linkages between nucleotides, but contains one, two, or three phosphorothioate linkages between terminal nucleotides on both the 5' and 3' ends and any inverted baseless residue terminal caps present therein. In some embodiments, a targeting ligand is connected to the sense strand via phosphorothioate linkages.

[0229] In some embodiments, the RNAi agonist antisense strand contains four phosphorothioate nucleoside linkages. In some embodiments, the four phosphorothioate nucleoside linkages are located between nucleotides at positions 1–3 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 from the 5’ end. In some embodiments, three phosphorothioate nucleoside linkages are located between positions 1–4 from the 5’ end of the antisense strand, and a fourth phosphorothioate nucleoside linkage is located between positions 20–21 from the 5’ end of the antisense strand. In some embodiments, the RNAi agonist contains at least three or four phosphorothioate nucleoside linkages within the antisense strand.

[0230] In some embodiments, the RNAi agonist contains one or more modified nucleotides and one or more modified nucleoside linkages. In some embodiments, the 2'-modified nucleoside is combined with the modified nucleoside linkages.

[0231] Exemplary modifications of oligonucleotide-based agents, such as RNAi agents, are provided in Tables 8 and 9 below, each of which is within the scope of the present disclosure.

[0232] C. CNS gene targets of oligonucleotide-based agonists

[0233] The oligonucleotide-based agonists described herein, such as RNAi agonists, include protein amyloid precursor protein (APP; Uniprot: P05067), ataxin-1, ataxin-2 (ATXN2; Uniprot: Q99700), ataxin-3 (ATXN3; Uniprot: P54252), ataxin-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 repeat serine / threonine-protein kinase 2 (LRRK2; Q5S007), complement C3 (C3; Uniprot: P01024), and microtubule association Protein tau (MAPT; Uniprot: P10636), alpha-2A adrenergic receptor (ADRA2A; Uniprot: P08913), sodium channel protein type 9 subunit alpha (SCN9A; Uniprot: Q15858), apolipoprotein E (APOE; Uniprot: P02649), alpha-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 It may have a complementary antisense sequence targeted for the mRNA encoding DYRK1A.Knockdown of the expression of these proteins is expected to be effective in treating or preventing selected CNS disorders 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, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy's disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy.

[0234] In some embodiments, oligonucleotide-based agonists targeting androgen receptor (AR) mRNA are selected from those presented in Tables 8 and 9.

[0235] For example, the ability of interfering RNA to knock down the level of endogenous target gene expression in HeLa cells can be evaluated in vitro as follows. HeLa cells are plated in standard growth medium (e.g., DMEM supplemented with 10% fetal bovine serum) 24 hours prior to transfection. Transfection is performed using, for example, Dharmafect 1 (Dharmacon, Lafayette, Colorado) at interfering RNA concentrations ranging from 0.1 nM to 100 nM according to the manufacturer's instructions. SiCONTROL™ non-targeting siRNA #1 and SiCONTROL™ cyclophyllin B siRNA (Dharmacon) are used as negative and positive controls, respectively. Target mRNA levels and cyclophyllin B mRNA (PPIB, NM_000942) levels are evaluated by qPCR 24 hours after transfection using, for example, a TAQMAN® gene expression assay (Applied Biosystems, Foster City, California) that preferably overlaps the target site. Positive control siRNA provides essentially complete knockdown of cyclophyllin B mRNA when the transfection efficiency is 100%. Therefore, target mRNA knockdown is corrected for transfection efficiency by reference to cyclophyllin B mRNA levels in cells transfected with cyclophyllin B siRNA. Target protein levels can be evaluated, for example, by Western blot at approximately 72 hours after transfection (actual time depending on protein conversion rate). Standard techniques for RNA and / or protein isolation from cultured cells are widely known to those skilled in the art. To reduce the chance of non-specific off-target effects, the lowest possible concentration of interfering RNA that produces the desired level of knockdown in target gene expression is used. Cultured human endothelial cells can be used to evaluate the ability of interfering RNA to knock down the levels of endogenous target genes.

[0236] D. Preparation of Anti-TfR1 Antibody Conjugates

[0237] The anti-TfR1 antibody conjugate provided herein may be prepared by commercial technology (e.g., chemical conjugation technology) or by the method disclosed herein (e.g., see Example 6 below). In some cases, oligonucleotide-based agents, such as RNAi agents, may be conjugated to a chemical linker, e.g., L20-p presented in Example 6, via a standard amide reaction. Modified oligonucleotide-based agents may be conjugated to an anti-TfR1 antibody (e.g., Fab format) via a commercial chemical reaction.

[0238] In some embodiments, the anti-TfR1 antibody may be modified (capped) with a chemical, e.g., CP-113 or N-ethyl maleimide, before or after conjugation with an oligonucleotide-based agent (see Example 6 below). In some cases, the antibody-oligonucleotide conjugate provided herein may include a chemical linker and / or cap.

[0239] III. Pharmaceutical Composition

[0240] Any of an anti-TfR1 antibody or an anti-TfR1 antibody conjugate may be formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers. Such pharmaceutical compositions may be used for therapeutic or diagnostic purposes, for example, for use in the therapeutic methods disclosed herein, depending on the payload conjugated to the antibody.

[0241] The "pharmaceutical composition" as used herein comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product) that is intentionally included in the drug delivery system. The excipient does not exert or is not intended to exert a therapeutic effect at the intended dosage. The excipient may function to a) assist in the processing of the drug delivery system during manufacturing and / or b) protect, support, or enhance the stability, bioavailability, or patient tolerance of the API and / or c) assist in product identification and / or d) enhance any other attribute of the overall safety and efficacy of the delivery of the API during storage or use. The pharmaceutically acceptable excipient may or may not be an inert substance.

[0242] Excipients include, but are not limited to, absorption promoters, anti-adhesion agents, defoaming agents, antioxidants, binders, buffers, carriers, coating agents, coloring agents, delivery promoters, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, lubricants, humectants, lubricants, oils, polymers, preservatives, brine, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, enteric agents, vehicles, water repellents, and wetting agents.

[0243] The pharmaceutical compositions described herein may contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional components are pharmaceutical-active substances. Pharmaceutical-active substances include, but are not limited to, antipruritics, astringents, local anesthetics or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0244] The pharmaceutical composition may also contain preservatives, solubilizers, stabilizers, humectants, emulsifiers, sweeteners, coloring agents, odorants, salts for osmotic pressure fluctuations, buffers, coating agents, or antioxidants. It may also contain other agents having known therapeutic benefits.

[0245] The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is preferred and the area to be treated. Administration may be made by any method orally known in the relevant art, such as, but not limited to, local (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including by a nebulizer, trachea, or nasal cavity), epidermis, transdermis, oral, or parenteral. Parenteral administration includes, but is not limited to, intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (e.g., via an implanted device), intracranial, parenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection. The pharmaceutical composition may be administered orally in the form of, for example, tablets, coated tablets, sugar-coated tablets, hard or soft gelatin capsules, solutions, emulsions, or suspensions. Administration may also be by rectal, for example, using suppositories; For example, it can be performed topically or transdermally using an ointment, cream, gel, or solution; or parenterally using an injectable solution, for example.

[0246] Pharmaceutical compositions suitable for injectable use comprise sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers comprise physiological saline, bacterial water, Cremophor® EL (BASF, Parsippany, NJ), or phosphate-buffered saline. These must be stable under manufacturing and storage conditions and preserved against the action of microorganisms, such as bacteria and fungi. The carrier may 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. Suitable fluidity may be maintained, for example, by coating, such as the use of lecithin; by maintaining the particle size required in the case of dispersions; and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0247] A sterile injectable solution may be prepared by incorporating the required amount of the active compound with one or a combination thereof of the components listed above into a suitable solvent, and then filtering and sterilizing. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For a sterile powder for the preparation of a sterile injectable solution, the preparation method comprises vacuum drying and freeze-drying to produce a powder of the active component plus any additional desired component from its previously sterile-filtered solution.

[0248] A formulation suitable for intra-articular administration may be in the form of a sterile aqueous formulation of any of the ligands described herein, which may be in a microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to present any of the ligands described herein for both intra-articular and ophthalmic administration.

[0249] The active compound may be prepared with a carrier that protects the compound against rapid removal from the body, such as a implant and a microencapsulated delivery system, as well as a controlled-release formulation. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Methods for preparing such formulations will be obvious to a person skilled in the art. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to a person skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0250] The pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. These additional components include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” refers to the amount of pharmaceutical active agent that produces a pharmacological, therapeutic, or prophylactic effect.

[0251] A medicine containing an anti-TfR1 antibody or antibody fragment conjugated or unconjugated to an oligonucleotide-based compound is also an object of the present invention, and a method for manufacturing such a medicine is likewise an amount comprising making one or more of the anti-TfR1 antibodies or antibody fragments disclosed herein, and, if desired, one or more other substances having known therapeutic benefits into a pharmaceutically acceptable form.

[0252] The described anti-TfR1 antibody and antibody fragment, and pharmaceutical compositions comprising the anti-TfR1 antibody and antibody fragment disclosed herein may be packaged in or contained in a kit, container, pack, or dispenser. The described anti-TfR1 antibody and antibody fragment, and pharmaceutical compositions comprising the described anti-TfR1 antibody and antibody fragment may be packaged in a pre-filled syringe or vial.

[0253] The pharmaceutical compositions provided herein may comprise more than one TfR1 antibody (e.g., Fab) conjugated to the same oligonucleotide-based agonist (e.g., interfering RNA), different antibodies reactive to TfR1, such as a cocktail of Fabs, or more than one oligonucleotide-based agonist, such as interfering RNA, targeting different locations on a single mRNA.

[0254] The pharmaceutical composition is a formulation comprising up to 99 weight percent of interfering RNA or its antibody conjugate mixed with a physiologically acceptable carrier medium, such as water, a buffer, a saline solution, glycine, hyaluronic acid, mannitol, etc. The pH of the formulation is about pH 4.0 to about pH 9.0, or about pH 4.5 to about pH 7.4. A method for manufacturing nanoparticles and its use in the delivery of pharmaceutical agents is described in U.S. Patent No. 6,632,671, the full text of which is incorporated by reference. A method for manufacturing nanoparticle-Fab conjugates and their use in the delivery of pharmaceutical agents is described in U.S. Patent No. 6,372,250, the full text of which is incorporated by reference.

[0255] IV. Treatment Methods

[0256] In some aspects, the present disclosure provides a method for attenuating the expression of a target mRNA in a patient's CNS, comprising: (a) providing an anti-TfR1 antibody (e.g., Fab)-conjugated oligonucleotide-based agonist, e.g., interfering RNA; and (b) administering the anti-TfR1 antibody conjugate to a patient's CNS, wherein the oligonucleotide-based agonist, e.g., interfering RNA molecule, attenuates the expression of the target mRNA in the CNS.

[0257] In certain embodiments, the present disclosure provides a method for preventing or treating CNS disorders in a patient, said method comprising administering an anti-TfR1 antibody conjugate (e.g., comprising an anti-TfR1 Fab conjugated to interfering RNA) to the patient. The antibody moiety binds to TfR1 and transports an oligonucleotide-based agonist, such as interfering RNA, into the CNS through the patient's brain endothelial cells.

[0258] Exemplary CNS disorders 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 tauopathy. CNS brain regions for systemic targeting may include the cerebellum, cortex, striatum, tuberous sclerosis complex (TCS), or thoracic spinal cord.

[0259] In certain embodiments, the patient has a CNS disorder associated with endothelial cells, astrocytes, perivascular cells, microglia, neurons or oligodendrons, or another type of cell in the CNS.

[0260] The method of the present disclosure is useful for attenuating the expression of specific genes in a patient's CNS using RNA interference. In certain embodiments, a TfR1-specific Fab-interfering RNA conjugate comprises an interfering RNA molecule that targets genes associated with CNS disorders. Examples of mRNA target genes designed to be targeted by the interfering RNA of the present invention include genes associated with CNS disorders.

[0261] In certain embodiments, a method for treating CNS disease involves targeting specific cell types within the CNS. In some embodiments, TfR1 Fab-interfering RNA conjugates or TfR1 Fab-conjugated nanoparticles are delivered to the CNS via brain endothelial cells.

[0262] Animal models are used to test the activity of systemically delivered interfering RNA molecules. The embodiments of this application demonstrate successful and effective in vivo delivery of interfering RNA molecules to the CNS. For example, targeted RNA interference can be tested in mice as described in the literature [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. Epub 2011 Mar 20. PMID: 21423189].

[0263] In another embodiment, a method for delivering an interfering RNA molecule comprises administering an antibody conjugated to a nanoparticle to a patient, wherein the interfering RNA molecule is encapsulated within the nanoparticle and the nanoparticle is linked to a TfR1 antibody, which transports an antisense oligonucleotide molecule into the patient's CNS via brain endothelial cell transport. Another embodiment of the present invention provides a method for preventing or treating a CNS disorder, said method comprising delivering an antisense oligonucleotide molecule to the patient's CNS via brain endothelial cells using Fab-conjugated nanoparticles.

[0264] TfR1 antibody (e.g., Fab)-conjugated oligonucleotide-based agonists (e.g., interfering RNA) and nanoparticles containing the same may be administered by intravenous injection, oral administration, intramuscular injection, nasal, intraperitoneal, intrathecal injection, inhalation, transdermal application, sublingual, or transmucosal application. The form and concentration (e.g., capsule, tablet, solution, emulsion) in which the TfR1 antibody (e.g., Fab)-conjugated oligonucleotide-based agonists (e.g., interfering RNA) and nanoparticles containing the same are administered will depend at least partially on the route of administration.

[0265] In certain embodiments, treatment of CNS disorders using interfering RNA molecules is achieved by directly administering nanoparticles containing such compositions, comprising a TfR1 antibody (e.g., Fab)-conjugated oligonucleotide-based agonist (e.g., interfering RNA) and the interfering RNA, to the CNS. Therapeutic treatment of patients using interfering RNA directed toward target mRNA is expected to be beneficial compared to small molecule therapy by increasing the duration of action, enabling less frequent administration and greater patient compliance, and by increasing target specificity, thereby reducing side effects.

[0266] The therapeutically effective dose of the formulation may depend on factors such as, for example, the patient's age, race and sex, severity of the disorder, target gene transcript / protein conversion rate, antibody (e.g., Fab) affinity, the number of oligonucleotide-based agonists (e.g., interfering RNA molecules) per antibody (e.g., Fab), the potency of the oligonucleotide-based agonist (e.g., interfering RNA), and the stability of the oligonucleotide-based agonist (e.g., interfering RNA). In one embodiment, the interfering RNA is delivered at a therapeutic dose to improve the target gene-associated disease process.

[0267] In certain embodiments, the present invention provides a specific pharmaceutical composition for preventing or treating CNS disorders in a patient, comprising a therapeutically effective amount of the interfering RNA-TfR1 Fab conjugate or nanoparticle-TfR1 Fab conjugate of the present invention within a carrier acceptable to the CNS. The therapeutically effective amount of interfering RNA used in the composition of the present invention produces extracellular concentrations of about 100 pM to 1 μM, or about 1 nM to 100 nM, or about 5 nM to about 50 nM, or up to about 25 nM at the surface of target cells. The dose required to achieve these local concentrations will depend on a number of factors, including, among other factors, the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, and whether the delivery is intracranial or local delivery through the systemic. For example, the composition may be delivered to the CNS by intravenous injection every other week, every month, or more, at the judgment of a skilled clinician. The interfering RNA-TfR1 Fab conjugate and the nanoparticle-TfR1 Fab conjugate of the present invention are administered as a solution, suspension, or emulsion. The TfR1 Fab-conjugated interfering RNA can be delivered as a solution, suspension, or via a bio-erosive or non-biodegradable delivery device.

[0268] V. Kit

[0269] In certain embodiments, the present disclosure also provides a kit comprising reagents for attenuating the expression of mRNA as described herein in cells. The kit contains oligonucleotide-based agonist-antibody conjugates (e.g., interfering RNA-Fab conjugates) and / or components necessary for the production of interfering RNA-Fab conjugates (e.g., interfering RNA molecules as well as materials necessary for Fab and ligation). The kit may also contain positive and negative control siRNA or shRNA expression vectors (e.g., non-targeting control siRNA or siRNA targeting unrelated mRNA). 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 target mRNA and / or antibodies against the corresponding protein for Western blot). Alternatively, the kit may include siRNA sequences or shRNA sequences, and instructions and materials necessary for generating siRNA by in vitro transcription or constructing shRNA expression vectors.

[0270] A pharmaceutical combination in the form of a kit is further provided, comprising a first container means comprising a carrier means configured to accommodate a container means in a tightly sealed manner within the packaged combination and a TfR1 antibody-conjugated interfering RNA composition. If desired, such a kit may further comprise, as will be readily apparent to a person skilled in the art, one or more of various conventional pharmaceutical kit components, such as a container containing one or more pharmaceutically acceptable carriers, additional containers, etc.

[0271] Printed instructions as an insert or label indicating the amount of the ingredient to be administered, guidelines for administration, and / or guidelines for mixing the ingredients may also be included in the kit.

[0272] References cited herein are included by reference specifically to the extent that they provide exemplary procedures or other details that supplement what is presented herein.

[0273] In light of this disclosure, a person skilled in the art will recognize that obvious variations of the embodiments disclosed herein may be made without departing from the spirit and scope of the invention. All embodiments disclosed herein may be made and practiced without excessive experimentation in light of this disclosure. The full scope of the invention is set forth in this disclosure and its equivalent embodiments. This specification should not be construed as unduly narrowing the full scope of protection granted by the invention.

[0274] General skills

[0275] Practice of the present disclosure will, unless otherwise indicated, utilize the ordinary techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the art of the relevant field. Such techniques are derived from the literature, e.g., [Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press; Animal Cell Culture (RI Freshney, 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, eds. 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel, et al., eds.1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., 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 J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (lRL Press, (1986); and B.This is sufficiently explained in Perbal, A Practical Guide to Molecular Cloning (1984); FM Ausubel et al., (eds.)].

[0276] Without further explanation, it is believed that a person skilled in the art will be able to make the full use of the present invention based on the foregoing description. Accordingly, the specific embodiments below are merely illustrative and should be interpreted as not limiting the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subjects mentioned herein.

[0277] Examples

[0278] Example 1. Humanization of TfR1 Fab

[0279] The human consensus sequences of heavy chain subgroup III (humIII) and light chain κ subgroup I (humκI) (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 ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) were used for the humanization of anti-hTfR1 antibodies. To generate the template pAY2 for future CDR swapping, the humIII and humκI genes were inserted into a phagemid designed to display human Fab on the surface of an M13 bacteriophage. Two open reading frames were used to individually code two peptide chains under the control of the phoA promoter. The first open reading frame coded the light chain, and the second open reading frame coded the heavy chain fused to the C-terminal domain of the M13 minor coat protein P3. The secretion of both peptide chains was directed by the N-terminal stII signal sequence.

[0280] pAY2 single-stranded DNA (ssDNA) template

[0281] Subsequently, chemically qualified CJ236 cells (uracil deglycosidase deficient) were transfected with pAY2 using a micropulsar electroporator (Bio-Rad). A single colony was inoculated into 1 ml of 2YT starting culture containing 100 μg / ml ampicillin and 10 μg / ml chloramphenicol, and the resulting culture was shaken at 37°C for 6 hours. M13KO7 helper phage (~10 10pfu) was added and shaken at 37°C for 10 minutes, after which 300 ml of the mixture was transferred to a 30 ml 2YT containing 100 μg / ml ampicillin and 0.25 μg / ml uridine. After growing at 37°C for 18 hours, the phages were purified, and uracil-containing ssDNA was isolated using the EZNA® M13 DNA Mini Kit (Omega Biotek Inc.).

[0282] Example 2. Incorporation of six CDRs into humIII in the case of VH and humκI in the case of VL to generate humanized hmab1a

[0283] A CDR-swapped version of the humanized anti-hTfR1 antibody hmab1a was constructed using Kunkel mutagenesis (Kunkel TA Proc Natl Acad Sci USA 82, 488-92 (1985); Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)). Six primers were designed to swap six CDRs of the mab0039 antibody with pAY2. The primers were individually phosphorylated using T4 polynucleotide kinase (NEB) at 37°C for 1 hour, then annealed on a uracil-containing ssDNA template at 90°C for 1 minute and at 50°C for 3 minutes, and placed on ice. 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. The DNA was desalted, purified by affinity using the Qiagen QiaQuick DNA Purification Kit, and transformed into XL-1 Blue cells (a uracil glycosidase-containing strain) by heat shock transformation. A small amount of DNA was purified using the Qiagen MiniPrep Kit and sent for sequencing to confirm the sequence. Subsequently, uracil-containing ssDNA for humanized library construction was prepared using plasmid hmab1a.

[0284] Humanized Library (Lib AY1 construction)

[0285] Based on published studies (Baca M, et al., J. Biol. Chem. 272, 10678-10684 (1997)) and other therapeutic antibody sequences, the library was designed to include mouse and frequent 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 axial neutral codons used for each site are presented 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, and D = A, G, or T. Two primers in VL and three primers in VH were designed to introduce these axially neutral codons into the target site via Kunkel mutagenesis. Phosphorylation of the primers and Kunkel mutagenesis were performed as described (Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)). The covalently closed circular DNA obtained as described in [Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)] was electroporated into electrically qualified SS320 cells to prepare an hmab1 humanized Fab library. The apparent variety measured by titer was 6.9 x 108, which was greater than the designed theoretical variety of 4.7 x 106.

[0286] Selection of Humanized hmab1 Clone

[0287] Selection was performed in a manner 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 onto magnetic beads (Promega) and incubated for 15 minutes with 10¹²–13 cfu of phages in 1 ml of PT (1 x PBS containing 0.05% Tween 20) supplemented with 0.5% BSA and 0.2 mg / ml streptavidin. Subsequently, the solution was removed, the beads were washed twice with PT, and amplified for selection in subsequent rounds. In subsequent rounds, the purified phage pool was first incubated with streptavidin beads for 15 minutes, and the supernatant was used for subsequent selection on a Kingfisher magnetic particle processor (Thermo Fisher). Phages (10¹⁰⁴⁴ cfu) were incubated for 15 minutes with decreasing concentrations of hTfR1-B (20 - 0.1 nM). Subsequently, streptavidin magnetic beads were added to the solution for 15 minutes to enable the 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 selection round, the recovered phages were amplified as described (Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)).

[0288] Phage ELISA screening using low antigen concentration (PESLA)

[0289] After selection in 4 rounds, individual clones were analyzed by phage ELISA (Ye JD, et al., Proc Natl Acad Sci USA 105, 82-87 (2008)). The procedure was modified to screen high-affinity binders in a high-throughput manner. More than 96 individual colonies were collected 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 grown overnight at 37°C while shaking at 300 rpm. Subsequently, the cells were pelleted by centrifuging the deep-well plates at 3500 rpm and 4°C for 15 minutes. The supernatant was diluted three-fold with phage dilution buffer (PBS, 0.5% BSA, and 0.05% Tween 20) to prepare the phage solution. 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). After removing the coating solution, the MaxiSorp plates were blocked with 200 μL / well of 1% (w / v) BSA in PBS for 1 hour. After removing the blocking solution, the MaxiSorp plates were washed four times with PBS (PT) containing 0.05% (v / v) Tween 20 and incubated with 100 μL / well of phage solution at room temperature for 1 hour. After washing 6 times with PT buffer, Maxisorb plates were incubated with 100 μL / well anti-M13 / horseradish peroxidase conjugate (diluted 3000x in phage dilution buffer) at room temperature for 30 minutes. After washing 6 times with PT buffer, Maxisorb plates were incubated with 100 μL / well Ultra TMB-ELISA substrate for 5–10 minutes, quenched with 100 μL / well 1 M phosphate, and read at 450 nm using a spectrophotometer on a microplate reader.Since low antigen concentrations act as limiting factors, higher signals correlate with tighter binding clones. These tighter binders were miniprepped and sequenced to identify unique clones.

[0290] Characterization of unique clones using competitive phage ELISA

[0291] Twenty-four unique clones were identified from library AY1. The binding affinities of these clones were characterized using a competitive phage ELISA. Maxisorb ELISA plates were coated with hTfR1 overnight at 4°C and blocked with BSA for 1 hour at room temperature. Serial dilutions of hTfR1 were incubated with phages at sub-saturated concentrations for 1 hour at room temperature, 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 color development with TMB for 5–10 minutes and quenching with 1 M phosphate. The binding signals were analyzed using a plate reader. Mab1G exhibited the best binding affinity (0.78 nM) for hTfR1.

[0292] Example 3. General method for affinity maturation of mab1G

[0293] Construction of the mab1G affinity-mature library (Lib AY2)

[0294] A uracil-containing single-stranded DNA template of mab1G was generated using a procedure similar to that described in the literature [Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)]. In this library, all six CDRs were screened simultaneously. A single amino acid CDR working strategy (Yang WP, et al., J. Mol. Biol. 254, 392-403 (1995)) was adopted. Randomized locations 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 using axially neutral codon NNS to code for all 20 amino acids in a given CDR. The randomizations were incorporated into the mab1G ssDNA template via Kunkel mutagenesis (Kunkel TA Proc Natl Acad Sci USA 82, 488-92 (1985); Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)). Primer phosphorylation and Kunkel mutagenesis were performed as described (Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)). Lib AY2 was prepared by electroporating the covalent closed circular DNA obtained as described in the literature [Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)] into electrically qualified SS320 cells. The titer-measured apparent variety was 1.1 x 10¹⁰.

[0295] Selection of Affinity-Matured Antibodies

[0296] Selection was performed in a manner 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 each round, excluding the first round, the purified phage pool was first incubated with streptavidin beads for 15 minutes, and the supernatant was used for subsequent selection on a Kingfisher magnetic particle processor (Thermo Fisher). Phages (10 10-11 cfu) was incubated with hTfR1-B (20, 0.3, 0.05, and 0.05 nM, respectively, during rounds 1–4) for 15 minutes to 1 hour. Subsequently, streptavidin magnetic beads were added to the solution for 1.5–15 minutes to enable the capture of hTfR1-B along with the bound phage. In the third round, a separate selection was performed in addition to the regular selection. After the capture of the antigen / antibody complex on the beads, the beads were washed with PBS (PT) supplemented with 0.05% Tween 20, and >1000 times competitive non-biotinylated hTfR1 was incubated with the beads at room temperature for 2.5 hours. The beads were then washed five times with PT and resuspended in 100 μl of PT. After each selection round, the recovered phages were amplified as described (Sidhu S. S et al., Methods Enzymol 328, 333-63 (2000)).

[0297] Example 4. Combination of beneficial mutations from different CDRs

[0298] PESLA was applied to the Round 3 products (no hTfR1 competition) and Round 4 products (with hTfR1 competition in Round 3). Unique mutations were identified by sequencing. These mutations were combined using two different approaches. In Method 1, individual clones with the combined mutations were produced via Kunkel mutagenesis. In Method 2, a mini-library was generated by combining all identified mutations and selected for hTfR1-B. Together, these methods generated 15 Fab clones (Fab0060-Fab0074).

[0299] Table 1-2 shows CDR1 and CDR2 variants from VL and VH with combined beneficial mutations of Fab 0060-0074. CDR3 is the same sequence in 15 Fab clones VL (QHFWGTPLT; sequence identification number: 13) and VH (GTRAYHY; sequence identification number: 24).

[0300] Table 1. Light chain complementarity determination region (CDR)

[0301]

[0302] Table 2. Heavy Chain Complementarity Determination Region (CDR)

[0303]

[0304] Table 3 shows exemplary light chain framework regions used in the exemplary anti-TfR1 antibody provided herein. An exemplary CL sequence for use in the Fab light chain is also provided in Table 3.

[0305] Table 3. Exemplary light chain framework areas and invariant areas

[0306]

[0307] Table 4 shows an exemplary heavy chain framework region used in the exemplary anti-TfR1 antibody provided herein. An exemplary CH1 sequence for use in the Fab heavy chain is also provided in Table 4.

[0308] Table 4. Exemplary reprint framework region and invariant region fragment

[0309]

[0310] Table 5 below lists the VH and VL sequences of the exemplary anti-TfR1 antibody provided herein, and its heavy and light chain sequences in the Fab format.

[0311] Table 5. Sequence of an exemplary anti-TfR1 Fab antibody

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320] Example 5. Affinity binding parameters of TfR1 antibody by competitive binding assay

[0321] cTfR BLI dynamics

[0322] Samples were analyzed via biolayer interferometrics (BLI) to determine the kinetics of synomolgus monkey transferrin receptor 1 (cTfR1). Briefly, Octet RH16 (Sartorius) was used for BLI measurements employing a streptavidin biosensor (Sartorius p / n 18-5019) to irreversibly immobilize biotinylated cTfR1 (AcroBio p / n TFR-C8249). Samples were serially diluted from 150 to 5 nM according to expected affinity, and five concentrations within this range were used for analysis. The sample diluent was prepared in-house and consists of 10 mM PBS pH 7.4 (150 mM NaCl), 0.01% BSA, 0.002% Tween 20, and 0.01% Carton. A 96-well plate was used for the analysis and filled with 10 μg / mL cTfR1 in one row for the required sample solution and the loading of the streptavidin sensor. For each sample, one well was reserved for the reference sensor control (evaluation of non-specific binding without cTfR1 on the surface at the highest sample concentration) and the sample reference (buffer alone on the cTfR1-loaded sample). One well was also reserved for a second replicate of one of five concentrations to evaluate sensor and method variability. Eight channels were used for the maximum sensitivity provided by Octet RH16. For each test performed, the steps were as follows: a sensor check for baseline evaluation of the sensor alone in the buffer was performed for 30 seconds, followed by loading cTfR1 for 60 seconds, and then a baseline check for 90 seconds to verify the stability of cTfR1 immobilized on the sensor surface, followed by sample aggregation for 180 seconds and finally sample dissociation for 300 seconds. A shaking rate of 1000 was used in all steps. The sample results were analyzed using octet analysis software.

[0323] hTfR BLI dynamics

[0324] Samples were analyzed via biolayer interferometrics (BLI) to determine the kinetics of human transferrin receptor 1 (hTfR1). Briefly, Octet RH16 (Sartorius) was used for BLI measurements employing a streptavidin biosensor (Sartorius p / n 18-5019) to irreversibly immobilize biotinylated hTfR1 (AcroBio p / n TFR-H82E5). Samples were serially diluted from 150 to 5 nM according to expected affinity, and five concentrations within this range were used for analysis. The sample diluent was prepared in-house and consists of 10 mM PBS pH 7.4 (150 mM NaCl), 0.01% BSA, 0.002% Tween 20, and 0.01% Carton. A 96-well plate was used for the analysis and filled with 10 μg / mL hTfR1 in one row for the required sample solution and the loading of the streptavidin sensor. For each sample, one well was reserved for the reference sensor control (evaluation of non-specific binding without hTfR1 on the surface at the highest sample concentration) and the sample reference (buffer alone on the hTfR1-loaded sample). One well was also reserved for a second replicate of one of five concentrations to evaluate sensor and method variability. Eight channels were used for the maximum sensitivity provided by Octet RH16. For each test performed, the steps were as follows: a sensor check for baseline evaluation of the sensor alone in the buffer was performed for 30 seconds, followed by loading cTfR1 for 60 seconds, and then a baseline check for 90 seconds to verify the stability of cTfR1 immobilized on the sensor surface, followed by sample aggregation for 180 seconds and finally sample dissociation for 300 seconds. A shaking rate of 1000 was used in all steps. The sample results were analyzed using octet analysis software.

[0325] cTfR1 SPR dynamics

[0326] Samples were analyzed via surface plasmon resonance (SPR) to determine the kinetics for synomolgus monkey transferrin receptor 1 (cTfR1). Briefly, a Viacor X100 (Citiva) was used for SPR measurements using a CM5 chip (carboxymethylated dextran surface, Citiva p / n BR100399) with anti-histidine antibodies irreversibly immobilized on the surfaces of both flow cells for the reversible capture of his-tagged cTfR1 (Acro Biosystems p / n TFR-C524a). The anti-histidine antibodies were irreversibly immobilized via amine coupling using the Citiva-provided amine coupling kit (p / n BR100050) and His capture kit (p / n 28995056). Samples were serially diluted to 150–5 nM according to expected affinity, and five concentrations within this range were used for analysis. The sample diluent was prepared in-house and consisted of 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20. Citivar caps and vials compatible with Viacore X100 were used for sample analysis. Each analysis cycle consisted of a capture step, followed by sample or blank injection, and finally a regeneration step to remove any sample and cTfR1 from the sensor surface. The capture step was performed at 5 μL / min with a contact time of 24 seconds only on an active flow cell, followed by a Fluidix wash with 0.1% Tween 20. Generally, 2 μg / mL hTfR1 was a sufficient concentration to observe adequately captured hTfR1 consistently between cycles. Sample or buffer was injected at 30 μL / min with a contact time of 180 seconds and a dissociation time of 180 seconds, followed by Fluidix washing with 0.1% Tween 20. A 10 mM glycine solution at pH 1.5 (provided in the His capture kit) was used for regeneration at 30 μL / min with a contact time of 60 seconds.One cycle was performed for each sample concentration analyzed, along with one cycle repeating a single concentration and 2–3 buffer cycles to evaluate variability. The results were analyzed using ViaCore evaluation software. The buffer cycles were averaged and subtracted from the sample injection prior to kinetic fitting. A monovalent or kinetic fit was identified as appropriate for the analyzed samples. The accuracy of the fit was evaluated, and data points were excluded where applicable.

[0327] hTfR1 SPR dynamics

[0328] Samples were analyzed via surface plasmon resonance (SPR) to determine the kinetics for human transferrin receptor 1 (hTfR1). Briefly, a Viacore X100 (Citiva) was used for SPR measurements using a CM5 chip (carboxymethylated dextran surface, Citiva p / n BR100399) with anti-histidine antibodies irreversibly immobilized on the surfaces of both flow cells for the reversible capture of his-tagged hTfR1 (Acro Biosystems p / n CD1-H5243). The anti-histidine antibodies were irreversibly immobilized via amine coupling using the Citiva-provided amine coupling kit (p / n BR100050) and His capture kit (p / n 28995056). Samples were serially diluted to 150–5 nM according to expected affinity, and five concentrations within this range were used for analysis. The sample diluent was prepared in-house and consists of 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20. Citivar caps and vials compatible with Viacore X100 were used for sample analysis. Each analysis cycle consists of a capture step, followed by sample or blank injection, and finally a regeneration step to remove any sample and hTfR1 from the sensor surface. The capture step was performed at 5 μL / min with a contact time of 24 seconds only on an active flow cell, followed by a Fluidix wash with 0.1% Tween 20. Generally, 2 μg / mL hTfR1 is a sufficient concentration to observe adequate hTfR1 consistently captured between cycles. Sample or buffer was injected at 30 μL / min with a contact time of 180 seconds and a dissociation time of 180 seconds, followed by Fluidix washing with 0.1% Tween 20. A 10 mM glycine solution at pH 1.5 (provided in the His capture kit) was used for regeneration at 30 μL / min with a contact time of 60 seconds.One cycle was performed for each sample concentration analyzed, along with one cycle repeating a single concentration and 2–3 buffer cycles for variability evaluation. The results were analyzed in ViaCore evaluation software. The buffer cycles were averaged and subtracted from the sample injection prior to kinetic fitting. A monovalent or kinetic fit was specified as appropriate for the analyzed samples. The accuracy of the fit was evaluated, and data points were excluded where applicable. Figure 4 shows the binding affinity of the selected TfR1 Fab normalized to Fab0002 as KdD. Fab0002 has the structure presented below:

[0329]

[0330] Tables 6 and 7 show the binding parameters as measured by competitive ELISA, SPR, and BLI with siRNA conjugates.

[0331] Table 6. Human transferrin receptor 1 (hTfR1) binding parameters as measured by competitive ELISA, SPR, and BLI

[0332]

[0333] Table 7. Synomolgus monkey transferrin receptor 1 (cTfR1) binding parameters as measured by competitive ELISA, SPR, and BLI

[0334]

[0335] Example 6. Synthesis of RNAi Agent and TfR1-Specific Conjugate

[0336] The RNAi agonist duplex disclosed herein was synthesized according to the following:

[0337] A. Synthetic.

[0338] Sense and antisense strands of RNAi agents were synthesized using phosphoramidite technology on a solid phase used for oligonucleotide synthesis. Depending on the scale, MerMade 96E® (Bioautomation), MerMade 12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was performed on a solid support prepared of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, Pennsylvania, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, Wisconsin, USA). Specifically, the 2'-O-methyl phosphoramidite used included the following: (5'-O-dimethoxytrityl-N 6 -(Benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, (5'-O-dimethoxytrityl-N 2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-deoxy-2'-fluoro-phosphoramidite possessed the same protecting group as 2'-O-methylRNAamidite. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia). Inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes (Wilmington, Massachusetts, USA). The following UNA phosphoramidites were used: 5'-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-Benzoyl-3'-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-Benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-Benzoyl-3'-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite. TFA aminolinked phosphoramidite was also commercially purchased (Thermo Fisher). Cyclopropyl phosphonate phosphoramidite is International Patent Application Publication No. WO Synthesized according to 2017 / 214112 (also see reference [Altenhofer et al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)]).

[0339] B. Cutting and deprotection of the oligomer bonded to the support.

[0340] After the completion of solid-phase synthesis, the dried solid support was treated with a 40 wt% methylamine and 28% to 31% ammonium hydroxide solution (Aldrich) in a 1:1 volume solution of water for 1.5 hours at 30°C. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0341] Conjugation of C. RNAi agents to Fab and capping by CP-1113.

[0342] L20-p using standard amide reaction chemistry after cleavage from the solid phase of the RNAi agent described herein containing free amine

[0343] It was conjugated to. A freshly prepared solution of (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 mixture was kept overnight at room temperature and shielded from light. The next day, the reaction mixture was loaded onto a PD-10 desalting column equilibrated with PBS and eluted with PBS to remove TCEP. The concentration of Fab in the eluent was determined using the theoretical absorption factor at 280 nm. A solution of the L20-modified sense strand in sodium phosphate buffer was prepared, and its concentration was determined using the theoretical absorption factor at 260 nm. The L20-modified sense strand (1-1.3 equivalents, 0.5-2.5 mM) was added to the desalted Fab solution, and the reaction mixture was mixed end-over-end. Analysis by SEC Method 1 and AIEX Method 1 shows a mixture of starting Fab, DAR1, and DAR2. After 1 hour, CP-1113-p in DMSO: A solution of was added to the reaction mixture (3 equivalents, 36 mM). After 1 hour, a solution of L-cysteine ​​in PBS was added to the reaction mixture (6-10 equivalents, 165 mM). Finally, the antisense strand (1.2-1.5 equivalents, 0.5-2.5 mM) was added to anneale the conjugate. The conjugate was purified by an AKTA Pure FPLC system equipped with a 5 x 200 mm column packed with 20 mM Tris pH 8 (buffer A), 20 mM Tris 1500 mM NaCl (buffer B), and Toso SuperQ 5PW (20 micrometers). The crude reaction mixture was pump-loaded onto the column and eluted with a gradient of 10-40% buffer B. DAR1 and DAR2 fractions were distinguished by SEC Method 1, AEX Method 1, and Nanodrop 260 / 280 readings. The DAR1 fraction was pooled and buffered with PBS using a PD-10 desalting column. The purified conjugate was analyzed by SEC method 1 and eluted as a monomer peak with a retention time of 13.2 minutes.

[0344] D. Conjugation of RNAi agents to Fab and capping by NEM.

[0345] L20-p using standard amide reaction chemistry after cleavage from the solid phase of the RNAi agent described herein containing free amine

[0346] It was conjugated to. 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 mixture was kept overnight at room temperature and shielded from light. The next day, the reaction mixture was loaded onto a PD-10 desalting column equilibrated to 20 mM Tris pH 8 and eluted with 20 mM Tris pH 8 to remove TCEP. The concentration of Fab in the eluent was determined using the theoretical absorption factor at 280 nm. A solution of the L20-modified sense strand in sodium phosphate buffer was prepared, and its concentration was determined using the theoretical absorption factor at 260 nm. The L20-modified sense strand (1–1.3 equivalents, 0.5–2.5 mM) was added to the desalted Fab solution, and the reaction mixture was mixed end-over-end. Analysis by SEC Method 1 and AIEX Method 1 shows a mixture of starting Fab, DAR1, and DAR2. After 1 hour, a solution of N-ethyl maleimide (NEM) in 20 mM Tris pH 8 was added to the reaction mixture (12 equivalents, 160 mM). After 1 hour, the antisense strand (1.2–1.5 equivalents, 0.5–2.5 mM) was added to anneale the conjugate. The conjugate was purified by an AKTA Pure FPLC system equipped with a 5 x 200 mm column packed with 20 mM Tris pH 8 (buffer A), 20 mM Tris 1500 mM NaCl (buffer B), and Toso SuperQ 5PW (20 micrometers). The crude reaction mixture was pump-loaded onto the column and eluted with a 10–40% buffer B gradient. DAR1 and DAR2 fractions were distinguished by SEC Method 1, AEX Method 1, and UV-Vis 260 / 280 measurements. The DAR1 fraction was pooled and buffered with PBS using a PD-10 column. The purified conjugate was analyzed by SEC Method 1, which eluted as a monomer peak with a retention time of 13.2 minutes.

[0347] SEC Method 1

[0348]

[0349] AIEX Method 1

[0350]

[0351] The RNAi agent described herein containing free amines was first prepared using standard solid-phase synthetic coupling techniques for L1026-p, i.e.:

[0352]

[0353] L-1026-modified sense strand joined to:

[0354] (NH2-C6)s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb) (i.e., CS915332, sequence identification number: 151) was generated. After cleavage from the solid phase, the RNAi agent described herein was synthesized according to the following procedure as exemplified by the synthesis of AC006448:

[0355] A freshly prepared solution of TCEP-HCl in PBS (5 equivalents, 70 mM, 42 μL) was added to a solution of Fab0070 (28 mg, 0.59 μmol, 5.55 mg / mL in PBS). The reduction mixture was mixed end-over-end for 15 minutes at ambient temperature and then maintained overnight at 5°C without stirring. The next day, the reaction mixture was loaded onto two PD-10 desalting columns (Sitiba) equilibrated with 20 mM Tris and 50 mM NaCl at pH 7.6 (alternatively, 20 mM Tris at pH 8 or PBS buffer may be used) and eluted with the same buffer to remove TCEP. The concentration of Fab in the eluent was determined using the theoretical absorption factor at 280 nm. A solution of L-1026-modified sense strand (CS915332) in 10 mM sodium phosphate buffer at pH 6.0–6.5 was prepared, and the concentration was determined using the theoretical absorption coefficient at 260 nm. L-1026-modified CS915332 (1.15 equivalents, 2.75 mM, 240 µL) was added to the desalted Fab solution, and the reactants were mixed end-over-end 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, a solution of L-cysteine ​​in 20 mM Tris and 50 mM NaCl at pH 7.6 (alternatively, some L-1026 conjugates were prepared in 20 mM Tris at pH 8 or PBS buffer solution) was added to the reaction mixture (10 equivalents, 165 mM, 36 µL). After 30 minutes, the conjugate was annealed by adding the antisense strand (CA003820) (1.3 equivalents, 1.45 mM in water, 529 µL). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM Tris at pH 8 (buffer A), 20 mM Tris and 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with a Toso SuperQ 5PW (20 micrometers).The crude reaction mixture was pump-loaded onto a column and eluted with a 10–40% buffer B gradient. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AIEX Method 1, and UV-Vis 260 / 280 measurements. The DAR1 fraction was pooled and buffer-exchanged with PBS using two PD-10 columns. The purified conjugate was analyzed by SEC Method 1, which eluted as a monomer peak with a retention time of 7.2 minutes.

[0356] SEC Method 1

[0357]

[0358] AIEX Method 1

[0359]

[0360] Conjugation of E. RNAi agents to Fab.

[0361] Synthesis of Fab0070-L-1288

[0362] A freshly prepared solution of TCEP-HCl in PBS (5 equivalents) was added to a solution of Fab0070 in PBS (82 mg, 8.2 mg / mL). The reaction mixture was maintained overnight at 5°C. The next day, the reaction mixture was buffer-exchanged with 20 mM Tris 50 mM NaCl pH 7.6 using four PD-10 columns equilibrated with the same buffer. The concentration was determined by nanodrops, and the solution was diluted to 2 mg / mL with 20 mM Tris 50 mM NaCl pH 7.6. The solution was diluted with DMSO (1.95 mL). A solution of L-1288 was prepared (2 mg / mL in DMSO) and charged over 15 minutes (1.6 mL, 3.19 mg, 3 equivalents). After 10 minutes, the reaction mixture was quenched with cysteine ​​(10 equivalents). Insoluble material was removed by centrifugation. The supernatant was removed, filtered, diluted 1:1 with PBS, and concentrated / desalted to a volume of 8 mL using a Pierce 10K MWCO spin column. Finally, the solution was loaded onto a HiPrep 26 / 10 desalting column and buffer exchanged by eluting with PBS. The final yield was approximately 80 mg.

[0363] L-1289 bioconjugation

[0364] Freeze-dried CS915332 (86 mg, 11.6 μmol) was placed 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 progress of the reaction was monitored by LCMS. After 1 hour, additional triethylamine (4 equivalents) and L-1289 solution (2 equivalents) were added. After 20 minutes, the LCMS indicated complete conversion. The reaction mixture was acidified with phosphoric acid (200 mg / mL, 5 equivalents in water). 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 pellet 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%).

[0365] Junction of Fab0070-L-1288 and L-1307 modified sense strands

[0366] L1307-modified sense strand in PBS in solution of Fab0070-L-1288 (12.5 mg, 6.97 mg / mL, 1.79 mL, 1 equivalent):

[0367] A solution (6.18 mg, 17.9 mg / mL, 345 μL, 3 equivalents) of L-1307s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb), (i.e., CS009529, sequence identification number: 152) was added. The combined solution was mixed end-over-end for 10 minutes and then stored at 5°C without stirring. The next day, the reaction mixture was heated to room temperature and annealed with antisense strand: cPrpusUfsgauuAfgcagGfuCfaAfaagsusg (i.e., AC003820, sequence identification number: 153), 6.23 mg, 10.4 mg / mL, 599 μL, 3.3 equivalents. The conjugate was purified by an AKTA Pure FPLC system equipped with a 5 x 200 mm column packed with 20 mM Tris pH 8 (Buffer A), 20 mM Tris 1500 mM NaCl (Buffer B), and Toso SuperQ 5PW (20 micrometers). The crude reaction mixture was diluted to 40 mL using MPA, loaded onto the column, and eluted with a 10–40% Buffer B gradient. The DAR1-containing fraction was pooled and buffer exchanged with PBS using two PD-10 columns. The conjugate was analyzed by SEC Method 1 and confirmed to be 99% pure with a retention time of 7.2 m. Additionally, the Fab-L-1288-L-1289-RNAi conjugate disclosed herein was generated according to this procedure.

[0368] SEC Method 1

[0369]

[0370] F. Synthesis of antibody-siRNA linkers

[0371] The synthesis of various Fab linkers used throughout this application is provided below.

[0372] Synthesis of 2,3,5,6-tetrafluorophenyl 16-((3,5-bis(5-(methylsulfonyl)-1,3,4-oxadiazole-2-yl)phenyl)amino)-16-oxo-4,7,10,13-tetraoxahexadecanoate (i.e., L-1026-p)

[0373]

[0374] Compound 6 (2.35 g, 7.31 mmol; prepared according to the literature [Sarbisheh et al., Bioconjugate Chemistry 2020 31 (12), 2789-2806]), EDC·HCl (2.38 g, 12.43 mmol), and K-oxyma (2.50 g, 13.9 mmol) were combined as solids and slurried in DMF (190 mL) at ambient temperature under N2. Compound 7 (1.192 g, 5.48 mmol) was added as a solution in DMF (10 mL). 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 with red oil under reduced pressure, slurried in DCM (250 mL), and washed with saturated aqueous sodium bicarbonate (200 mL). The layers were separated, and the aqueous layer was further extracted with DCM (100 mL). The combined organic phase was washed with water (200 mL) and brine (200 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated. The residue was purified by normal SiO2 chromatography using an ethyl acetate gradient (0–100%) in DCM. Yield of Compound 8: 1.77 g (49%), partially contaminated with Compound 6. Calculated mw for Compound 8: 653.77 g / mol, found m / z (ESI, positive mode): 654.83.

[0375]

[0376] 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 SiO2 chromatography using a gradient of DCM containing 0.1% formic acid and methanol (0-7%). Yield of Compound 9: 1.30 g (80%). Calculated mw for Compound 9: 597.66 g / mol, found m / z (ESI, positive mode): 598.79.

[0377]

[0378] Compound 9 (1.30 g, 2.18 mmol) was dissolved in DCM (50 mL) and cooled to 0°C. A 100 mg / mL m-CPBA solution was prepared by dissolving 10.38 g m-CPBA (77 wt%) in 80 mL of DCM and dried with sodium sulfate until clear. 58 mL of m-CPBA (5.85 g, 26.1 mmol) was added dropwise to the solution of Compound 9 at 0°C. The reaction mixture was heated to ambient temperature and allowed to proceed overnight. The reaction mixture was concentrated, slurried in 0.1% formic acid (50 mL) in DCM, and filtered. The filtrate was purified by normal SiO2 chromatography using a gradient of DCM containing 0.1% formic acid and methanol (0-10%). Yield of Compound 10: 1.03 g (72%). Calculated mw for Compound 10: 661.65 g / mol, found m / z (ESI, positive mode): 662.65.

[0379]

[0380] At 0°C, a solution of Compound 10 (1.03 g, 1.56 mmol) in DCM:ACN [4:1] (15 mL) was mixed with EDC (0.448 g, 2.34 mmol), 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 and dried. The crude material was purified using a reverse-phase HPLC (Phenomenex Gemini C18 50 mm x 250 mm, 10 µm) with a water / acetonitrile gradient containing 0.1% TFA. The product-containing fraction was concentrated under reduced pressure. Yield of L-1026-p: 1.10 g (87%). Calculated mw for compound L-1026-p: 809.71 g / mol, found m / z (ESI, positive mode): 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).

[0381] Synthesis of L20-p

[0382]

[0383] Methyl iodide (4.20 mL, 67.50 mmol) was added at room temperature 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 and concentrated over anhydrous Na2SO4. Compound 2 was obtained as a pale yellow solid, 5.41 g, 96%. Compound 2 was used directly without further purification. LC-MS: [M+H] Calculated 251.05, Found 251.18.

[0384]

[0385] LiOH (2.59 g, 108.08 mmol) was added at room temperature 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 using [C] HCl. Subsequently, extraction was performed using EtOAc (3 x 60 mL). The organic layers were combined, washed with brine, dried over anhydrous Na2SO4, and concentrated. Compound 3 was obtained 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.

[0386]

[0387] 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) at room temperature. The reaction mixture was stirred overnight at room temperature. After removing the solvent under vacuum, the residue was loaded onto a 120 g column, and Compound 5 was eluted with 0-50% EtOAc in hexane. Compound 5 was obtained as a white solid, 9.36 g, 99%. LC-MS: Calculated [M+H] 385.03, Found 385.46.

[0388]

[0389] 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) at 0 °C. The reaction mixture was stirred at room temperature for 6 hours. Another 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 obtained as white needle-shaped crystals, 1.93 g, 78%. LC-MS: Calculated [M+H] 417, Found 417.

[0390] Example 7. Relative expression of mRNA to target androgen receptor (AR) mRNA after delivery of TfR1 Fab-conjugated siRNA antisense

[0391] The siRNA duplexes are presented 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 presented in Table 8 below.

[0392] Table 8. siRNA sense sequence for targeting AR mRNA.

[0393]

[0394]

[0395] *Fab0044 is identical to Fab0002 except that the valine at position 205 of the light chain is mutated to cysteine.

[0396] **Fab0046 is identical to Fab0002 except that the alanine at position 117 of the heavy chain is mutated to cysteine.

[0397] Symbol description: (a) represents 2'-O-methyladenosine, (c) represents 2'-O-methylcytidine, (g) represents 2'-O-methylguanosine, (u) represents 2'-O-methyluridine; (Af) represents 2'-fluoroadenosine, (Cf) represents 2'-fluorocytidine, (Gf) represents 2'-fluoroguanosine, (Uf) represents 2'-fluorouridine; (s) represents a phosphorothioate linkage; (invAb) and (invAb)s represent inverted abasic nucleotides (structures in Table 13 below); cPrpus represents 5'-cyclopropylphosphonate-2'-O-methyluridine-3'-phosphorothioate (structures in Table 13 below); (NH-C6)s represents the structure in Table 13 below; L20 represents the structure in Table 13 below; [CP-1113] indicates that Fab was capped using CP-1113p according to the procedure of Example 6C; and [NEM] indicates that Fab was capped using N-ethyl maleimide according to the procedure of Example 6D.

[0398] Table 9. siRNA antisense sequences for targeting AR mRNA

[0399]

[0400] Symbol description: (a) represents 2'-O-methyl adenosine, (c) represents 2'-O-methyl cytidine, (g) represents 2'-O-methyl guanosine, (u) represents 2'-O-methyl uridine; (Af) represents 2'-fluoro adenosine, (Cf) represents 2'-fluoro cytidine, (Gf) represents 2'-fluoro guanosine, (Uf) represents 2'-fluoro uridine; (s) represents a phosphorothioate linkage; cPrpus represents 5'-cyclopropyl phosphonate-2'-O-methyluridine-3'-phosphorothioate (structure in Table 13 below).

[0401] Table 10. Shows the structures for cPrpus, (invAb)s, (invAb), L20, and NH-C6, defined in the symbol descriptions above for sense and antisense sequences in Tables 8 and 9.

[0402]

[0403]

[0404]

[0405] Example 8. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice

[0406] On Day 1 of the study, Tg B-hTfr1 mice were intravenously injected with a dosage of 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab-conjugated siRNA (Groups 2-7) at a dosage of 250 μL / 25g PBS according to Table 11 below, and the same preparations were administered again on Day 2:

[0407] Table 11: Dosage group for mice in Example 8.

[0408]

[0409] On day 15, mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. The samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 1 shows the relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0410] Example 9. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice

[0411] On Day 1 of the study, Tg B-hTfr1 mice were intravenously injected with a dosage of 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab-conjugated siRNA (Groups 2-10) at a dosage of 250 μL / 25g PBS according to Table 12 below, and the same preparations were administered again on Day 2:

[0412] Table 12: Dosage group for mice in Example 9.

[0413]

[0414] On day 15, mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. The samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 2 shows the relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0415] Example 10. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice

[0416] On Day 1 of the study, Tg B-hTfr1 mice were intravenously injected with a dosage of 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab-conjugated siRNA (Groups 2-6) at a dosage of 250 μL / 25g PBS according to Table 13 below, and the same preparations were administered again on Day 2:

[0417] Table 13: Dosage group for mice in Example 10.

[0418]

[0419] On day 15, mice were euthanized, and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. The samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 3 shows the relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0420] Example 11. In vivo AR mRNA gene expression in different antibody conjugate groups compared to the AC003313 (Fab0002) control group in selected brain regions

[0421] In vivo AR mRNA gene knockdown of the Fab-duplex was measured in selected brain regions. AR mRNA gene expression in each group was normalized relative to the AC003313 (Fab0002-conjugated siRNA) group. Figure 4 shows a graph of relative expression values. The Fab0070-siRNA duplex group appears to have the lowest relative expression (highest gene knockdown).

[0422] Example 12. In vivo knockdown of AR mRNA gene expression in synomolgus monkeys

[0423] On Day 0 of the study, cynomolgus monkeys were injected subcutaneously with PBS or a compound preparation containing 3 mg / kg of anti-TfR1 Fab-siRNA conjugate at a concentration of 1.5 mg / mL, according to Table 17 below. On Day 7 of the study, groups 2, 3, and 4 were administered the same preparation as on Day 0. All animals were administered a volume of 2 mL / kg.

[0424] Table 14: Dosage group for non-human primates of Example 12.

[0425]

[0426] Three monkeys (n=3) were administered to Groups 1 (control), 3, and 4 (trigger-treated), and two monkeys (n=2) were administered to Group 2. On Day 28 of the study, animals from all groups were euthanized, and various tissues were collected from each animal, including the frontal cortex, temporal cortex, caudate, cerebellum (cortex), putamen, motor cortex, medulla, pons, hippocampus, thalamus, hypothalamus, midbrain, substantia nigra, cervical dorsal root ganglion (DRG), thoracic DRG, lumbar DRG, cervical spinal cord, lumbar spinal cord, thoracic spinal cord, visual cortex, gastrocnemius muscle, triceps 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 presented in Table 15 below:

[0427] Table 15. Relative expression of AR mRNA in various tissues analyzed by qPCR for each administration group of Example 12.

[0428]

[0429]

[0430] As shown in Table 15 above, a sustained decrease in AR mRNA expression was observed in multiple tissues in non-human primates treated with anti-TfR1 antibody-siRNA conjugates. Groups 3 (Fab0061-siRNA conjugates) and 4 (Fab0070-siRNA conjugates) outperformed Group 2 (Fab002-siRNA conjugates) in almost all tissues analyzed, with the exception of the thoracic and lumbar spine DRGs.

[0431] Table 15 also shows that Fab0061 and Fab0070 can deliver siRNA to skeletal muscle tissue, such as the gastrocnemius and triceps muscles. Delivery to cardiac tissue using Fab0061 and Fab0070 is also possible, and deep knockdown of AR mRNA is presented in each section of the collected heart.

[0432] Example 13. In vivo knockdown of MAPT in cynomolgus monkeys

[0433] As shown in Table 15 above, a sustained decrease in AR mRNA expression was observed in multiple tissues in non-human primates treated with anti-TfR1 antibody-siRNA conjugates. Groups 3 (Fab0061-siRNA conjugates) and 4 (Fab0070-siRNA conjugates) outperformed Group 2 (Fab002-siRNA conjugates) in almost all tissues analyzed, with the exception of the thoracic and lumbar spine DRGs.

[0434] On Day 0 of the study, cynomolgus monkeys were injected with compound preparations containing AC007414 in artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or in aCSF at a concentration of 1.5 mg / mL and a volume of 2 mL / kg, 3 mg / kg at a concentration of 7.5 mg / mL and a volume of 2 mL / kg, or 15 mg / kg at a concentration of 7.5 mg / mL and a volume of 4 mL / kg, according to Table 16 below:

[0435] Table 16: Dosage group for non-human primates of Example 13.

[0436]

[0437] Four monkeys (n=4) were administered to each group. Monkeys were injected subcutaneously on Day 0, Day 7, and Day 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 by qPCR for MAPT mRNA knockdown. Samples were analyzed by JESS for protein knockdown. The mean mRNA knockdowns for the frontal cortex, hippocampus, and thoracic spinal cord for each group relative to Group 1 are presented in Table 17 below:

[0438] Table 17. Relative expression of MAPT mRNA in various tissues analyzed by qPCR for each administration group of Example 13

[0439]

[0440] Figure 5 presents the average protein knockdown for the frontal cortex, hippocampus, and thoracic spinal cord for each group relative to Group 1:

[0441] As can be seen in Table 17 and Figure 5, the MAPT RNAi agonist conjugated to anti-transferrin Fab070 achieved dose-dependent and deep knockdown in CNS tissues when injected subcutaneously. The results demonstrate that the treatment of MAPT-related diseases and disorders can be alleviated by RNAi agonists administered subcutaneously.

[0442] The foregoing disclosure highlights specific embodiments of the invention, and all variations or alternative equivalents thereof should be understood to be within the spirit and scope of the invention as set forth in the appended claims.

[0443] Example 14. In vivo administration of an HTT RNAi agonist in synomolgus monkeys.

[0444] HTT RNAi agonists containing anti-Tfr Fab were evaluated in vivo in cynomolgus monkeys. On days 1, 8, and 15, four male cynomolgus monkeys (n=4) in each test group were administered HTT RNAi agonists formulated in PBS at doses of 3.0 mg / kg (adjusted to individual animal body weight), 2.0 ml / kg, and 1.5 mg / ml, or PBS. Each dose was administered via subcutaneous (SC) injection, based on the most recent body weight of each animal. Administration followed Table 18 below.

[0445] Table 18. Administration of Example 14 to synomolgus monkeys.

[0446]

[0447] The test animals were non-human primates, male, non-naive, 3–5 year old Cynomorgus facicularius monkeys. The test animals were acclimatized to laboratory accommodation according to facility and acclimatization standard operating procedures for at least 14 days prior to the start of administration. The RNAi agonist test product was administered via subcutaneous (SC) administration using a syringe and needle in the mid-shoulder region.

[0448] The administration site was shaved prior to administration. The Day 1 dose was delivered to the animal's left shoulder blade, the Day 8 dose to the right shoulder blade, and the Day 15 dose to the left shoulder blade. Each dose was administered using a syringe with a 23-25 ​​gauge needle.

[0449] The individual body weight of the test animals was recorded once on day 7 before treatment, then weekly throughout the duration of the study, and once before necropsy.

[0450] ~0.5 mL of cerebrospinal fluid (CSF) was collected from all groups on Day 7. On Day 43 (the day of necropsy), ~2.0 mL of CSF was collected from all groups. For the CSF collection procedure, all test animals were anesthetized by intravenous (IV) injection of ketamine (10 mg / kg, IV) and intramuscular injection of dexmedetomidine (0.02 mg / kg, IM) and positioned in a lateral recumbent position. The skin covering the insertion site was shaved and wiped several times with an individual chlorhexidine scrub. The animal's head was gently bent to a position where the jaw was nearly touching the chest but the airway was not obstructed. The specialist palpated the laryngeal process and the wing of the atlas (C1). The needle was inserted through the skin perpendicularly over the atlas to access the subarachnoid space. If it struck the bone, the needle was redirected anteriorly or posteriorly until the designated access point was found. Once proper placement was achieved, the CSF would flow freely through the needle and be collected within the freezing tube. The needle was removed, and direct pressure was applied to the puncture site for at least 2 minutes. Once the procedure was completed, the animal was administered 2-4 mg / kg of carprofen subcutaneously every 12 hours for 1 day. Atipamazole (0.225 mg / kg, IM) was administered as needed.

[0451] 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 ganglion (DRG). Tissues other than those mentioned above may also be collected, and if such additional tissues were collected, their biological data are presented similarly below. The collected tissues were analyzed for biological parameters.

[0452] From the collected tissues, cHTT mRNA transcript levels were quantified by qPCR and normalized to group 1 sinos administered PBS using cPPIB as the endogenous control gene. The cHTT expression data are presented in Table 19 below.

[0453] Table 19. cHTT expression in the CNS tissue of Example 14.

[0454]

[0455]

[0456] In the temporal cortex, frontal cortex, motor cortex, caudate, and hippocampus, groups 2–5 showed a decrease in HTT transcripts for at least 43 days after administration. In the putamen, groups 2, 4, and 5 showed a decrease in HTT transcripts for at least 43 days after administration. Most notably, the most significant decrease in HTT transcripts was observed in the hippocampus, where three doses of 3.0 mg / kg AC007867 showed ~69% HTT transcript inhibition (0.306), and three doses of 3.0 mg / kg AC007865 showed ~70% HTT transcript inhibition (0.302).

[0457] HTT protein expression was analyzed in Sino tissues using the Jess protein assay and normalized to Group 1 Sino administered PBS. HTT protein was quantified using an anti-huntingtin protein antibody (Sigma-Aldrich®, Cat. MAB2166). HTT protein expression data are presented in Table 20 below.

[0458] Table 20. HTT protein expression in the CNS tissue of Example 14.

[0459]

[0460] In the temporal cortex, frontal cortex, motor cortex, caudate, putamen, hippocampus, and cerebellum, groups 2-5 showed a decrease in HTT protein by at least day 43 after administration. In the liver, groups 2, 3, and 5 showed a decrease in HTT protein by at least day 43 after administration. Most notably, the most significant decrease in HTT protein was observed in the motor cortex, where three doses of 3.0 mg / kg AC007867 showed ~86% HTT protein inhibition (0.143), and three doses of 3.0 mg / kg AC007865 showed ~85% HTT protein inhibition (0.146).

[0461] Example 15. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0462] AR RNAi agonists were evaluated in vivo in mice. On days 1 and 2, 5 female B-hTFR1 mice (n=5) were administered saline or AR RNAi agonists (formulated in saline, 0.75 or 1.5 mg / kg animal body weight) via subcutaneous (SC) injection at a total administration volume of 250 μL / 25g. Administration followed Table 21 below.

[0463] Table 21. Dosage group for mice in Example 15.

[0464]

[0465] C57BL / 6-Tfrc tm1(TFRC)Bcgen The B-hTFR1 mouse, also known as the / Bcgen mouse (Biocytogen), has exons 4-19 of the mouse Tfr1 gene coding for the extracellular region replaced by human TFR1 exons 4-19.

[0466] Each AR RNAi agonist included a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding moiety having a modified sequence as presented in the duplex structure of the present invention.

[0467] On day 15, the mice were euthanized. Thoracic spinal cords, cerebellum, striatum, and cortex were collected from the mice for analysis. mAR mRNA transcriptome expression was analyzed by qPCR and normalized to Group 1 mice administered saline using mPPIA as the endogenous gene. The mAR expression data are presented in Table 22 below.

[0468] Table 22. Relative expression of mAR in the mouse thoracic spinal cord, cerebellum, striatum, and cortex of Example 15.

[0469]

[0470] In the thoracic spinal cord, right cerebellum, right cortex, and right striatum, groups 2-12 showed a decrease in mAR by at least day 15. In the thoracic spinal cord, right cerebellum, right cortex, and right striatum, dose-response was observed for AC004791, AC004792, and AC004798.

[0471] Example 16. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0472] AR RNAi agonists were evaluated in vivo in mice. On days 1 and 2, 5 female B-hTFR1 mice (n=5) were administered saline or AR RNAi agonists (formulated in saline, 1.5 mg / kg animal body weight) via subcutaneous (SC) injection at a dose concentration of 0.15 mg / mL and a dose volume of 10 mL / kg. Administration followed Table 23 below.

[0473] Table 23. Dosage group for mice in Example 16.

[0474]

[0475] C57BL / 6-Tfrc tm1(TFRC)BcgenThe B-hTFR1 mouse, also known as the / Bcgen mouse (Biocytogen), has exons 4-19 of the mouse Tfr1 gene coding for the extracellular region replaced by human TFR1 exons 4-19.

[0476] Each AR RNAi agonist included a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding moiety having a modified sequence as presented in the duplex structure of the present invention.

[0477] On day 15, the mice were euthanized. CNS and muscle tissues were collected from the mice for analysis. mAR mRNA transcriptome expression was analyzed by qPCR and normalized to Group 1 mice administered saline using mPPIA as the endogenous gene. The mAR expression data are presented in Table 24 below.

[0478] Table 24. Relative expression of mAR in mouse CNS and muscle tissue of Example 16.

[0479]

[0480] In the cerebellum, cortex, triceps, gastrocnemius, thoracic spinal cord, and right striatum, groups 2 and 3 showed a decrease in mAR by at least day 15. Most notably, a double dose of 1.5 mg / kg AC011951 achieved ~84% inhibition (0.157) in the cerebellum.

[0481] Example 17. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0482] AR RNAi agonists were evaluated in vivo in mice. On Day 1, Day 1 & Day 2, or Day 1 & Day 2 & Day 3 & Day 4, 5 female B-hTFR1 mice (n=5) were administered saline or AR RNAi agonists (formulated in saline, 0.75, 1.5, 3.0, and 12.0 mg / kg animal body weight) via subcutaneous (SC) injection at a total administration volume of 250 μL / 25g. Administration followed Table 25 below.

[0483] Table 25. Dosage group for mice in Example 17.

[0484]

[0485] C57BL / 6-Tfrc tm1(TFRC)Bcgen The B-hTFR1 mouse, also known as the / Bcgen mouse (Biocytogen), has exons 4-19 of the mouse Tfr1 gene coding for the extracellular region replaced by human TFR1 exons 4-19.

[0486] Each AR RNAi agonist included a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding moiety having a modified sequence as presented in the duplex structure of the present invention.

[0487] On day 15, the mice were euthanized. Thoracic spinal cords, cerebellum, striatum, and cortex were collected from the mice for analysis. mAR mRNA transcriptome expression was analyzed by qPCR and normalized to Group 1 mice administered saline using mPPIA as the endogenous gene. The mAR expression data are presented in Table 26 below.

[0488] Table 26. Relative expression of mAR in the mouse thoracic spinal cord, cerebellum, striatum, and cortex of Example 17.

[0489]

[0490]

[0491] In the thoracic spinal cord, cerebellum, cortex, striatum, and midbrain, groups 2–10 showed a reduction in mAR by at least day 15. Most notably, four doses of 3.0 mg / kg AC006372 achieved ~85% inhibition (0.153) in the cerebellum. In the thoracic spinal cord and cerebellum, dose-responses were observed for single, double, and quadruple doses of AC006372. In the cortex, dose-responses were observed for single and double doses of AC006372. In the striatum, dose-responses were observed for single and quadruple doses of AC006372. In the midbrain, dose-responses were observed for double and quadruple doses of AC006372.

[0492] Example 18. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0493] AR RNAi agonists were evaluated in vivo in mice. On days 1 and 2, 5 female B-hTFR1 mice (n=5) were administered saline or AR RNAi agonists (formulated in saline, 1.5 mg / kg animal body weight) via subcutaneous (SC) injection at a total administration volume of 250 μL / 25g. Administration followed Table 27 below.

[0494] Table 27. Dosage group for mice in Example 18.

[0495]

[0496] C57BL / 6-Tfrc tm1(TFRC)Bcgen The B-hTFR1 mouse, also known as the / Bcgen mouse (Biocytogen), has exons 4-19 of the mouse Tfr1 gene coding for the extracellular region replaced by human TFR1 exons 4-19.

[0497] Each AR RNAi agonist included a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding moiety having a modified sequence as presented in the duplex structure of the present invention.

[0498] On day 15, the mice were euthanized. Thoracic spinal cords, cerebellum, striatum, and cortex were collected from the mice for analysis. mAR mRNA transcriptome expression was analyzed by qPCR and normalized to Group 1 mice administered saline using mPPIA as the endogenous gene. The mAR expression data are presented in Table 28 below.

[0499] Table 28. Relative expression of mAR in the mouse thoracic spinal cord, cerebellum, striatum, and cortex of Example 18.

[0500]

[0501] In the thoracic spinal cord, cerebellum, cortex, and striatum, groups 2-7 showed a decrease in mAR by at least day 15.

[0502] Example 19. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0503] AR RNAi agonists were evaluated in vivo in mice. On days 1, 2, and 3, 4 female B-hTFR1 mice (n=4) were administered saline or AR RNAi agonists (formulated in saline, 3.0 mg / kg animal body weight) via subcutaneous (SC) injection at a total administration volume of 250 μL / 25g. Administration followed Table 29 below.

[0504] Table 29. Dosage group for mice in Example 19.

[0505]

[0506] C57BL / 6-Tfrc tm1(TFRC)BcgenThe B-hTFR1 mouse, also known as the / Bcgen mouse (Biocytogen), has exons 4-19 of the mouse Tfr1 gene coding for the extracellular region replaced by human TFR1 exons 4-19.

[0507] Each AR RNAi agonist included a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand comprising an antigen-binding moiety having a modified sequence as presented in the duplex structure of the present invention.

[0508] Mice were euthanized on days 4, 10, 17, 31, 45, 59, 80, 100, or 122 according to Table 58 above. Thoracic spinal cords, cerebellum, striatum, and cortex were collected from the mice for analysis. mAR mRNA transcriptome expression was analyzed by qPCR using mPPIA as the endogenous gene; groups 3–7 were normalized to mice in saline group 1, and groups 9–11 were normalized to mice in saline group 8. mAR expression data are presented in Tables 30 and 31 below.

[0509] Table 30. Relative expression of mAR in the mouse thoracic spinal cord, cerebellum, striatum, and cortex of Example 19.

[0510]

[0511] In the thoracic spinal cord, cerebellum, left cortex, striatum, and brainstem, groups 3-7 showed a decrease in mAR by at least day 59. Most notably, three doses of 3.0 mg / kg AC006448 achieved ~85% inhibition (0.147) in the cerebellum on day 59.

[0512] Table 31. Relative expression of mAR in the mouse thoracic spinal cord, cerebellum, striatum, and cortex of Example 19.

[0513]

[0514] In the thoracic spinal cord, cerebellum, and striatum, groups 9-11 showed a reduction in mAR by at least day 122. Most notably, a three-dose regimen of 3.0 mg / kg AC006448 achieved ~39% inhibition (0.612) in the striatum on day 122.

[0515] Other embodiments

[0516] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Accordingly, unless otherwise expressly stated, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0517] From the foregoing description, a person skilled in the art can easily identify the essential features of the present invention and, without departing from the spirit and scope thereof, make various changes and variations of the present invention to suit various uses and conditions. Accordingly, other embodiments are also within the scope of the claims.

[0518] Equivalents

[0519] Although various embodiments of the present invention have been described and illustrated herein, a person skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or obtain results and / or one or more advantages, and each such modification and / or variation is considered to be within the scope of the embodiments of the present invention described herein. More generally, a person skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific applications or uses in which the teachings of the present invention are applied. A person skilled in the art will be able to recognize or identify many equivalents to the specific embodiments of the present invention described herein by using only commercial experimentation. Accordingly, the embodiments are presented merely as examples, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced differently from those specifically described and claimed. The embodiments of the present invention in this disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more of these features, systems, articles, materials, kits and / or methods is included within the scope of the invention of this disclosure, provided that these features, systems, articles, materials, kits and / or methods are not mutually contradictory.

[0520] All definitions as defined and used herein shall be understood to take precedence over dictionary definitions, definitions in the literature incorporated by reference, and / or the ordinary meaning of the defined terms.

[0521] All references, patents, and patent applications disclosed herein are incorporated by reference in relation to the subject matter for which they are cited, and in some cases, may encompass the entirety of the literature.

[0522] The singular form used herein in the specification and claims should be understood to mean "at least one" unless otherwise clearly indicated.

[0523] The phrase “and / or” as used herein in the specification and claims should be understood to mean “either one or both” of the elements thus combined, that is, elements that exist jointly in some cases and separately in others. Multiple elements enumerated with “and / or” should be interpreted in the same way, that is, “one or more” of the elements thus combined. Whether or not they relate to the elements specifically identified, other elements other than those specifically identified by the “and / or” clause may arbitrarily exist. Accordingly, as a non-limiting example, references to “A and / or B,” when used with open language such as “comprising,” may refer, in one embodiment, to A alone (optionally including elements other than B); in another embodiment, to B alone (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), and so on.

[0524] The term “or” as used herein in the specification and claims should be understood to have the same meaning as “and / or” as defined above. For example, when separating items from a list, “or” or “and / or” should be interpreted as inclusive, that is, including not only at least one of a number of elements or a list of elements, but also more than one, and optionally additional unlisted items. Only terms otherwise explicitly indicated, such as “only one of” or “exactly one of”, or “consisting of” when used in the claims, will refer to including exactly one of a number of elements or a list of elements. In general, the term “or” as used herein shall be interpreted to indicate an exclusive alternative (i.e., “one or the other, but not both”) only when preceded by an exclusive term such as “either one,” “one of”, “only one of”, or “exactly one of”. “Consisting of essentially” will have its ordinary meaning as used in the field of patent law when used in the claims.

[0525] With respect to a list of one or more elements, the phrase “at least one” as used herein in the specification and claims means at least one element selected from any one or more of the elements in the list of elements, but it should be understood that it does not necessarily include at least one of each and all of the elements specifically enumerated within the list of elements, nor exclude any combination of elements in the list of elements. This definition also allows that elements other than the specifically identified elements may exist within the list of elements referred to by the phrase “at least one,” whether or not they relate to the specifically identified elements. Accordingly, 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”) may refer, in one embodiment, at least one A (and optionally including an element other than B) including optionally more than one where B is absent; in another embodiment, at least one B (and optionally including an element other than A) including optionally more than one where A is absent; in yet another embodiment, at least one A including optionally more than one, and at least one B including optionally more than one (and optionally including other elements), etc.

[0526] Furthermore, unless otherwise clearly indicated, in any method claimed herein comprising more than one step or act, it should be understood that the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are mentioned.

Claims

Claim 1 An antibody that binds to a transferrin receptor (TfR1), comprising a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL); wherein VH is: heavy chain complementarity determining region 1 (HC CDR1) presented as GX1TFX2SYWX3H, where X1 is F or Y; X2 is K, N, or T; and X3 is M or V; heavy chain complementarity determining region 2 (HC CDR2) presented as EINPTNGRX4NYIEKFKS (Sequence Identification No.: 23), where X4 is F, S, T, or V; An antibody comprising a heavy chain complementarity determining region 3 (HC CDR3) presented as GTRAYHY (SEQ ID No.: 24); wherein VL is: a light chain complementarity determining region 1 (LC CDR1) presented as RASDX5LYX6NLA (SEQ ID No.: 5), where X5 is G, K, or N and X6 is R or S; a light chain complementarity determining region 2 (LC CDR2) presented as DAX7X8LAS, where X7 is F, K, R, or T; and X8 is K, L, N, or R; and a light chain complementarity determining region 3 (LC CDR3) presented as QHFWGTPLT (SEQ ID No.: 13). Claim 2 An 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. Claim 3 In claim 1, comprising any one of HC CDR1, HC CDR2, and HC CDR3 as presented in Table 2: Fab0070, Fab0061, Fab0060, Fab0062, Fab0063, Fab0064, Fab0065, Fab0066, Fab0067, Fab0068, Fab0069, Fab0071, Fab0072, Fab0073, Fab0074, Fab0165, Fab0166, Fab0167, or Fab0168; An antibody comprising 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 presented in Table 1. Claim 4 In paragraph 3, (a) HC CDR1, HC CDR2, and HC CDR3 presented as sequence identification numbers: 16, 19, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 presented as sequence identification numbers: 4, 11, and 13, respectively; or (b) HC CDR1, HC CDR2, and HC CDR3 presented as sequence identification numbers: 14, 20, and 24, respectively, and LC CDR1, LC CDR2, and LC CDR3 presented as sequence identification numbers: 2, 7, and 13, respectively, comprising antibodies. Claim 5 An antibody according to any one of claims 1 to 4, wherein the antibody is a humanized antibody; optionally comprising a light chain framework region presented in Table 3 and / or a heavy chain framework region presented in Table 4. Claim 6 An antibody according to claim 5 comprising any one of the VH sequences presented in Table 5 and / or any one of the VL sequences presented in Table 5. Claim 7 An antibody according to any one of claims 1 to 6, which is a full-length antibody, its antigen-binding fragment, or a single-chain variable fragment (scFv) antibody. Claim 8 An antibody according to any one of claims 1 to 7, wherein the Fab molecule; wherein the heavy chain comprises VH and a CH1 heavy chain constant region fragment; and wherein the light chain comprises VL and a light chain constant region (CL). Claim 9 An antibody according to claim 8, wherein CH1 comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (Sequence Identification No.: 34) and / or CL comprises the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence Identification No.: 29). Claim 10 An antibody according to claim 8 or 9, wherein VH comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTLVTVSS (Sequence Identification No.: 59) and / or VL comprises the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (Sequence Identification No.: 60). Claim 11 An antibody according to claim 8 or 9, wherein (a) the heavy chain comprises the amino acid sequence of sequence identification number: 61 and the light chain comprises the amino acid sequence of sequence identification number: 62; or (b) the heavy chain comprises the amino acid sequence of sequence identification number: 45 and the light chain comprises the amino acid sequence of sequence identification number:

46. Claim 12 An antibody according to any one of claims 1 to 11, comprising an Fc fragment and optionally a Fab-Fc molecule. Claim 13 A conjugate comprising an antibody that binds to a transferrin receptor (TfR1) (anti-TfR1 antibody) and an oligonucleotide-based agonist, wherein the anti-TfR1 antibody is the conjugate presented in any one of claims 1 to 12. Claim 14 In paragraph 13, a conjugate in which an oligonucleotide-based agent is directly conjugated to an anti-TfR1 antibody via a covalent or non-covalent bond. Claim 15 In paragraph 13, a conjugate in which an oligonucleotide-based agent is conjugated to an anti-TfR1 antibody through a linker that is optionally a chemical linker, a peptide linker, a polymer linker, or a nucleic acid-binding polypeptide. Claim 16 In paragraph 13, a conjugate in which the linker is a peptide linker that is a glycine spacer of 1-4 glycine residues. Claim 17 A conjugate according to any one of claims 13 to 16, wherein the oligonucleotide-based agent comprises one or more chemically modified nucleotides. Claim 18 In paragraph 17, a conjugate chemically modified by substitution of nucleotides by alkyl, alkoxy, amino, deaza, halo, hydroxyl and / or thiol groups; sugar modification, one or more non-nucleotides; one or more deoxyribonucleotides; one or more non-phosphodiester linkages; or a combination thereof. Claim 19 A conjugate according to claim 18, wherein the sugar modification comprises a 2' OH group replaced by a 2' amino group, a 2' O-methyl group, a 2' fluoro group, a 2' methoxyethyl group, or a 2'-O, 4'-C methylene crosslink; and one or more non-nucleotides comprise xanthine, hypoxanthine, azapurine, methylthioadenine, 7-deaza-adenosine, or O- and N-modified nucleotides; and one or more non-phosphodiester linkages comprise one or more nitrogen or sulfur substitutions for oxygen in the phosphate group. Claim 20 A conjugate of an RNAi agent comprising a sense strand and an antisense strand forming a double strand, wherein the oligonucleotide-based agent in any one of claims 13 to 19. Claim 21 In paragraph 20, the RNAi agonist attenuates the expression of a target gene selected from the group consisting of APP, ataxin-1, ataxin-2, ataxin-3, ataxin-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich repeat serine / threonine-protein kinase 2, complement C3, microtubule-associating protein tau, alpha-2A adrenergic receptor, sodium channel protein type 9 subunit alpha, apolipoprotein E, alpha-synuclein, putative 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 A conjugate that is. Claim 22 A conjugate according to claim 21, wherein the target gene is selected from the group consisting of APP, ATXN2, ATXN3, AR, SOD1, HTT, C9ORF72, LRRK2, C3, MAPT, ADRA2A, SCN9A, APOE, SNCA, GPR75, and FUS. Claim 23 In any one of claims 20 to 22, the sense strand is conjugated to an anti-TfR1 antibody, wherein the anti-TfR1 antibody is a conjugate in which the Fab molecule. Claim 24 In any one of claims 13 to 23, the anti-TfR1 antibody is a Fab molecule; wherein the oligonucleotide-based agent is conjugated to a linker of the following chemical formula: ,here represents the attachment site for the Fab molecule, and A conjugate that represents the attachment site of the linker to the oligonucleotide-based agonist portion of the conjugate. Claim 25 A conjugate according to any one of claims 13 to 23, wherein the anti-TfR1 antibody is a Fab molecule comprising a cap moiety; wherein the oligonucleotide-based agonist is conjugated to a linker, and wherein the oligonucleotide-based agonist is conjugated to the Fab molecule through the linker. Claim 26 In paragraph 25, the cap moiety has the following chemical formula: ; The linker has the following structure , here represents the attachment site for the Fab molecule, and A conjugate that represents the attachment site of the linker to the oligonucleotide-based agonist portion of the conjugate. Claim 27 In paragraph 25 or 26, the oligonucleotide-based agonist is an RNAi agonist comprising a sense strand and an antisense strand forming a double strand; wherein the sense strand is a conjugate joined to a linker. Claim 28 A pharmaceutical composition comprising an anti-TfR1 antibody or a conjugate thereof and a pharmaceutically acceptable carrier, wherein the anti-TfR1 antibody is one of claims 1 to 12. Claim 29 A pharmaceutical composition according to claim 27, comprising a conjugate of an anti-TfR1 antibody, wherein the conjugate is one of claims 13 to 27. Claim 30 A method for attenuating the expression of a target gene in the brain of a subject, comprising administering an effective amount of a conjugate or a pharmaceutical composition containing the same to a subject requiring attenuation of the expression of a target gene in the brain, wherein the conjugate comprises an anti-TfR1 antibody conjugated to an oligonucleotide-based agonist, wherein the anti-TfR1 antibody is presented in any one of claims 1 to 12; wherein the oligonucleotide-based agonist is a method for attenuating the expression of a target gene. Claim 31 In paragraph 30, the method in which the composite is presented in any one of paragraphs 12 through 27. Claim 32 A method according to paragraph 30 or 31 in which the subject is a human patient who has or is suspected of having a central nervous system (CNS) disease. Claim 33 A method according to claim 32, wherein the CNS disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy. Claim 34 A method according to any one of claims 30 to 33, wherein the conjugate or the pharmaceutical composition containing the same is administered to a subject by intravenous, subcutaneous, intramuscular, parenchymal, or intrathecal injection. Claim 35 A method for alleviating CNS disease comprises administering an effective amount of a conjugate presented in any one of claims 12 to 26 or a pharmaceutical composition containing the same to a subject requiring alleviation of CNS disease, wherein the conjugate comprises APP, ataxin-1, ataxin-2, ataxin-3, ataxin-7, androgen receptor, SOD1, huntingtin, chromosome 9 open reading frame 72, leucine-rich repeat serine / threonine-protein kinase 2, complement C3, microtubule-associating protein tau, alpha-2A adrenergic receptor, sodium channel protein type 9 subunit alpha, apolipoprotein E, alpha-synuclein, putative G-protein coupled receptor 75, RNA-binding protein FUS, GFAP, KCNT1, PRNP, MSH3, RAGE, SNC9A, SCN10A, SCN11A, SORT1, VCP, A method comprising an oligonucleotide-based agonist that attenuates the expression of a target gene selected from the group consisting of PIKFYVE, CHMP7, PDE2A, SARM1, NLRP3, GSDME, and DYRK1A; wherein the subject has or is suspected of having a CNS disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, cognitive impairment, frontotemporal dementia, Lewy body disease, cerebral amyloid angiopathy, tauopathy, spinocerebellar ataxia, Alexander disease, Kennedy disease, childhood epilepsy, fatal familial insomnia, prion disease, pain, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, TDP43 proteinopathy, traumatic brain injury, Down syndrome, neuropathic pain, hereditary dysphagia, paroxysmal pain disorder, and hereditary small fiber neuropathy. Claim 36 A method for weakening the expression of a target gene in the skeletal muscle of a subject, comprising administering an effective amount of a conjugate or a pharmaceutical composition containing the same to a subject requiring weakening of the expression of the target gene in the skeletal muscle, wherein the conjugate comprises an anti-TfR1 antibody conjugated to an oligonucleotide-based agonist, wherein the anti-TfR1 antibody is presented in any one of claims 1 to 12; wherein the oligonucleotide-based agonist is a method for weakening the expression of the target gene. Claim 37 A method according to claim 36, wherein the target gene is selected from the group consisting of DUX4, DM1, ACVR2A, ACVR2B, MSTN, and GYS1. Claim 38 A method according to paragraph 36 or 37 in which the subject is a human patient who has or is suspected of having a skeletal muscle disease. Claim 39 A method according to claim 38, wherein the skeletal muscle disease is selected from the group consisting of facialoscapulohumeral dystrophy (FSHD), Duchenne muscular dystrophy, and myotonic dystrophy of type 1, spinal muscular atrophy, or metabolic myopathy, e.g., Pompe disease, to the non-limiting extent, or neuromuscular junction disease, e.g., myasthenia gravis (MG), to the non-limiting extent, or metabolic syndrome. Claim 40 A nucleic acid or set of nucleic acids comprising a nucleotide sequence encoding the heavy chain and light chain of an anti-TfR1 antibody presented in any one of claims 1 to 12. Claim 41 In paragraph 40, a nucleic acid or nucleic acid set that is a vector or vector set. Claim 42 In paragraph 41, a nucleic acid or nucleic acid set in which a vector or vector set is an expression vector or expression vector set. Claim 43 A host cell comprising the nucleic acid or set of nucleic acids presented in any one of paragraphs 40 to 42. Claim 44 A method for producing an anti-TfR1 antibody comprising: (iii) culturing the host cells of claim 43 under conditions that allow the expression of the anti-TfR1 antibody; and (iv) collecting the anti-TfR1 antibody produced thereby.

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