Oligonucleotide conjugates targeted to the transferrin receptor

Abstract

Provided herein are conjugates comprising a protein that binds to the transferrin receptor and an oligonucleotide capable of modulating expression of a target gene or sequence, as well as methods of use thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 217,743, filed July 1, 2021, U.S. Provisional Application No. 63 / 298,193, filed January 10, 2022, and U.S. Provisional Application No. 63 / 333,449, filed April 21, 2022, each of which is incorporated by reference herein.

[0002] Sequence Listing The sequence listing in file 582256_SeqListing.xml is 574 kilobytes, was created on June 29, 2022, and is incorporated herein by reference. [Background technology]

[0003] In vivo delivery of nucleic acid-based molecules, such as antisense oligonucleotides or RNAi agents, often requires specific targeting to reach specific tissues or cell types. In particular, delivery to non-liver tissues remains an obstacle, limiting the use of such therapies. Delivery of oligonucleotides to the central nervous system (CNS) represents a distinct challenge due to the blood-brain barrier (BBB). One means of delivering oligonucleotides to the CNS is by intrathecal delivery. However, intrathecal delivery is invasive, carries a high risk of side effects, and often leads to uneven distribution.

[0004] Thus, there is a continuing need for new and improved methods for delivering nucleic acid-based therapeutics in vivo, particularly to CNS tissues. Summary of the Invention

[0005] Thus, certain embodiments described herein provide conjugates comprising at least one oligonucleotide (e.g., an antisense oligonucleotide (ASO) or an RNA interference (RNAi) agent) and at least one protein engineered to bind to the transferrin receptor (TfR). In certain aspects, the protein contained within such conjugates comprises a modified constant domain with a substitution that creates a TfR binding site. TfR is highly expressed at the blood-brain barrier (BBB), and TfR naturally transports transferrin from the blood into the brain. Because these proteins bind to TfR, they can also be transported across the BBB, and these proteins can be further utilized to transport bound oligonucleotides across the BBB. This approach can greatly improve the brain uptake and / or biodistribution of these therapeutic agents, and thus is extremely useful for treating disorders and diseases where delivery to the brain is advantageous. The conjugates described herein can also be used to enhance the delivery of oligonucleotides, such as ASOs and RNAi, to certain peripheral tissues, such as peripheral tissues that express TfR (e.g., muscle). Also disclosed are polypeptides and proteins that contain modifications that add conjugation sites or facilitate conjugation, including cysteine ​​substitutions as described herein.

[0006] Thus, in certain embodiments, the subject matter described herein is directed to a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein that contains an engineered binding site that specifically binds to the transferrin receptor (TfR) with an affinity of about 3 nM to about 600 nM; each y is independently at least 1 (e.g., 1, 2, 3, or 4); n is at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0007] In certain embodiments, the subject matter described herein is an Fc polypeptide dimer, comprising: (a) a first Fc polypeptide comprising a modified constant domain that specifically binds to the transferrin receptor; (b) a second Fc polypeptide dimerized to the first Fc polypeptide of (a); and or a Fab-Fc dimeric fusion thereof, The Fab-Fc dimeric fusion further comprises a first Fab and a second Fab, The Fc polypeptide dimer, or Fab-Fc dimer fusion, contains one or more cysteine ​​substitutions.

[0008] In certain embodiments, the subject matter described herein is an Fc polypeptide dimer, comprising: (a) a first Fc polypeptide comprising a modified constant domain that specifically binds to the transferrin receptor; (b) a second Fc polypeptide dimerized to the first Fc polypeptide of (a); and The present invention relates to an Fc polypeptide dimer comprising The first and / or second Fc polypeptide comprises one or more substitutions selected from the group consisting of N297A and N297G.

[0009] In certain embodiments, the present disclosure is directed to a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising a transferrin receptor (TfR) binding polypeptide described herein; each y is independently at least 1 (e.g., 1, 2, 3, or 4); n is at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0010] In one embodiment, the proteins described herein comprise a modified constant domain that binds (e.g., specifically binds) TfR.

[0011] In one aspect, the proteins described herein further comprise one or more modification sites that facilitate the attachment of P to each L. Thus, certain embodiments provide a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein that contains 1) a modified constant domain that specifically binds to the transferrin receptor, and 2) one or more modification sites that facilitate binding of P to each L; each y is independently at least 1 (e.g., 1, 2, 3, or 4); n is at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0012] In certain aspects, two or more oligonucleotides are attached to the protein, for example, through the attachment of two or more linking groups. Thus, certain embodiments provide a conjugate of formula I comprising two or more oligonucleotides, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1; n is 2 or more (e.g., 2 or 4).

[0013] In certain other aspects, two or more oligonucleotides are attached to a single linking group. Thus, certain embodiments provide a conjugate of formula I comprising two or more oligonucleotides, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1, and at least one y is 2 or greater; n is at least 1.

[0014] As described herein, the TfR binding affinity of the protein (P) can vary. Thus, certain embodiments provide low affinity (e.g., about 3 nM to about 600 nM) transferrin receptor binding conjugates of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein that includes a modified constant domain that specifically binds to the transferrin receptor with low affinity (e.g., about 40 nM to 1200 nM); each y is independently at least 1; n is at least 1.

[0015] Certain embodiments provide conjugates of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; Each L is [ka] [ka] is a linking group independently selected from the group consisting of During the ceremony, Each A is independently (C 1 -C 15 ) alkyl, Each D is -(CH 2 -CH 2 -O) m - and each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1; n is at least 1.

[0016] As described herein, the protein (P) may or may not include a Fab fragment or portion thereof. For example, in certain embodiments, the protein does not include a Fab fragment or portion thereof. In other embodiments, the protein includes one or more non-targeting Fab fragments or portions thereof. Thus, certain embodiments provide a conjugate of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising a modified constant domain that specifically binds to the transferrin receptor, the protein not comprising a Fab fragment or portion thereof; each y is independently 1 or greater; n is 1 or greater. In certain embodiments, the size of such conjugates is greater than 50 kDa. In some embodiments, P does not include the antigen-binding portion or variable domain portion of a Fab fragment. In some embodiments, P includes an Fc dimer fusion but does not include an antibody antigen-binding domain or an antibody variable domain.

[0017] Another embodiment provides a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising 1) a modified constant domain that specifically binds to the transferrin receptor, and 2) one or more non-targeting Fab fragments or portions thereof; each y is independently 1 or greater; n is 1 or greater.

[0018] Certain embodiments also provide a conjugate of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; Each L is independently [ka] In the formula, A is (C 1 -C 15 ) alkyl, P is a protein comprising a Fab-Fc dimeric fusion, comprising 1) a modified constant domain that specifically binds to the transferrin receptor with an affinity of about 100 nm, and 2) one or more modification sites that facilitate binding of P to each L and are selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering, or A114C according to Kabat numbering; each y is independently at least 1; n is at least 1.

[0019] In certain aspects, the modified constant domain is a modified CL domain. In some embodiments, the modified constant domain is a modified CH1 domain. In some embodiments, the modified constant domain is a modified CH2 domain. In some embodiments, the modified constant domain is a modified CH3 domain. In one aspect, the protein described herein comprises a modified CH3 domain that specifically binds to a transferrin receptor, the modified CH3 domain comprising 4, 5, 6, 7, 8, or 9 substitutions at a set of amino acid positions including 157, 159, 160, 161, 162, 163, 186, 189, and 194, the substitutions and positions being determined with reference to amino acids 114-220 of SEQ ID NO: 1. In some embodiments, the modified CH3 domain further comprises 1, 2, 3, or 4 substitutions at positions including 153, 164, 165, and 188. In one embodiment, the modified CH3 domain further comprises 1, 2, 3, 4, 5, 6, or 7 substitutions at positions including 153, 164, 165, 187, 188, 197, and 199.

[0020] In certain embodiments, the modified constant domain has, numbered relative to SEQ ID NO:1, at position 153, Trp, Tyr, Leu, Gln, or Glu; at position 157, Leu, Tyr, Met, Val, Phe, or Trp; at position 159, Leu, Thr, His, Pro, or Phe; at position 160, Val, Pro, or an acidic amino acid; at position 161, Trp; at position 162, Val, Ser, Ala, or Gly; at position 163, Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Thr, or Glu; and at position 164, Ser, Thr, Gln, Phe, Tyr, or Val. , 165 is Gln, Phe, or His; 186 is Glu, Ala, Ser, Leu, Thr, Pro, or Asp; 187 is Lys, Arg, Gly, or Pro; 188 is Glu or Ser; 189 is Thr, Asn, or an acidic amino acid; 194 is Trp, Tyr, His, or Phe; 197 is Ser, Thr, Glu, Lys, or Trp; and 199 is Ser, Trp, Gly, Cys, Pro, or Met.

[0021] In certain embodiments, the protein comprises a first Fc polypeptide or Fab-Fc fusion comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 279, 281, 361-366, 491-494, 702-718, 632, 645-649, 738-746, 804, and a second Fc polypeptide or Fab-Fc fusion comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 557-561, 627-631, 635-644, 719 and a second Fc polypeptide or Fab-Fc fusion comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of sequences 1 to 723, 724 to 731, 733 to 737, and 810, wherein the first Fc polypeptide or Fab-Fc fusion and / or the second Fc polypeptide or Fab-Fc fusion comprise one or more modification sites that promote binding of P to each L. In a specific embodiment, the first Fc polypeptide or Fab-Fc fusion comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser or Ala at position 162, Asn at position 163, Thr or Ser at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO:1.

[0022] In certain embodiments, the protein is an Fc polypeptide dimer or Fab-Fc dimer fusion comprising a first Fc polypeptide or Fab-Fc fusion and a second Fc polypeptide or Fab-Fc fusion, wherein the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362 and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 702 and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 708 and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 709. the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO:718 and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO:559; the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO:362 and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO:560; the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO:362 and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO:561; or the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:804 and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:810.

[0023] Certain embodiments also include Also provided is a first Fc polypeptide or Fab-Fc fusion comprising 1) a modified constant domain that specifically binds to the transferrin receptor, and 2) one or more cysteine, alanine, or glycine substitutions (e.g., S239C, S442C, A330C, K149C (light chain), T289C, N297A and / or N297G according to EU numbering, and / or A114C according to Kabat numbering).

[0024] Certain embodiments also include (a) a first Fc polypeptide comprising a modified constant domain that specifically binds to the transferrin receptor; (b) a second Fc polypeptide capable of dimerizing with the first Fc polypeptide of (a); and An Fc polypeptide dimer comprising: Also provided are Fc polypeptide dimers or Fab-Fc dimer fusions thereof, wherein the first and / or second Fc polypeptide comprises one or more cysteine ​​substitutions or the first and / or second Fab-Fc fusion comprises one or more cysteine ​​substitutions (e.g., S239C, S442C, A330C, K149C, A114C, and T289C).

[0025] Another aspect is (a) a first Fc polypeptide comprising a modified constant domain that specifically binds to the transferrin receptor; (b) a second Fc polypeptide capable of dimerizing with the first Fc polypeptide of (a); and An Fc polypeptide dimer comprising: The Fc polypeptide dimer is provided, wherein the first and / or second Fc polypeptide comprises one or more substitutions selected from the group consisting of N297A and N297G.

[0026] In a specific embodiment, the first and second Fc polypeptides are each linked to a non-targeting Fab fragment or portion thereof to generate a Fab-Fc dimeric fusion.

[0027] A non-targeting Fab (NTF) does not specifically bind to an epitope that naturally occurs in a subject. In some embodiments, the NTF comprises a non-binding variable region (NBVR). The NBVR comprises a light chain variable domain and a heavy chain variable domain, and does not specifically bind to an epitope that naturally occurs in a subject. The NBVR can be, but is not limited to, an scFv.

[0028] In some embodiments, the NBVR comprises three Kabat heavy chain CDRs and three Kabat light chain CDRs from NBVR1 or NBVR2. In some embodiments, the NBVR comprises CDR-H1, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 from NBVR1 or NBVR2, and CDR-H2 comprising SEQ ID NO:869.

[0029] In some embodiments, the NBVR does not specifically bind to an epitope naturally occurring in a subject and comprises three light chain CDRs and three heavy chain CDRs, each CDR having at least 90% sequence identity or 100% sequence identity to the corresponding CDRs from the heavy and light chain variable regions of NBVR1 or NBVR2. The heavy chain CDRs CDR-H1, CDR-H2, and CDR-H1 from NBVR1 comprise SEQ ID NOs: 825, 827, or 869, and 829, respectively. The light chain CDRs CDR-L1, CDR-L2, and CDR-L1 from NBVR1 comprise SEQ ID NOs: 819, 821, and 823, respectively. The heavy chain CDRs CDR-H1, CDR-H2, and CDR-H1 from NBVR2 comprise SEQ ID NOs: 826, 828, or 869, and 829, respectively. The light chain CDRs, CDR-L1, CDR-L2, and CDR-L1 from NBVR2, comprise SEQ ID NOs: 820, 822, and 824, respectively. In some embodiments, CDR-H2 of NBVR1 or NBVR2 is replaced with SEQ ID NO:869.

[0030] In some embodiments, the NBVR comprises a heavy chain variable region comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 837, and a light chain variable region comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 832. In some embodiments, the NBVR comprises a heavy chain variable region comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 853, and a light chain variable region comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 851.

[0031] In some embodiments, the NBVR comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:837 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:832, or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:853 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:851.

[0032] In some embodiments, the NTF comprises a heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 838, 839, 840, 841, 844, 845, 846, 847, 854, 855, 856, 857, 859, 860, 861, and 862, and a light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 833, 835, 850, and 852.

[0033] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:833, the heavy chain can comprise the amino acid sequence of SEQ ID NO:838, 839, 840, or 841.

[0034] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:833, the heavy chain can comprise the amino acid sequence of SEQ ID NO:844, 845, 846, or 847.

[0035] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:835, the heavy chain can comprise the amino acid sequence of SEQ ID NO:838, 839, 840, or 841.

[0036] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:835, the heavy chain can comprise the amino acid sequence of SEQ ID NO:844, 845, 846, or 847.

[0037] If the NTF light chain comprises the amino acid sequence of SEQ ID NO: 850, the heavy chain can comprise the amino acid sequence of SEQ ID NO: 854, 855, 856, or 857.

[0038] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:850, the heavy chain can comprise the amino acid sequence of SEQ ID NO:859, 860, 861, or 862.

[0039] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:852, the heavy chain can comprise the amino acid sequence of SEQ ID NO:854, 855, 856, or 857.

[0040] If the NTF light chain comprises the amino acid sequence of SEQ ID NO:852, the heavy chain can comprise the amino acid sequence of SEQ ID NO:859, 860, 861, or 862.

[0041] Also described are nucleic acids encoding the heavy and / or light chains of any of the NBVRs described herein. In some embodiments, the nucleic acid encodes a heavy chain of an NBVR comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identity to SEQ ID NO: 865 or 867. In some embodiments, the nucleic acid encodes a light chain of an NBVR comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% identity to SEQ ID NO: 864 or 866.

[0042] In certain embodiments, an Fc polypeptide, an Fc polypeptide dimer, a Fab-Fc fusion, or a Fab-Fc fusion dimer is incorporated into a conjugate of Formula (I), as described herein.

[0043] Certain embodiments also provide a pharmaceutical composition comprising a conjugate described herein and a pharma- ceutically acceptable carrier.

[0044] In certain embodiments, the subject matter described herein relates to a method for targeting delivery of an oligonucleotide to muscle and / or brain tissue of a patient, comprising administering to the subject any of the conjugates or pharmaceutical compositions comprising the conjugates described herein.

[0045] In certain embodiments, the subject matter described herein provides a method of modulating gene expression in a brain cell or a plurality of brain cells, comprising: (i) a protein that specifically binds to TfR; (ii) an oligonucleotide; administering to a patient a conjugate comprising The method relates to a method in which the protein binds with low affinity to the TfR and the conjugate is transported across the BBB, where within the cell the oligonucleotide modulates expression of a target gene.

[0046] In certain embodiments, the subject matter described herein provides a method of delivering an oligonucleotide to a deep brain region, comprising: (i) a protein that specifically binds to TfR; (ii) an oligonucleotide; administering to a patient a conjugate comprising The method relates to a method in which the protein binds with low affinity to TfR and the conjugate is transported across the BBB and within brain cells in deep brain regions where the oligonucleotide modulates expression of a target gene.

[0047] In certain embodiments, the subject matter described herein provides a method of delivering antisense oligonucleotides across a brain region, comprising: (i) a protein that specifically binds to TfR; (ii) an oligonucleotide; administering to a patient a conjugate comprising The method relates to a method in which the protein binds with low affinity to the TfR and the conjugate is transported across the BBB and within cells of the brain region, the oligonucleotide modulates expression of a target gene.

[0048] In certain embodiments, the subject matter described herein relates to a method of generating a neuronal cell having reduced expression of a target gene, comprising delivering an oligonucleotide to a neuronal cell, where the oligonucleotide is transported across the BBB as part of a conjugate with a protein that binds to TfR with low affinity, where the oligonucleotide reduces the expression level of the target gene.

[0049] In certain embodiments, the subject matter described herein relates to a method of modifying a neuronal cell to reduce target gene expression, comprising delivering an oligonucleotide to the neuronal cell, where the oligonucleotide is transported across the BBB as part of a conjugate with a protein that binds to TfR with low affinity, where the oligonucleotide reduces the expression level of the target gene.

[0050] Certain embodiments provide a method for transcytosis of an oligonucleotide (e.g., an ASO or an RNAi agent) across the endothelium, the method comprising contacting the endothelium (e.g., the blood-brain barrier) with a conjugate described herein.

[0051] Certain embodiments provide a method of transporting an oligonucleotide (e.g., an ASO or an RNAi agent) across the blood-brain barrier (BBB) ​​of a patient, the method comprising administering to the patient a conjugate described herein. For example, certain embodiments provide a method of transporting an oligonucleotide across the BBB of a patient, the method comprising administering to the patient a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein that contains 1) a modified constant domain that specifically binds to the transferrin receptor, and 2) one or more modification sites that facilitate binding of P to each L; each y is independently at least 1; n is at least 1; The method is provided.

[0052] Certain embodiments also provide a method of transporting an oligonucleotide across the BBB in a patient, comprising administering to the patient a conjugate of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; Each L is independently [ka] In the formula, A is (C 1 -C 15 ) alkyl, P is a protein comprising a Fab-Fc dimeric fusion, comprising: 1) a modified constant domain that specifically binds to the transferrin receptor with an affinity of about 100 nM; and 2) one or more modification sites that facilitate binding of P to each L and are selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering, and A114C according to Kabat numbering; each y is independently at least 1; The method further comprises administering to the patient a conjugate comprising: n is at least 1; In certain embodiments, the conjugate is administered intravenously to the patient; In certain embodiments, the oligonucleotide is distributed throughout the central nervous system; In certain embodiments, the oligonucleotide is distributed across brain regions; In certain embodiments, the oligonucleotide is distributed to deep brain regions; In certain embodiments, the oligonucleotide is distributed to the spinal cord; In certain embodiments, the conjugate is administered intravenously to the patient; In certain embodiments, the oligonucleotide modulates expression of a target gene; In certain embodiments, the modulation of target gene expression is gene knockdown or gene knockout.

[0053] Certain embodiments provide conjugates described herein that are used to transport an oligonucleotide (e.g., an ASO or an RNAi agent) across the blood-brain barrier (BBB) ​​of a patient.

[0054] Certain embodiments provide for the use of a conjugate described herein in the preparation of a medicament for transporting an oligonucleotide (e.g., an ASO or RNAi agent) across the blood-brain barrier (BBB) ​​of a patient.

[0055] Certain embodiments provide a method of modulating expression of a target gene in a patient, comprising administering to the patient an effective amount of a conjugate described herein. In particular, certain embodiments provide a method of modulating expression of a target gene in a cell in the brain of a patient, comprising administering to the patient a conjugate described herein. For example, certain embodiments provide a method of modulating expression of a target gene in a cell in the brain of a patient, comprising administering to the patient a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein that contains 1) a modified constant domain that specifically binds to the transferrin receptor, and 2) one or more modification sites that facilitate binding of P to each L; each y is independently at least 1; and n is at least 1. Certain embodiments also provide a method of modulating expression of a target gene in a cell in the brain of a patient, comprising administering to the patient a conjugate of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; Each L is independently [ka] In the formula, A is (C 1 -C 15 ) alkyl, P is a protein comprising a Fab-Fc dimeric fusion, comprising: 1) a modified constant domain that specifically binds to the transferrin receptor with an affinity of about 100 nM; and 2) one or more modification sites that facilitate binding of P to each L and are selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering, and A114C according to Kabat numbering; each y is independently at least 1; wherein n is at least 1.

[0056] In certain embodiments, the conjugate is administered to the patient intravenously.

[0057] In certain embodiments, the cell in the brain is a neuron, an endothelial cell, an oligodendrocyte, an astrocyte, or a microglia.

[0058] In certain embodiments, such methods modulate expression of a target gene in a plurality of cells in the brain of a patient. In certain embodiments, some cells of the plurality of cells are a single type of cell. In other embodiments, the plurality of cells comprises different types of cells. For example, in certain embodiments, the plurality of cells comprises at least two, three, four, or five cell types selected from the group consisting of neurons, endothelial cells, oligodendrocytes, astrocytes, and microglia. In certain embodiments, the neurons are excitatory neurons. In certain embodiments, the neurons are inhibitory neurons.

[0059] In certain embodiments, the modulation of target gene expression is a gene knockdown or gene knockout.

[0060] Certain embodiments provide a conjugate as described herein for use in modulating expression of a target gene in a patient.

[0061] Certain embodiments provide for the use of a conjugate as described herein in the preparation of a medicament for modulating expression of a target gene in a patient.

[0062] Certain embodiments also include a method of administering an effective amount of an oligonucleotide to the CNS of a patient, comprising administering to the patient a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1; n is at least 1; The method provides that the administered oligonucleotide modulates gene expression throughout the CNS.

[0063] Certain embodiments also provide a method of delivering an effective amount of an oligonucleotide to the CNS of a patient, comprising administering to the patient a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1; n is at least 1; The method provides that the administered oligonucleotide modulates gene expression throughout the CNS.

[0064] In certain embodiments, the administered oligonucleotides modulate gene expression throughout brain regions and spinal cord of a patient.

[0065] In certain embodiments, the brain region comprises a deep brain region.

[0066] In certain embodiments, the brain regions include the frontal lobe, the parietal lobe, the temporal lobe, the occipital lobe, and the cerebellum.

[0067] In certain embodiments, the brain region comprises endothelial cells, neurons, astrocytes, oligodendrocytes, and microglia.

[0068] In certain embodiments, the oligonucleotides modulate gene expression in endothelial cells, neurons, astrocytes, oligodendrocytes, and microglia.

[0069] In certain embodiments, an effective amount reduces gene expression by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to expression in the absence of administration of the oligonucleotide.

[0070] In certain embodiments, gene expression is reduced by at least about 50%.

[0071] In certain embodiments, gene expression is reduced by at least about 70%.

[0072] In certain embodiments, the conjugate is administered to the subject intravenously.

[0073] Certain embodiments provide a conjugate as described herein for use in a method of administering an effective amount of an oligonucleotide to the CNS of a patient, the method comprising administering to the patient a conjugate as described herein, wherein the administered oligonucleotide modulates gene expression throughout the CNS.

[0074] Certain embodiments provide a conjugate as described herein for use in a method of delivering an effective amount of an oligonucleotide to the CNS of a patient, the method comprising administering to the patient a conjugate as described herein, wherein the administered oligonucleotide modulates gene expression throughout the CNS.

[0075] Certain embodiments provide the use of a conjugate as described herein in the preparation of a medicament for administering an effective amount of an oligonucleotide to the CNS of a patient, by administering the medicament to the patient, wherein the administered oligonucleotide modulates gene expression throughout the CNS. [Brief description of the drawings]

[0076] [Figure 1] 1 is an exemplary embodiment of a conjugate described herein that includes a Fab-Fc dimer fusion. The Fc polypeptide on the left contains a modified CH3 domain that specifically binds to the transferrin receptor and a "knob" substitution for heterodimerization. The Fc polypeptide on the right contains a "hole" substitution for heterodimerization and is attached to an oligonucleotide via a linking group. [Diagram 2] FIG. 1 shows the time course of plasma in Hu Tfr mice between OTV variants from days 1 to 7 of a multiple-dose study. [Diagram 3] 1 shows huIgG in plasma of Hu Tfr mice at 24 hours across OTV variants in a multiple dose study. [Figure 4] Shown are unchanged drug and total ASO among OTV variants from days 1 to 7 of a multiple-dose study. [Diagram 5] FIG. 1 shows the plasma time course of unchanged drug and total ASO among OTV variants in wild-type mice. [Figure 6] The proportion of huIgG and unchanged drug among OTV variants in wild-type mice is shown. [Figure 7] A summary of tissue PK 24 hours after a single dose is shown. For each tissue type of "HuIgG", the bars represent from left to right RSV-ASO, S239C, OTR2 (1.23), OTR4 (2.5), low affinity, A330C, S442C, and SansFab. For each tissue type of "Unaltered Drug", the bars represent from left to right RSV-ASO, S239C, OTR2 (1.23), OTR4 (2.5), low affinity, A330C, S442C, and SansFab. For each tissue type of "Total ASO", the bars represent from left to right RSV-ASO, S239C, OTR2 (1.23), OTR4 (2.5), low affinity, A330C, S442C, SansFab, and naked ASO. [Figure 8]Figure 1 shows huIgG in tissues 24 hours post-dose in single dose studies normalized to plasma AUC. For each tissue type, the bars represent, from left to right, RSV-ASO, S239C, OTR2 (1.23), OTR4 (2.5), low affinity, A330C, S442C, and no Fab. Normalizing huIgG concentrations to plasma AUC increases comparability between OTV variants, suggesting that most of the concentration differences are due to plasma clearance kinetics. [Figure 9] huIgG, unchanged drug, percent unchanged drug, and total ASO in the CNS, i.e., cortex, spinal cord, for increased OTR / ASO burden variants in a single dose study are shown. [Figure 10] huIgG, unchanged drug, percent unchanged drug, and total ASO in the CNS, i.e., cortex, spinal cord, for low affinity variants in a single dose study are shown. [Figure 11] huIgG, unchanged drug, percent unchanged drug, and total ASO in the CNS, i.e., cortex, spinal cord, for conjugation site variants in a single dose study are shown. [Figure 12] huIgG, unchanged drug, percent unchanged drug, and total ASO in the CNS, i.e., cortex, spinal cord, for the SansFab (i.e., no Fab) variant in a single dose study are shown. [Figure 13] Tissue PK and knockdown of increased OTR / ASO loading variants in the CNS, i.e., cortex, spinal cord, 72 hours after the final dose of a multiple dose study. [Figure 14] Figure 1 shows tissue PK and knockdown of the low affinity variant in the CNS, i.e., cortex and spinal cord, 72 hours after the final dose of a multiple dose study. [Figure 15] Tissue PK and knockdown of conjugation site variants in the CNS, i.e., cortex and spinal cord, 72 hours after the final dose of a multiple dose study are shown. [Figure 16] 1 shows tissue PK and knockdown of SansFab (i.e., Fab-less) variant in the CNS, i.e., cortex, spinal cord, 72 hours after the last dose of a multiple dose study. [Figure 17] Peripheral tissue PK 72 hours after the last dose for huIgG, unchanged drug, and total ASO is shown. For each tissue type, the bars represent (left to right) RSV-ASO, S239C, OTR2 (1.23), OTR4 (2.5), low affinity, A330C, S442C, and no Fab. [Figure 18] Shown is knockdown in kidney, diaphragm, liver, and quadriceps 72 hours after the final dose. [Figure 19] Knockdown in the liver 72 hours after the final administration is shown by comparing OTR, TfR affinity, different conjugation sites, and Fab removal. [Figure 20] (A) shows the biodistribution of ASO in a single-dose study in non-human primates, and (B) shows knockdown of Malat1 in the frontal lobe of the brain. [Figure 21] ASO biodistribution following intrathecal versus OTV IV administration in non-human primates is shown via ASO immunofluorescence in hemibrain coronal sections (neocortex inset). [Figure 22] A shows brain dissected regions for snRNAseq analysis. B shows targeted knockdown across CNS cell types using single nucleus RNAseq analysis of mouse brains treated with multiple doses. [Figure 23] Plasma PK of OTV linker variants in wild-type mice: (A) total ASO, (B) total huIgG, (C) ASO to huIgG (linear), and (D) ASO to huIgG (log). [Figure 24] Shown are total ASO concentrations in the brain, spinal cord, and liver of OTV linker variants from a multiple-dose study using TfRms / hu knock-in mice. [Diagram 25] 1 shows Malat1 knockdown in brain, spinal cord, and liver of OTV linker variants from a multiple-dose study using TfRms / hu knock-in mice. [Figure 26]Knockdown efficiency (knockdown normalized to ASO concentration) of OTV linker variants in a multiple-dose study using TfRms / hu knock-in mice is shown. [Figure 27] 1 shows the plasma PK profiles of the S239C, A114C, and T289C conjugation site variants. [Figure 28] Shows brain ASO uptake for the S239C, A114C, and T289C conjugation site variants in single-dose and multiple-dose studies. [Figure 29] 1 shows hepatic ASO uptake for the S239C, A114C, and T289C conjugation site variants in single-dose and multiple-dose studies. [Diagram 30] 1 shows renal ASO uptake for the S239C, A114C, and T289C conjugation site variants in single-dose and multiple-dose studies. [Diagram 31] 1 shows brain Malat1 knockdown for the S239C, A114C, and T289C conjugation site variants in single and multiple dose studies. [Diagram 32] 1 shows liver and kidney Malat1 knockdown for the S239C, A114C, and T289C conjugation site variants in single and multiple dose studies. [Diagram 33] Shown are huIgG, unchanged drug, % unchanged drug, and total ASO in the CNS, i.e., cortex, spinal cord, for TfR affinity variants 24 hours after a single dose. [Diagram 34] Shown is knockdown of huIgG, unchanged drug, total ASO, and Malat1 in the CNS, i.e., cortex, spinal cord, for TfR affinity variants 72 hours after the last dose in a multiple dose study. [Diagram 35] MCV measurements for TfR variants are shown (A) 24 hours after a single dose and (B) 72 hours after the final dose in a multiple dose study. [Diagram 36](A) Tfr degradation, (B) Malat1 knockdown, and (C) ASO concentration of TfR affinity variants. [Figure 37] Shown are total ASOs in the brain and periphery in non-human primate studies. [Figure 38] ASO biodistribution following single and multiple doses of naked ASO IV vs. OTV IV in non-human primates is shown via ASO immunofluorescence in hemi-brain coronal sections (neocortex inset). [Figure 39] In a multiple-dose study in non-human primates, (A) IV delivery of naked ASO and (B) OTV-mediated delivery show Malat1 knockdown in the CNS and periphery. [Diagram 40] (A) Biodistribution of ASO immunofluorescence in the brain and lumbar spinal cord via intrathecal administration of ASO and (B) IV administration of OTV, and ASO levels to the lumbar spinal cord are shown. [Diagram 41] Malat1 knockdown in the CNS (frontal lobe, striatum, lumbar spine) and periphery (liver and kidney) in non-human primate studies. [Diagram 42] Shows Malat1 knockdown in the CNS and periphery in a mouse single-dose study via IV administration of (A) naked ASO and (B) OTV. [Diagram 43] ASO PK / PD. A: Total ASO concentration after a single dose, B: Total RNA knockdown. [Diagram 44] ASO PK / PD: A, total ASO concentration after multiple dose loading window; B, total RNA knockdown. [Diagram 45] A shows huIgG in the brain of mice administered affinity variants, and B shows the results of huIgG ELISA after brain capillary depletion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] V. Introduction Oligonucleotide therapies for CNS disorders caused by genetic abnormalities or increased protein accumulation have received increasing attention as an approach to modulate gene expression in such neurological disorders. The blood-brain barrier (BBB) ​​poses a challenge to the delivery of systemically administered oligonucleotides to their relevant sites of action within the CNS. Intrathecal (IT) delivery, in which drugs are administered directly into the cerebrospinal fluid (CSF) space, allows for bypass of the BBB. However, one limitation of this approach is that delivery of these oligotherapies directly to the CSF via an IT approach does not achieve uniform distribution throughout the CNS.

[0078] Here, we use human transferrin receptor binding molecules, termed oligonucleotide transport vehicles (OTVs), as a way to transport oligonucleotides across the BBB. OTVs delivered intravenously can cross the BBB to reach CNS cells and ultimately provide targeted knockdown in the brain and spinal cord. OTV molecules can provide knockdown across CNS regions including deep brain regions, entire brain regions including frontal, parietal, temporal, occipital lobes, and cerebellum, as well as across all types of CNS cells including endothelial cells, neurons, astrocytes, oligodendrocytes, and microglia. OTVs not only provided comparable muscle knockdown levels in the periphery to bivalent molecules such as antibodies, but also resulted in significantly higher levels of knockdown across the brain, spinal cord, and diaphragm. In summary, OTVs provide an interesting new potential therapeutic route for transporting oligonucleotides across the BBB via systemic administration. In addition, OTVs can be used for targeted delivery of oligotherapies to skeletal and cardiac muscles in neuromuscular disorders. This targeted delivery allows for the use of lower doses of oligotherapy compared to administration of oligotherapy alone, i.e., non-targeted delivery to the periphery.

[0079] Described herein are conjugates that include a protein that binds to the transferrin receptor (TfR) and at least one oligonucleotide (e.g., an ASO or an RNAi agent). In particular, provided herein are conjugates of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising a TfR-binding polypeptide as described herein; each y is independently at least 1; n is at least 1. In certain embodiments, y is 1 or more, 2 or more, 3 or more, or 4 or more. In certain embodiments, y is 1 or 2. In certain embodiments, y is 1 to 3. In certain embodiments, y is 1 to 4. In certain embodiments, y is 2 to 4. In certain embodiments, y is 3 or 4. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4.

[0080] In certain embodiments, at least one y is 2 or more (e.g., 2 or 4, e.g., 2). In certain embodiments, at least one L is separately attached to two oligonucleotides. In certain other embodiments, two or more oligonucleotides are attached to a single L. For example, the oligonucleotides can be linked to each other in series. In certain embodiments, L is attached to the 5' end of a first oligonucleotide and a second oligonucleotide is linked to the 3' end of the first oligonucleotide. In certain embodiments, the oligonucleotides can be linked via a nucleic acid linker or a non-oligonucleotide cleavable linker.

[0081] In certain embodiments, n is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more. In certain embodiments, n is 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In certain embodiments, n is 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In certain embodiments, n is 1 to 4. In certain embodiments, n is 2 to 4. In certain embodiments, n is 2 to 3. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6, and in certain embodiments, n is 7. In certain embodiments, n is 8.

[0082] In certain embodiments, the TfR binding protein comprises a constant domain or Fc polypeptide, and certain amino acids are modified to generate a specific binding site for TfR. Taking advantage of the fact that TfR is highly expressed on the blood-brain barrier (BBB) ​​and that TfR naturally transports transferrin from the blood into the brain, these proteins can be used to transport oligonucleotides, such as ASOs and RNAi agents, across the BBB. This approach can greatly improve the brain uptake and / or biodistribution of these therapeutic agents, and is therefore extremely useful for treating disorders and diseases where delivery to the brain is advantageous. TfR is also expressed in certain peripheral tissues, such as skeletal and cardiac muscles. Thus, the conjugates described herein can also be used to enhance delivery of oligonucleotides to certain peripheral tissues, such as peripheral tissues that express TfR (e.g., skeletal and cardiac muscles).

[0083] In some embodiments, the TfR binding protein comprises a CL domain, a CH1 domain, a CH2 domain, and / or a CH3 domain with substitutions at a particular set of amino acids to generate a binding site specific for the TfR. In some embodiments, the TfR binding protein comprises a CL domain with one or more substitutions to generate a binding site specific for the TfR. In some embodiments, the TfR binding protein comprises a CH1 domain with one or more substitutions to generate a binding site specific for the TfR. In some embodiments, the TfR binding protein comprises a CH2 domain with one or more substitutions to generate a binding site specific for the TfR. In some embodiments, the TfR binding protein comprises a CH3 domain with one or more substitutions to generate a binding site specific for the TfR. Exemplary CH2 and CH3 domains that specifically bind to the TfR are described, for example, in WO2018 / 152326, which is incorporated herein by reference in its entirety.

[0084] In one embodiment, the TfR binding protein comprises a CH3 domain with substitutions at a particular set of amino acids. Thus, in one embodiment, the transferrin binding protein comprises multiple substitutions at the set of amino acids at positions (i) 157, 159, 160, 161, 162, 163, 186, 189, and 194, or (ii) 118, 119, 120, 122, 210, 211, 212, and 213, numbered with reference to SEQ ID NO: 1. Any position from four positions to all positions of the set of amino acids may be substituted. For the purposes of this disclosure, unless otherwise specified, substitutions are determined with reference to SEQ ID NO: 1. Thus, if an amino acid differs from the corresponding amino acid at a position of SEQ ID NO: 1, that amino acid is considered to be a substitution, even if that amino acid is present at that position in a naturally occurring CH3 domain protein.

[0085] In further aspects, provided herein are therapeutic methods and methods of using the conjugates described herein to target oligonucleotides (e.g., ASOs or RNAi agents) to transferrin receptor-expressing cells, e.g., to deliver oligonucleotides to those cells, or to deliver molecules across endothelia, such as the blood-brain barrier.

[0086] VI.Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0087] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that the numerical value and reasonable deviations from that value known to one of ordinary skill in the art, e.g., ±20%, ±10% or ±5%, are within the intended meaning of the stated value.

[0088] The term "halo" means fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. refer to both straight and branched chain groups. However, when referring to individual radicals such as propyl, only the straight chain radical is included; the branched chain isomer, such as isopropyl, is specifically referred to.

[0089] The term “alkyl,” by itself or as part of another substituent, has the number of carbon atoms indicated, unless otherwise stated (i.e., C 1-6 means 1 to 6 carbons), a straight or branched chain hydrocarbon radical. Examples include (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkyl, and (C 3 -C 6Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the higher homologs and isomers.

[0090] The term "alkoxy" refers to an alkyl group linked to the remainder of the molecule via an oxygen atom ("oxy").

[0091] The term "alkylthio" refers to an alkyl group linked to the remainder of the molecule via a thio group.

[0092] The term "alkoxycarbonyl" as used herein refers to the group (alkyl)-OC(=O)-, where the term alkyl has the meaning defined herein.

[0093] The term "alkanoyloxy" as used herein refers to the group (alkyl)-C(=O)-O-, where the term alkyl has the meaning defined herein.

[0094] The term "aryloxy" refers to an aryl group linked to the remainder of the molecule through an oxygen atom (aryl-O-).

[0095] The term "heteroaryloxy" refers to a heteroaryl group linked to the remainder of the molecule through an oxygen atom (heteroaryl-O-).

[0096] As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0097] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) all-carbon ring (i.e., (C3-C6) carbocycle) having from three to six carbon atoms. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0098] The term "aryl" as used herein refers to a single all-carbon aromatic ring or a multiple condensed all-carbon ring system, where at least one of the rings is aromatic. For example, in certain embodiments, an aryl group has 6-20 carbon atoms, 6-14 carbon atoms, 6-12 carbon atoms, or 6-10 carbon atoms. Aryl includes phenyl radicals. Aryl also includes multiple condensed carbocyclic ring systems (e.g., ring systems containing 2, 3, or 4 rings) having about 9-20 carbon atoms, where at least one ring is aromatic and the other rings may or may not be aromatic (i.e., cycloalkyl). The rings of the multiple condensed ring system may be connected to each other via fused, spiro, and bridged bonds, where valence requirements permit. It should be understood that the point of attachment of the multiple condensed ring system may be at any position of the ring system, including the aromatic or carbocyclic portions of the ring, as defined above. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0099] The term "heterocycle" refers to a single saturated or partially unsaturated ring having at least one atom other than carbon in the ring, where the atom is selected from the group consisting of oxygen, nitrogen, and sulfur, and the term also includes multiple condensed ring systems having at least one such saturated or partially unsaturated ring, which are further described below. Thus, the term includes a single saturated or partially unsaturated ring (e.g., a 3-, 4-, 5-, 6-, or 7-membered ring) having about 1-6 carbon atoms and about 1-3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. The sulfur and nitrogen atoms can also be present in oxidized form. Exemplary heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, and piperidinyl. The term "heterocycle" also includes multiple condensed ring systems (e.g., ring systems containing 2, 3, or 4 rings), where a single heterocycle (as defined above) can be fused with one or more groups selected from cycloalkyl, aryl, and heterocycle to form a multiple condensed ring system. The rings of a multiple condensed ring system can be connected to each other through fused, spiro, and bridged bonds, where valence requirements permit. It should be understood that the individual rings of a multiple condensed ring system can be connected to each other in any order. It should also be understood that the point of attachment of a multiple condensed ring system (as defined above for heterocycle) may be at any position of the multiple condensed ring system, including the heterocyclic, aryl, and carbocyclic portions of the ring. In one embodiment, the term heterocycle includes 3-12 membered heterocycles. In one embodiment, the term heterocycle includes 3-7 membered heterocycles. In one embodiment, the term heterocycle includes 3-6 membered heterocycles. In one embodiment, the term heterocycle includes 4-6 membered heterocycles. In one embodiment, the term heterocycle includes 3- to 12-membered monocyclic or bicyclic heterocycles containing 1-3 heteroatoms. In one embodiment, the term heterocycle includes 3- to 6-membered monocyclic heterocycles containing 1-2 heteroatoms. In one embodiment, the term heterocycle includes 4- to 6-membered monocyclic heterocycles containing 1-2 heteroatoms.Exemplary heterocycles include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, and the like. ranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, 1,4-dioxane, and. [ka] These include, but are not limited to:

[0100] In one embodiment, the heterocycle is divalent, i.e., it can be attached to the remainder of the molecule or to a linking group at two positions in the heterocycle (-heterocycle-). 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1 -C 6 ) substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), and carboxy.

[0101] As used herein, a wavy line "" that intersects a bond in a chemical structure [ka] " indicates the point of attachment of the bond that the wavy bond crosses to the remainder of the molecule in a chemical structure.

[0102] "Transferrin receptor" or "TfR" as used herein refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 235. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee: Accession No. XP_003310238.1, rhesus monkey: NP_001244232.1, dog: NP_001003111.1, cow: NP_001193506.1, mouse: NP_035768.1, rat: NP_073203.1, and chicken: NP_990587.1). The term "transferrin receptor" also encompasses exemplary reference sequences encoded by genes at the transferrin receptor protein 1 chromosomal locus, e.g., allelic variants of the human sequence. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO:107.

[0103] A "new binding site" or "non-native binding site" refers to a site on a variant protein that specifically recognizes and binds an antigen, such as a transferrin receptor protein, where the unmodified or native protein does not specifically bind to the antigen. For example, a non-native antigen-binding site can be introduced by amino acid substitution, deletion, and / or insertion into the native protein sequence, resulting in specific recognition and binding at the mutated site.

[0104] As used herein, the term "constant domain" refers to the light chain constant region domain polypeptide (CL) and the CH1, CH2, and CH3 domain polypeptides of the heavy chain.

[0105] As used herein, the terms "CH1 domain", "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of an IgG antibody, a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme, a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme, and a CH1 domain polypeptide refers to the segment of amino acids from about position 118 to about position 215 according to the EU numbering scheme. CH1, CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the CH1 domain is numbered 1-98, the CH2 domain is numbered 1-110 and the CH3 domain is numbered 1-107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc polypeptide of an immunoglobulin. In the context of an IgG antibody, the Fc polypeptide refers to the segment of amino acids from about position 231 to about position 447, numbered according to the EU numbering scheme.

[0106] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide typically includes a constant region sequence including at least a CH2 domain and / or a CH3 domain, and may include at least a portion of a hinge region. Exemplary hinge region sequences or portions thereof are set forth in SEQ ID NOs: 232-234.

[0107] As used herein, the term "CL domain" refers to a light chain immunoglobulin constant domain. In the context of an IgG antibody, a kappa CL domain polypeptide refers to the segment of amino acids from about position 108 to about position 214, numbered according to the EU numbering scheme. Alternatively, kappa and lambda CL domains may be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where kappa CL domains are numbered 1-107 and lambda CL domains are numbered 1-106 according to the IMGT Scientific chart numbering (IMGT website).

[0108] As used herein, the term "Fab" or "Fab fragment" refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. Fab or Fab fragments may or may not include part or all of the antibody hinge region.

[0109] As used herein, the term "non-targeting Fab fragment" or "NTF" refers to a Fab fragment that does not specifically bind to an antigen through a heavy or light chain variable domain, or does not specifically bind to an antigen expressed in a particular mammal, such as a primate, e.g., human and non-human primates, or rodents, e.g., mice, or in a particular tissue within such a mammal, through a heavy or light chain variable domain. In certain embodiments, the Fab used in the Fab-Fc fusions or Fab-Fc dimer fusions described herein does not specifically bind to transferrin through its heavy or light chain variable domain. Non-limiting examples of non-targeting Fab fragments include (a) RSV (palivizumab) Fab fragments, which are non-targeting in mice and non-human primates, and (b) Fab fragments against dinitrophenyl hapten (DNP) (see Leahy, PNAS 3661-3665, 1988).

[0110] The terms "wild-type," "native," and "naturally occurring" with respect to a CH3 or CH2 domain are used herein to refer to a domain having a sequence that occurs in nature.

[0111] As used herein, the term "mutant" with respect to mutant polypeptides or mutant polynucleotides is used interchangeably with "variant". Variants relative to a particular wild-type CH3 or CH2 domain reference sequence may include naturally occurring allelic variants. A "non-natural" CH3 or CH2 domain refers to a variant or mutant domain of a native CH3 or CH2 domain polynucleotide or polypeptide that is not present in a natural cell and is created by genetic modification, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain that contains at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

[0112] As used herein, the term "modification site" refers to a specific position within a polypeptide that includes a mutant or variant with respect to a corresponding wild-type polypeptide (e.g., a wild-type CL, CH1, CH2, or CH3 domain). In certain embodiments, the mutant or variant is not naturally occurring. A modification site may include, for example, an insertion or substitution. As used herein, the term "substitution" refers to an alteration that replaces an amino acid with another amino acid. For example, a "cysteine ​​substitution" refers to replacing an amino acid with a cysteine.

[0113] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0114] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0115] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of natural α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0116] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0117] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a single chain of polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. As used herein, the term "protein" refers to a polypeptide or either a dimer (i.e., two) or multimer (i.e., three or more) of single-chain polypeptides. The single-chain polypeptides of a protein can be linked by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0118] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to alterations that result in the substitution of an amino acid with another amino acid that can be classified as having similar characteristics. Examples of classifications of conservative amino acid groups defined in this way include the "charged / polar group" which includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), the "aromatic group" which includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), and the "aliphatic group" which includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His, a "negatively charged subgroup" consisting of Glu and Asp, and a "polar subgroup" consisting of Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp, and a "phenyl subgroup" consisting of Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups, such as an "aliphatic non-polar subgroup" consisting of Val, Leu, Gly, and Ala, and an "aliphatic slightly polar subgroup" consisting of Met, Ser, Thr, and Cys. Examples of conservative mutation classifications include amino acid substitutions of amino acids within the aforementioned subgroups, such as substitution of Lys with Arg to maintain a positive charge, or vice versa; substitution of Glu with Asp to maintain a negative charge, or vice versa; substitution of Ser with Thr to maintain a free -OH, or vice versa; substitution of a free -NH2 In some embodiments, a hydrophobic amino acid is substituted for a naturally occurring hydrophobic amino acid, for example in the active site, to retain hydrophobicity.

[0119] The terms "identical" or percent "identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that are the same over a particular region or have a particular percentage (%) of amino acid residues that match, e.g., at least 60% identical, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, when compared and aligned using a sequence comparison algorithm or as measured by manual alignment and visual inspection for maximum correspondence over a comparison window or designated region.

[0120] In the sequence comparison of polypeptides, typically, one amino acid sequence serves as a reference sequence to be compared with candidate sequences. Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using public software with known algorithms to achieve maximum alignment. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for alignment to obtain maximum alignment can be determined by those skilled in the art. In the sequence comparison of polypeptide sequences for the purpose of this application, the standard protein BLAST of the BLASTP algorithm is used to align two protein sequences with default parameters.

[0121] The phrases "corresponding to," "determined in accordance with," or "numbered in accordance with," when used in the context of identifying a given amino acid residue in a polypeptide or protein sequence, refer to the position of the residue in a particular reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a given polypeptide "corresponds to" an amino acid in the region from amino acid 114 to amino acid 220 of SEQ ID NO:1 when that residue is optimally aligned with SEQ ID NO:1, when that residue and that amino acid in SEQ ID NO:1 are aligned.

[0122] The term "binding affinity" as used herein refers to the strength of a non-covalent interaction between two molecules, e.g., between a single binding site on a polypeptide / protein and the target to which it binds, e.g., the transferrin receptor. Thus, for example, unless otherwise stated or clear from the context, the term may refer to a 1:1 interaction between a polypeptide / protein and its target. Binding affinity is measured by the dissociation rate constant (k d ,time -1 ) to the binding rate constant (k a ,time -1 M -1 ) is the equilibrium dissociation constant (K D ) can be quantified by measuring the K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods, such as the Biacore™ system; equilibrium exclusion methods, such as KinExA®; and biolayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, "binding affinity" includes not only formal binding affinity, such as that reflecting a 1:1 interaction between a polypeptide / protein and its target, but also K, which may reflect strong binding. DThe term "low affinity" refers to an affinity sufficient for targeting and release. In embodiments, low affinity is about 600 nM to about 3 nM, or about 500 nM to about 3 nM, or about 400 nM to about 20 nM, or about 300 nM to about 30 nM, or about 200 nM to about 40 nM, or about 150 nM to about 50 nM, or about 3 nM, 4 nM, 5 nM, 5 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 15 ... M, 160nM, 170nM, 180nM, 190nM, 200nM, 210nM, 220nM, 230nM, 240nM, 250nM, 260nM, 270nM, 280nM, 290nM, 300nM, 310nM, 320nM, 330nM, 340nM, 350nM, 360nM, 370nM, 380nM, 390nM, 400nM, 410nM, 420nM, 430nM, 440nM, 450nM, 460nM, 470nM, 480nM, 490nM, 500nM.

[0123] The phrases "specifically bind" or "selectively bind" to a target, e.g., transferrin receptor, when referring to a protein comprising a modified constant domain as described herein, refer to a binding reaction in which the protein binds to the target with higher affinity, higher avidity, and / or longer duration than it binds to a structurally different target, e.g., a target that is not in the transferrin receptor family. In typical embodiments, the protein has at least 5-fold, 10-fold, 100-fold, 1000-fold, or 10,000-fold or more higher affinity for the transferrin receptor compared to an unrelated target when assayed under the same affinity assay conditions. In some embodiments, the modified CH3 domain specifically binds to an epitope on the transferrin receptor that is conserved between species, e.g., between non-human primates and human species. In some embodiments, the protein may bind exclusively to the human transferrin receptor.

[0124] As used herein, the terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) that contain a sugar moiety, a phosphate, and a nucleobase. Unless otherwise specified, the terms encompass both modified and unmodified nucleic acids.

[0125] As used herein, the term "nucleobase" refers to a nitrogen-containing compound that can be bound to a sugar moiety to form a nucleoside, in other words, a building block of a nucleotide. The ability of nucleobases to base pair and stack with each other directly influences the long-chain helical structure of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Nucleobases can be naturally occurring (i.e., adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)) or modified.

[0126] As used herein, the term "nucleoside" refers to a compound that includes a nucleobase and a sugar moiety (e.g., deoxyribose or ribose, or modified variants thereof). The term nucleoside includes both modified and unmodified nucleosides.

[0127] As used herein, the term "nucleotide" refers to a compound that contains a nucleobase, a sugar moiety, and one or more phosphate groups. The term nucleotide includes both modified and unmodified nucleotides.

[0128] As used herein, the term "internucleoside linkage" refers to a covalent bond between two nucleosides in an oligonucleotide. The nucleosides may be linked via a natural bond (i.e., a phosphodiester (PO) bond) or a modified bond.

[0129] The terms "chemical modification," "modification," or "modified" may refer to a chemical change in a compound when compared to its naturally occurring counterpart. For example, a nucleobase, a sugar moiety, or an internucleoside linkage may be chemically modified.

[0130] The terms "nucleotide sequence" and "nucleic acid sequence" and "nucleic acid strand" refer to a sequence of bases (purines and / or pyrimidines, or synthetic derivatives thereof) within a polymer of DNA or RNA, which may be single-stranded or double-stranded, optionally including synthetic, non-natural, or modified nucleotides, and / or backbone modifications (e.g., modified oligomers) that can be incorporated into the DNA or RNA polymer. The terms "oligo", "oligonucleotide", and "oligomer" are used interchangeably and refer to a sequence of purines and / or pyrimidines. For example, an oligonucleotide may comprise a chemically modified or unmodified nucleic acid molecule (RNA or DNA) having a length of, for example, less than about 200 nucleotides (e.g., less than about 100 or 50 nucleotides). An oligonucleotide may be, for example, a single-stranded DNA or RNA (e.g., ASO), a double-stranded DNA or RNA (e.g., small interfering RNA (siRNA)), a double-stranded DNA or RNA with a hairpin loop, or a DNA / RNA hybrid. In one embodiment, the oligonucleotides have a length ranging from about 5 to about 60 nucleotides, or from about 10 to about 50 nucleotides. In another embodiment, the oligonucleotides have a length ranging from about 5 to about 30 nucleotides, or from about 15 to about 30 nucleotides. In yet another embodiment, the oligonucleotides have a length ranging from about 18 to about 24 nucleotides.

[0131] The terms "modified oligo", "modified oligonucleotide", or "modified oligomer" may also be used interchangeably and refer to sequences that contain synthetic, non-natural, or altered base, sugar, and / or backbone modifications.

[0132] The oligonucleotides described herein can be synthesized using standard solid-phase or liquid-phase synthesis techniques known in the art. In certain embodiments, oligonucleotides are synthesized using solid-phase phosphoramidite chemistry with an automated synthesizer (US Pat. No. 6,773,885). Chemical synthesis of nucleic acids allows the production of nucleic acids with modified linkages, chimeric compositions, and various forms of non-standard bases or modified groups attached at selected locations throughout the length of the nucleic acid.

[0133] The term "complementary" as used herein refers to the broad concept of complementary base pairing between two nucleic acids aligned at antisense positions.When a nucleotide position in both molecules is occupied by a nucleotide that can normally base pair with each other, the nucleic acids are considered to be complementary to each other at that position.Thus, two nucleic acids are substantially complementary to each other when at least about 50%, at least about 60%, or at least about 80% of the corresponding positions in each molecule are occupied by nucleotides that normally base pair with each other (e.g., A:T (A:U in RNA) and G:C nucleotide pairs).

[0134] The terms "identical" or percent "identity" in the context of two or more nucleotide sequences refer to two or more sequences or subsequences that are the same over a particular region or have a particular percentage (%) of nucleotides that match, e.g., at least 60% identical, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0135] In the sequence comparison of oligonucleotides (e.g., to determine identity or complementarity), typically, one nucleotide sequence serves as a reference sequence to be compared with candidate sequences.Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using public software with known algorithms to achieve maximum alignment.Such programs include BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR).The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art.

[0136] As used herein, "hybridize" or "hybridization" refers to the pairing of complementary nucleotide sequences (e.g., between an antisense compound and its target nucleic acid, or an antisense strand and a sense strand). As used herein, "specifically hybridize" refers to the ability of a reference nucleic acid to hybridize to a nucleic acid molecule with greater affinity than it hybridizes to other nucleic acid molecules.

[0137] "Expression" refers to the transcription and / or translation of an endogenous gene, a heterologous gene or nucleic acid segment, or a transgene in a cell. For example, expression can refer to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression can also refer to the production of a protein.

[0138] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or its precursor.

[0139] The phrase "modulating expression of a target gene or sequence" refers to altering (e.g., increasing or decreasing) the expression of the target gene or sequence (e.g., via target degradation or translational inhibition). For example, this includes inhibiting, decreasing, or reducing expression of the target gene or sequence. This also includes alterations in alternative splicing, which may result in changes in the absolute or relative amounts of particular splice variants.

[0140] The terms "subject," "individual," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0141] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" may refer to any indication of successful treatment or improvement of an injury, disease, or condition, including any objective or subjective parameter, such as palliative, remission, improved patient survival, increased survival time or survival rate, alleviation of symptoms, or increasing the patient's tolerance to the injury, disease, or condition, slowing the rate of degeneration or decline, or improving the patient's physical or mental health. Additionally, "treating" or "treatment" may refer to modulation of target gene expression, such as gene knockdown or gene knockout. For example, expression of a target gene or sequence is inhibited or reduced, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, compared to expression in a control. Treatment or improvement of symptoms may be based on objective or subjective parameters. The effect of treatment can be compared to untreated individuals or pools of individuals, or to the same patient at different time points before or during treatment.

[0142] The term "pharmaceutical acceptable excipient" refers to non-active pharmaceutical ingredients, such as, but not limited to, buffers, carriers, or preservatives, that are biologically or pharmacologically compatible for use in humans or animals.

[0143] As used herein, a "therapeutic amount" or "therapeutically effective amount" of a drug is an amount of drug that treats, relieves, alleviates, or reduces the severity of symptoms of a disease in a subject. A "therapeutic amount" or "therapeutically effective amount" of a drug can improve a patient's survival, increase the length or rate of survival, reduce symptoms, make an injury, disease, or condition more tolerable, slow the rate of degeneration or decline, or improve the physical or mental well-being of a patient.

[0144] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired biological site of action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the proteins described herein are administered intravenously.

[0145] The term "MCV volume" is a standard measure and is referred to herein as an index of conjugate resistance.

[0146] VII. Variable "X": Oligonucleotide As described herein, one or more oligonucleotides (e.g., ASOs or RNAi agents) can be attached to a transferrin receptor binding protein described herein via "L" to form a conjugate.

[0147] The length of the oligonucleotide may vary, but in certain embodiments, the oligonucleotide is about 10 to about 60 nucleotides in length, or about 10 to about 30 nucleotides in length, or about 18 to about 30 nucleotides in length, or about 15 to about 25 nucleotides in length, or about 16 to about 20 nucleotides in length. Also, as described below, the oligonucleotide may contain certain chemical modifications, such as modified internucleoside linkages, modified nucleobases, modified sugars, or combinations thereof. In certain embodiments, one or more oligonucleotides are linked to the transferrin receptor binding protein (i.e., via a linking group "L"). In certain embodiments, two or more oligonucleotides are linked to the transferrin receptor binding protein (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 or more). In certain embodiments, one oligonucleotide is linked to the transferrin receptor binding protein. In certain embodiments, two oligonucleotides are linked to the transferrin receptor binding protein. In certain embodiments, four oligonucleotides are linked to the transferrin receptor binding protein.

[0148] In certain embodiments, one oligonucleotide is linked to a single linking group (L). In certain embodiments, two oligonucleotides are linked to a single linking group (L). For example, the oligonucleotides can be linked to each other in series. In certain embodiments, L is linked to the 5' end of the first oligonucleotide, and the second oligonucleotide is linked to the 3' end of the first oligonucleotide. In certain embodiments, the oligonucleotides can be linked via a nucleic acid linker or a non-oligonucleotide cleavable linker.

[0149] In other embodiments, the linking group is a branched linking group, where two or more oligonucleotides are separately attached to a single linking group (L) (ie, y is 2 or greater).

[0150] When more than one oligonucleotide binds to a TfR binding protein, the oligonucleotides may be the same or different. In certain embodiments, the oligonucleotides are the same.

[0151] ASO In one embodiment, each oligonucleotide is independently an ASO. The term "antisense oligonucleotide (ASO)" refers to a single strand of DNA-like or RNA-like molecule (e.g., modified nucleotides such as those described herein) that is complementary or partially complementary to a selected target polynucleotide sequence, such as mRNA. ASO can modify or regulate gene expression through multiple mechanisms, such as by modifying splicing (exon exclusion or exon inclusion), by recruiting RNase H to cause target degradation, by translation inhibition, and by small RNA inhibition, by binding to complementary target sequence.

[0152] Typically, ASOs are in the range of about 10 to 30 base pairs (bp) in length, but may be longer or shorter. For example, in certain embodiments, ASOs are about 10 to about 60 nucleotides in length, or about 10 to about 50 nucleotides in length, or about 10 to about 40 nucleotides in length. In certain embodiments, ASOs are about 10 to about 30 nucleotides in length, or about 12 to about 30 nucleotides in length, or about 14 to about 30 nucleotides in length, or about 15 to about 30 nucleotides in length, or about 16 to about 30 nucleotides in length, or about 17 to about 30 nucleotides in length, or about 18 to about 30 nucleotides in length, or about 18 to about 28 nucleotides in length, or about 18 to about 26 nucleotides in length, or about 18 to about 24 nucleotides in length, or about 15 to about 25 nucleotides in length, or about 16 to about 20 nucleotides in length. In certain embodiments, the ASO is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0153] Selection of antisense oligonucleotide sequences specific for a given target sequence is based on analysis of the selected target sequence and determination of a number of factors, such as secondary structure, Tm, binding energy, relative stability, etc. Additionally, antisense oligonucleotides may be selected based on their relative reduced ability to form dimers, hairpins, or other secondary structures that reduce or prohibit specific binding to the target mRNA in the host cell. Target regions of mRNA include regions at or near the AUG translation initiation codon, and sequences that are substantially complementary to the 5' region of the mRNA. Secondary structure analysis and target site selection considerations can be performed using software and algorithms known in the art, such as version 4 of the OLIGO primer analysis software (Molecular Biology Insights), and / or the BLASTN 2.0.5 algorithm software (Altschul et al, Nucleic Acids Res. 1997, 25(17):3389-402).

[0154] RNAi agents In certain other embodiments, each oligonucleotide is independently an RNAi agent (e.g., siRNA or shRNA). The term "RNA interference (RNAi) agent" refers to an RNA agent, or a molecule that can be cleaved into an RNA agent, that can inhibit the expression of a target gene or sequence (e.g., mRNA, tRNA, or viral RNA) in a sequence-specific manner (e.g., via Dicer / RISC). An RNAi agent can be single-stranded or double-stranded. If the RNAi agent is single-stranded, it can include 5' modifications, such as one or more phosphate groups or one or more analogs of a phosphate group. In one embodiment, the RNAi agent is double-stranded and includes a sense strand and an antisense strand (e.g., a short interfering RNA (siRNA)).

[0155] RNAi agents typically contain a region with sufficient homology to target gene and have sufficient length to mediate the downregulation of target gene.The complementarity between RNAi agents and target sequence must be sufficient to allow RNAi agents or their cleavage products to direct sequence-specific silencing.In certain embodiments, RNAi agents are at least partially complementary to target RNA or contain such a region.In other specific embodiments, RNAi agents are completely complementary to target RNA or contain such a region.

[0156] In some embodiments, the RNAi agent comprises an unpaired region at one or both ends of the molecule. For example, a double-stranded RNAi agent may have its strands paired with an overhang, e.g., a 5' and / or 3' overhang, such as an overhang of 1-3 nucleotides. In certain embodiments, the RNAi agent will comprise an unpaired overhang of 1, 2, 3 or 4 nucleotides in length at each end. The overhangs can occur by one strand being longer than the other, or by two strands of the same length being staggered.

[0157] The length of the duplex region in an RNAi agent can vary, but typically ranges from about 5 to about 30 nucleotides in length. In certain embodiments, the length of the duplex region is about 15-60, or about 15-50, or about 15-40, or about 15-30, or about 15-25, or about 19-25 nucleotides. In certain embodiments, the length of the duplex region is about 20-24 nucleotides, or about 21-23 nucleotides. In certain embodiments, the length of the duplex region is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more nucleotides.

[0158] As used herein, a "single-stranded RNAi agent" or "ssRNAi agent" is composed of a single molecule. It may include a duplex region formed by intrastrand pairing, for example, may be or may include a hairpin or panhandle structure. A single-stranded RNAi agent may be antisense with respect to a target molecule. A single-stranded RNAi agent may be long enough to enter RISC and participate in RISC-mediated cleavage of target mRNA. In certain embodiments, a single-stranded RNAi agent is at least 10, 15, 20, 25, 30, 35, 40, or 50 nucleotides in length. In certain embodiments, a single-stranded RNAi agent is less than 200, 100, 80, or 60 nucleotides in length.

[0159] Small hairpin RNA (shRNA) agents typically have a duplex region less than 200, 100, or 50 nucleotides in length. In certain embodiments, the length of the duplex region is within the range of about 15-60, or about 15-50, or about 15-40, or about 15-30, or about 15-25, or about 19-25 nucleotides in length. In certain embodiments, the length of the duplex region is about 17-23, or about 19-23, or about 20-23, or about 21-23, or about 19-21 nucleotides. In certain embodiments, the length of the duplex region is at least about 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The hairpin may have a single-stranded overhang or a terminal unpaired region. In certain embodiments, the overhang is 2-3 nucleotides in length. In some embodiments, the overhang is on the sense side of the hairpin, and in some embodiments, the overhang is on the antisense side of the hairpin.

[0160] As used herein, a "double-stranded RNAi agent" or "dsRNAi agent" comprises two or more strands that can form a double-stranded region within the molecule by interstrand hybridization (e.g., hybridization between a sense strand and an antisense strand). In certain embodiments, the RNAi agent is large enough that it can be cleaved by an endogenous molecule, such as Dicer, to produce smaller molecules.

[0161] In certain embodiments, the RNAi agent is a siRNA molecule comprising a sense strand and an antisense strand.

[0162] As used herein, the term "antisense strand" refers to a strand of an RNAi agent that is sufficiently complementary to a target polynucleotide, e.g., a target mRNA. In certain embodiments, the antisense strand of a double-stranded RNAi agent is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 60 nucleotides in length. In certain embodiments, the antisense strand of a double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides in length. In certain embodiments, the antisense strand is within the range of about 17-25 nucleotides, or about 19-23 nucleotides, or about 19-21 nucleotides in length.

[0163] As used herein, the term "sense strand" refers to a strand of an RNAi agent that is sufficiently complementary to the antisense strand. In certain embodiments, the sense strand of a double-stranded RNAi agent is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 60 nucleotides in length. In certain embodiments, the sense strand of a double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides in length. In certain embodiments, the sense strand is within the range of about 17-25 nucleotides in length, or about 19-23 nucleotides, or about 19-21 nucleotides in length.

[0164] In certain embodiments, the double-stranded portion of the double-stranded RNAi agent is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 60 nucleotides in length. In certain embodiments, the sense strand of the double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides in length. In certain embodiments, the sense strand is within the range of about 17-25 nucleotides, or about 19-23 nucleotides, or about 19-21 nucleotides in length.

[0165] In certain embodiments, the sense strand and the antisense strand can be selected so that the dsRNAi agent comprises an unpaired region at one or both ends of the molecule.Thus, the dsRNAi agent can comprise a paired sense strand and an antisense strand to comprise an overhang, for example, a 5' and / or 3' overhang that is 1, 2, 3, or 4 nucleotides in length.An overhang can occur because one strand is longer than the other, or because two strands of the same length are staggered.In certain embodiments, the dsRNAi agent comprises at least one 3' overhang.In certain embodiments, both ends of the dsRNAi agent comprise a 3' overhang (for example, 2 nucleotides in length).

[0166] The length of the duplex region in a dsRNAi agent can vary, but typically ranges from about 5 to about 30 nucleotides in length. In certain embodiments, the length of the duplex region ranges from about 5 to 60, about 15 to 60, or about 15 to 50, or about 15 to 40, or about 15 to 30, or about 15 to 25, or about 19 to 25 nucleotides in length. In certain embodiments, the length of the duplex region is about 17 to 23, or about 19 to 23, or about 20 to 23, or about 21 to 23, or about 19 to 21 nucleotides. In certain embodiments, the length of the duplex region is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more nucleotides.

[0167] Methods for producing RNAi agents, such as siRNA and shRNA, are known in the art and can be easily adapted to produce RNAi agents that target any polynucleotide sequence.In certain embodiments, RNAi agents are chemically synthesized.For example, oligonucleotides can be synthesized using a variety of techniques, such as those described in Usman et al., J.Am.Chem.Soc., 109:7845 (1987); Scaringe et al., Nucl.Acids Res., 18:5433 (1990); Wincott et al., Nucl.Acids Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol.Bio., 74:59 (1997).

[0168] Exemplary Oligonucleotide Modifications In certain embodiments, the oligonucleotides described herein may contain at least one nucleic acid modification, such as those selected from the group consisting of modified internucleoside linkages, modified nucleic acid bases, modified sugars, and combinations thereof. Such modifications may be used to modify pharmacokinetics (improved nuclease resistance resulting in extended half-life), pharmacodynamics (better affinity for target RNA), or endocytic uptake. However, many modifications prevent cleavage by RNase H, which is the desired mechanism of action for many ASOs. Thus, certain RNase H ASOs may be designed as chimeras, where different bases are mixtures of different chemicals, or as gapmers, where some modifications are placed in the "wings" and not the central base. In contrast, for RNAi agents and ASOs that are intended to modify splicing or translation of mRNA, there is no need to consider RNase H.

[0169] Thus, the oligonucleotides described herein may contain one or more nucleic acid modifications.In certain embodiments, the oligonucleotides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 or more modifications.

[0170] In certain embodiments, the oligonucleotides described herein contain one or more nucleotide modifications (e.g., to the nucleobase or sugar moiety). In certain embodiments, 25% or more of the nucleotides present in the oligonucleotide are modified. In certain embodiments, 50% or more of the nucleotides present in the oligonucleotide are modified. In certain embodiments, 75% or more of the nucleotides present in the oligonucleotide are modified. In certain embodiments, 100% of the nucleotides present in the oligonucleotide are modified.

[0171] In certain embodiments, the oligonucleotide comprises one or more nucleobase modifications. In certain embodiments, the oligonucleotide comprises one or more modifications to the sugar moiety (e.g., furanosyl containing substituents at the 2', 3', 4', and / or 5' positions). In certain embodiments, the substituted sugar moiety comprises a bicyclic sugar moiety.

[0172] In certain embodiments, the nucleic acid modification by the oligonucleotide is included in the pattern. In certain embodiments, the oligonucleotide is a gapmer. The modification pattern of a gapmer oligonucleotide generally has the formula 5'-Xa-Ya-Za-3', where Xa and Za are adjacent regions around the gap region Ya. In certain embodiments, the Ya region is a continuous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides that can recruit an RNAse, such as RNAse H. In certain embodiments, the Ya region comprises at least 8 DNA nucleotides. In certain embodiments, the gapmer binds to the target nucleic acid, at which point an RNAse can be recruited and then cleave the target nucleic acid. In certain embodiments, the Ya region is flanked at both 5' and 3' by regions Xa and Za that contain high affinity modified nucleotides, e.g., 1-6 modified nucleotides at each of Xa and Za. In certain embodiments, modified nucleotides are present in the 5' and 3' regions of the oligonucleotide, although certain modified nucleotides and / or modified linkages may or may not be present in the central portion of the molecule. In certain embodiments, modified nucleotides are present in the 5' and 3' regions of the oligonucleotide, and certain modified nucleotides are absent in the central portion of the molecule (e.g., LNA residues are absent in the central portion, but the central region may contain modified linkages such as PS linkages). In certain embodiments, Xa and Za each comprise three modified nucleotides. In certain embodiments, three modified nucleotides are arranged in tandem with each of Xa and Za.

[0173] Modified nucleosides / nucleotides are known in the art and include, but are not limited to, 2'-O methyl (2'OMe) residues, 2'O-methoxyethyl (MOE) residues, constrained nucleic acid residues (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE), peptide nucleic acid (PNA) residues, locked nucleic acid (LNA) residues, and 5'-methylcytidine residues (methylated cytosine residues) (see also Scoles, et al., Neurol Genet Apr 2019, 5(2)e323). In certain embodiments, the oligonucleotide comprises one or more MOE residues. In certain embodiments, the oligonucleotide comprises one or more OMe or F residues (e.g., 2'-F or 2'OMe). In certain embodiments, the oligonucleotide comprises one or more constrained residues (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE) and / or LNA residues. A nucleic acid is considered "locked" when a methylene bridge bond is formed between the 2'-oxygen and the 4'-carbon of the ribose sugar molecule. In certain embodiments, the oligonucleotide is morpholino (i.e., contains certain modifications to the sugar moiety). In certain embodiments, the oligonucleotides described herein contain one or more LNA residues and one or more 5'-methylcytidine residues.

[0174] In certain embodiments, the oligonucleotide comprises one or more modifications to the internucleoside backbone (i.e., the natural phosphodiester (PO) linkage is modified). In certain embodiments, such modifications are made, for example, to reduce nuclease activity. Thus, in certain embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more modified internucleoside linkages. In certain embodiments, 25% or more of the internucleoside linkages are modified. In certain embodiments, 50% or more of the internucleoside linkages are modified. In certain embodiments, 75% or more of the internucleoside linkages are modified. In certain embodiments, 100% of the internucleoside linkages present in the oligonucleotide are modified.

[0175] Backbone modifications are known in the art, and include, but are not limited to, phosphorothioate bond, phosphoramidate bond, and phosphorodiamidate bond.For example, in certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) internucleoside bonds in an oligonucleotide are replaced with phosphorothioate (PS) bonds.In certain embodiments, oligonucleotides contain a mixture of PO and PS bonds.In certain embodiments, oligonucleotides contain only PS bonds. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) internucleoside bonds in the oligonucleotide are replaced with phosphorodiamidate bonds. In certain embodiments, the oligonucleotide is a phosphorodiamidate morpholino (PMO).

[0176] In certain embodiments, the internucleoside linkages are stereorandom with respect to the chiral centers (Rp and Sp). In certain other embodiments, the Rp and Sp configurations in the oligonucleotide are optimized in a particular configuration.

[0177] In certain embodiments, the oligonucleotide is a gapmer comprising LNA and PS modifications. For example, in certain embodiments, the oligonucleotide is a gapmer having a modification pattern of the formula 5'-Xa-Ya-Za-3', where Xa and Za are adjacent regions surrounding the gap region Ya, each of which comprises three LNA modified nucleotides (e.g., three consecutive LNA modified nucleotides), and the gap region Ya comprises a PS linkage. In certain embodiments, the oligonucleotide further comprises one or more 5'-methylcytidine residues. In certain embodiments, the gap region Y a does not contain any LNA residues.

[0178] VIII. Variable "L": Linking Group In certain embodiments, L is a linking group that links each oligonucleotide (X) to a TfR binding protein (P). The linking group can be any group suitable for binding an oligonucleotide to a TfR binding protein.

[0179] The linking group may be attached to any region of the TfR binding protein, including the TfR binding polypeptide (e.g., the N-terminal region, the C-terminal region, or an amino acid within the protein, such as a cysteine ​​or glutamine residue), as long as the oligonucleotide does not interfere with the binding of the TfR binding protein to the transferrin receptor. Similarly, the linking group may be attached to any region of the oligonucleotide (e.g., the 5' end, the 3' end, or a nucleic acid residue within the molecule), as long as the TfR binding protein does not interfere with the function of the oligonucleotide (e.g., complementary binding to a target nucleic acid). For example, the linker may be attached to the oligonucleotide through any number of points that can be synthesized that are located throughout the oligo, such as the 3' or 5' terminal residues of the oligo, the sugar moiety, the base moiety, or a residue located within the backbone.

[0180] In certain embodiments, the linker is attached to the oligonucleotide at the 5'-terminal residue of the oligonucleotide. In certain embodiments, the linker is attached to the oligonucleotide at the 3'-terminal residue of the oligonucleotide. In certain embodiments, the linker is attached to the oligonucleotide at a residue within the oligonucleotide. In certain embodiments, the oligonucleotide is a double-stranded RNAi molecule, and the linker is attached to the sense strand (e.g., at the 5'- or 3'-terminal residue). In certain embodiments, the oligonucleotide is a double-stranded RNAi molecule, and the linker is attached to the antisense strand (e.g., at the 5'- or 3'-terminal residue). In certain embodiments, the oligonucleotide is an siRNA, and the linker is attached to the 3'-end of the sense strand. In certain embodiments, the 3'-end of the sense strand of the siRNA is modified with a C6 amine.

[0181] In certain embodiments, the linking group comprises a spacer. In certain embodiments, the spacer is a hydrophilic spacer. In certain embodiments, the hydrophilic spacer is polyethylene glycol (PEG).

[0182] In certain embodiments, the linking group is a homobifunctional linker or a heterobifunctional linker.

[0183] In some embodiments, the linking group is cleavable (e.g., a nuclease-cleavable linker, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Pat. No. 5,208,020). In certain embodiments, the linking group comprises one or more nucleotides (e.g., 1, 2, 3, or more) or one or more nucleosides (e.g., 1, 2, 3, or more). In certain embodiments, one or more nucleotides or one or more nucleosides are unmodified. In certain embodiments, the linking group comprises one or more nucleotides having an unmodified base, an unmodified sugar group, and / or an unmodified phosphate group. In certain embodiments, the linking group comprises one or more nucleosides having an unmodified base and / or an unmodified sugar group. In certain embodiments, the linking group comprises a nuclease linker, TCA. In certain embodiments, the TCA is modified with a C6 amine at the T position. In certain embodiments, the linking group does not comprise a TCA. In certain embodiments, the linking group is enzymatically cleavable. In certain embodiments, The linking group is cleavable by enzymes present in the central nervous system or muscle. In certain embodiments, a cleavable linking group is selected for a conjugate containing an ASO (e.g., to allow the ASO to dissociate from the remainder of the conjugate for transport to the nucleus). In certain embodiments, the cleavable linking group is a cleavable dipeptide linker. In certain embodiments, the cleavable dipeptide linker is a val-cit cleavable linking group or a val-ala cleavable linker. In certain embodiments, the cleavable linking group is an acid cleavable linker. In certain embodiments, the acid cleavable linker is a carbonate linker or a hydrazone linker. In certain embodiments, the cleavable linking group comprises a PEG spacer. In certain embodiments, the cleavable linking group is a disulfide, such as SPDP (succinimidyl 3-(2-pyridyldithio)propionate) or a lys-conjugated acid cleavable hydrazide.

[0184] In certain embodiments, the linking group is a non-cleavable linking group. In certain embodiments, the linking group is a covalent linking group. In certain embodiments, the covalent linking group is derivable from APN or acrylamide. In certain embodiments, the covalent linking group is a group -CH 2 CH 2 In certain embodiments, the covalent linking group comprises: [ka] The group represented by the formula: In certain embodiments, the covalent linking group can be derived from a haloacetamide, for example, bromoacetamide, chloroacetamide, iodoacetamide.

[0185] In certain embodiments, the linking group has the formula -(CH 2 ) 6 -NH-C 6 Contains an amine group.

[0186] In certain embodiments, the linking group is derivable from a maleimide. For example, in certain embodiments, the linking group is [ka] The group represented by the formula: In certain embodiments, the linking group may be attached to P (eg, to a sulfur atom at the modification site within P) at the valence marked with *.

[0187] In certain embodiments, the maleimide is a modified maleimide. In certain embodiments, the modified maleimide is an alkyl-, aryl-, cycloalkyl-, or exocyclic-maleimide. In certain embodiments, the linking group comprises a protected maleimide. For example, in certain embodiments, the linking group has the formula [ka] The protected maleimide includes a protected maleimide represented by the formula:

[0188] In certain embodiments, the protected maleimide is removed after the bioconjugation reaction.

[0189] In certain embodiments, the linking group is an autohydrolyzable linking group.

[0190] Certain specific non-limiting embodiments (abbreviated as linker embodiments LE1-LE42) are described below.

[0191] In linker embodiment LE1, the linking group has a molecular weight of about 20 daltons to about 5,000 daltons. In linker embodiment LE2, the linking group has a molecular weight of about 20 daltons to about 1,000 daltons. In linker embodiment LE3, the linking group has a molecular weight of about 20 daltons to about 200 daltons. In linker embodiment LE4, the linking group has a length of about 5 angstroms to about 60 angstroms.

[0192] In linker embodiment LE5, the linking group separates the peptide from the remainder of the conjugate of Formula I by a length of about 5 angstroms to about 40 angstroms, inclusive.

[0193] In linker embodiment LE6, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced with (-O-), (-NH-), (-S), an amino acid, a hydrazone (-C(R')=N=N(R')-), a nucleotide, or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are each independently selected from the group consisting of (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1-C 6 ) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), hydrazone (=N=N(R')-) carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently selected from the group consisting of H or (C 1 -C 6 ) alkyl.

[0194] In linker embodiment LE7, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced with (-O-), (-NH-), or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are each independently selected from the group consisting of (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1 -C 6 ) optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0195] In linker embodiment LE8, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced with (-O-), (-NH-), (-S), an amino acid, a hydrazone (-C(R')=N=N(R')-), a nucleotide, or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are selected from the group consisting of (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1 -C 6 ) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), hydrazone (=N=N(R')-) carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently selected from the group consisting of H or (C 1 -C 6 ) alkyl.

[0196] In linker embodiment LE9, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced with (-O-), (-NH-), or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are each independently selected from the group consisting of (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6) alkoxycarbonyl, (C 1 -C 6 ) optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0197] In linker embodiment LE10, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, the chain containing at least one of the following carbons: (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1 -C 6 ) optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0198] In linker embodiment LE11, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, the chain containing at least one of the following carbons: (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1 -C 6) optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0199] In linker embodiment LE12, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0200] In linker embodiment LE13, the linking group is a divalent, branched or unbranched, saturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0201] In linker embodiment LE14, the linking group is a divalent, unbranched, saturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0202] In linker embodiment LE15, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain contains one or more disulfide bonds.

[0203] In linker embodiment LE16, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain contains one or more hydrazone groups in the chain or attached to carbon atoms in the chain.

[0204] In linker embodiment LE17, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes one or more amino acids in the chain.

[0205] In linker embodiment LE18, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes a dipeptide in the chain.

[0206] In linker embodiment LE19, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes a dipeptide val-cit in the chain.

[0207] In linker embodiment LE20, the linking group comprises one or more nucleotides in the chain.

[0208] In linker embodiment LE21, the linking group comprises two or more nucleotides in the chain.

[0209] In linker embodiment LE22, the linking group comprises a trinucleotide group in the chain.

[0210] In linker embodiment LE23, the linking group is attached to two or more oligonucleotides (eg, in the case of compounds of formula (I), at least one "y" is greater than 1).

[0211] In linker embodiment LE24, only one linking group is attached to two or more oligonucleotides (eg, in the case of compounds of formula (I), one "y" is greater than 1).

[0212] In linker embodiment LE25, at least two linking groups are attached to two or more oligonucleotides (eg, for compounds of formula (I), at least two "y"s are greater than 1).

[0213] In linker embodiment LE26, at least two linking groups are attached to two oligonucleotides (eg, for compounds of formula (I), "y" is 2 and n is greater than 1).

[0214] In linker embodiment LE27, the linking group is attached to the oligonucleotide via a phosphate of the oligonucleotide (eg, attached to the 5' terminal residue).

[0215] In linker embodiment LE28, the linking group is attached to the oligonucleotide via a phosphorothioate of the oligonucleotide (eg, attached to the 5' terminal residue).

[0216] In linker embodiment LE29, the linking group comprises a polyethyleneoxy chain. In other embodiments of the invention, the polyethyleneoxy chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethyleneoxy repeat units.

[0217] In linker embodiment LE30, the linking group comprises a 5-membered divalent heterocycle.

[0218] In linker embodiment LE31, the linking group has the structure: [ka] wherein L' is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced with (-O-), (-NH-), (-S), an amino acid, a hydrazone (-C(R')=N=N(R')-), a nucleotide, or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are each independently selected from the group consisting of (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 ) alkoxycarbonyl, (C 1 -C 6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), hydrazone (=N=N(R')-) carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently selected from the group consisting of H or (C 1 -C 6 ) alkyl, where in formula (I) the valence marked with * is attached to P and the valence marked with ** is attached to X. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2-25 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes one or more disulfide bonds. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2-25 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes one or more hydrazone groups in the chain or includes one or more hydrazone groups attached to a carbon atom in the chain. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2-35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes one or more amino acids in the chain. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2-35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain comprises a dipeptide in the chain. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated chain having 2-35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain comprises a dipeptide val-cit in the chain. In another embodiment, L' comprises one or more nucleotides. In another embodiment, L' comprises two or more nucleotides. In another embodiment, L' comprises a trinucleotide group. In another embodiment, L' comprises one or more nucleotides having an unmodified base, an unmodified sugar group, and / or an unmodified phosphate group.

[0219] In linker embodiment LE32, L' has the structure: [ka] In the formula, t is 1, 2, 3, 4, 5, 6, 7, or 8; z is 0, 1, 2, 3, 4, 5, 6, 7, or 8; R 1 , R 2 , and R 3 Each of is independently a nucleotide.

[0220] In linker embodiment LE33, L' has the structure: [ka]

[0221] In linker embodiment LE34, the linking group has the structure: [ka] wherein t is 1, 2, 3, 4, 5, 6, 7, or 8; and z is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0222] In linker embodiment LE35, the linking group has the structure: [ka] wherein t is 1, 2, 3, 4, 5, 6, 7, or 8, z is 0, 1, 2, 3, 4, 5, 6, 7, or 8, and in formula (I), the valence marked with * is attached to P and the valence marked with ** is attached to X. In certain embodiments, the valence marked with ** is attached to X via a phosphate of the oligonucleotide (e.g., attached to the 5' terminal residue).

[0223] In linker embodiment LE36, the linking group has the structure: [ka]

[0224] In linker embodiment LE37, the linking group has the structure: [ka] wherein in formula (I), the valence marked with * is attached to P and the valence marked with ** is attached to X. In certain embodiments, the valence marked with ** is attached to X via a phosphate of the oligonucleotide (e.g., attached to the 5' terminal residue). Thus, the A group in the linker structure can be, in embodiments, [ka] So, -O-PO 3 which can itself be covalently attached to an oligonucleotide.

[0225] In linker embodiment LE38, the linking group has the structure: [ka]

[0226] In the linker embodiment LE39, the linker is a peptide linker or is formed from a protein, peptide, or amino acid. For example, in certain embodiments, the linking group is a divalent radical formed from a protein. In other embodiments, the linking group is a divalent radical formed from a peptide. In other embodiments, the linking group is a divalent radical formed from an amino acid.

[0227] In linker embodiment LE40, the linking group can be configured to allow rotation of the oligonucleotide and the TfR binding protein relative to one another and / or to be resistant to digestion by proteases. In some embodiments, the linking group can be a flexible linker that includes amino acids such as, for example, Gly, Asn, Ser, Thr, Ala, etc. Such linking groups are designed using known parameters. For example, the linking group can have repeats, such as repeating Gly-Ser.

[0228] In the linker embodiment LE41, the linking group has or includes a formula selected from the group consisting of: [ka] [ka] During the ceremony, Each A is independently (C 1 -C 15 ) alkyl, Each D is -(CH 2 -CH 2 -O) m - and Each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0229] In linker embodiment LE42, the linking group has or includes a formula selected from the group consisting of: [ka] [ka]

[0230] In various embodiments, the conjugates can be made using well-known chemical crosslinkers and protocols. For example, there are numerous chemical crosslinkers known to those skilled in the art that are useful for crosslinking proteins with agents of interest. For example, the crosslinker is a heterobifunctional crosslinker that can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers allow for the design of more specific coupling methods for protein conjugation, thereby reducing the occurrence of undesired side reactions such as homoprotein polymers. A wide variety of heterobifunctional crosslinkers are known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analogue N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-iodoacetyl ... These include succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)-propionate]hexanoate (LC-SPDP). These crosslinkers with N-hydroxysuccinimide moieties can be obtained as N-hydroxysulfosuccinimide analogs, which generally have higher water solubility. Crosslinkers with disulfide bridges in the linking chain can also be synthesized instead as alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinkers, numerous other crosslinkers exist, including homobifunctional and photoreactive crosslinkers.Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethyl pimelimidate·2HCl (DMP) are examples of useful homobifunctional crosslinkers, and bis-[B-(4-azidosalicylamido)-ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinkers.

[0231] In embodiments, the linking group L comprises a moiety having the structure: [ka] where * indicates the point of attachment to the sulfur atom of the modification site in P, and specifically refers to any such linker described in this section and to the linker embodiments described herein.

[0232] In embodiments involving conjugates of formula (I) described herein, each L specifically refers to any of the linkers described in this section and linker embodiments described herein.

[0233] IX. Variable "P": Transferrin Receptor Binding Protein This section describes proteins that bind to the transferrin receptor and can be transported across the blood-brain barrier (BBB). Certain proteins that bind to the transferrin receptor are described herein and in WO2018 / 152326. Certain methods of making TfR-binding proteins are described in WO2018 / 152326, which is incorporated herein by reference for all purposes.

[0234] The TfR binding proteins described herein may have a variety of binding affinities. For example, in some embodiments, the proteins have affinities for TfR in the range of 1 pM to 10 μM. In some embodiments, the affinity for TfR is in the range of 1 nM to 5 μM, or 10 nM to 1 μM. In some embodiments, the proteins have affinities for TfR in the range of 3 nM, 4 nM, 5 nM, 5 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, 300 nM, 310 nM, 320 nM, 330 nM, 340 nM, 350 nM, 360 nM, 370 nM, 380 nM, 390 nM, 400 nM, 410 nM, 420 nM, 430 nM, 440 nM, 450 nM, 460 nM, 470 nM, 480 nM, 490 nM, 500 nM, 500 nM, 510 nM, 520 nM, 530 nM In some embodiments, the affinity for TfR is in the range of about 40 nM to about 1200 nM, 50 nM to about 500 nM, or about 75 nM to about 300 nM, or about 100 nM to about 250 nM. In some embodiments, the protein binds (e.g., specifically binds) to TfR with an affinity of about 40, 50, 80, 100, 130, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 nM. In some embodiments, the protein binds to TfR with an affinity of about 50 nM to about 300 nM, about 80 nM to about 300 nM, 100 nM to about 300 nM, or about 150 nM to about 250 nM, or about 200 nM to about 250 nM.In some embodiments, the protein binds to TfR with an affinity of about 40 nM to about 500 nM, about 50 nM to about 500 nM, about 50 nM to about 400 nM, about 50 nM to about 300 nM, about 50 nM to about 200 nM, about 50 nM to about 100 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, or about 100 nM to about 200 nM. In some embodiments, the protein binds to TfR with an affinity of about 5 nM to about 500 nM, about 5 nM to about 400 nM, about 5 nM to about 300 nM, about 5 nM to about 200 nM, about 5 nM to about 100 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, or about 10 nM to about 200 nM. In some embodiments, the protein binds to TfR with an affinity of about 3 nM to about 600 nM. In some embodiments, the protein binds to TfR with an affinity of about 80 nM to about 180 nM or about 50 nM to about 250 nM. In some embodiments, the protein binds to TfR with an affinity of about 10 nM, about 100 nM, or about 500 nM.

[0235] In one embodiment, the TfR binding proteins described herein may have a novel or non-naturally occurring TfR binding site.

[0236] In one aspect, a protein is provided that includes a constant domain having a modification that causes the protein to specifically bind to the transferrin receptor. In some embodiments, the protein has a modification in the CL domain. In some embodiments, the protein has a modification in the CH1 domain. In some embodiments, the protein has a modification in the CH2 domain. In some embodiments, the protein has a modification in the CH3 domain. Exemplary CH2 and CH3 domains with modifications that cause specific binding to TfR are described, for example, in WO2018 / 152326, which is incorporated by reference in its entirety.

[0237] In one aspect, a protein is provided that includes a constant domain with a modification that allows the protein to specifically bind to the transferrin receptor. The modification is introduced to a specific set of amino acids present on the surface of the constant (e.g., CH3) domain. In some embodiments, the protein that includes the modified constant domain specifically binds to an epitope in the apical domain of the transferrin receptor. In some embodiments, the protein binds to the transferrin receptor without inhibiting the binding of transferrin to the transferrin receptor. In some embodiments, the protein binds to an epitope that includes the 208th amino acid of the transferrin receptor sequence.

[0238] Those skilled in the art will appreciate that the constant (e.g., CH3) domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, IgD, etc., can be similarly modified by identifying amino acids in those domains that correspond to sets (i)-(ii) described herein. Modifications can also be made to corresponding domains of immunoglobulins from other species, such as non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, chickens, etc.

[0239] CH3 Transferrin Receptor Binding Protein In one aspect, a protein is provided that comprises a CH3 domain having a modification that allows the protein to specifically bind to the transferrin receptor. In some embodiments, the modified domain is a human Ig CH3 domain. The CH3 domain can be from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the segment of amino acids from about position 341 to about position 447, numbered according to the EU numbering scheme. Positions in the CH3 domain for purposes of identifying corresponding position sets of amino acids for transferrin receptor binding are determined with reference to SEQ ID NO:3, unless otherwise indicated, or with reference to amino acids 114-220 of SEQ ID NO:1. Substitutions are also determined with reference to SEQ ID NO:1, i.e., an amino acid is considered to be a substitution for an amino acid at the corresponding position of SEQ ID NO:1. SEQ ID NO:1 includes a partial hinge region sequence, PCP, as amino acids 1-3. The numbering of positions in the CH3 domain with reference to SEQ ID NO:1 includes the first three amino acids.

[0240] As indicated above, the sets of residues in a CH3 domain that can be modified as described herein are numbered herein with reference to SEQ ID NO: 1. However, any CH3 domain, e.g., the CH3 domain of an IgG1, IgG2, IgG3, or IgG4, can have a modification, e.g., an amino acid substitution, at one or more sets of residues that correspond to the residues at the indicated positions in SEQ ID NO: 1. The respective positions in the IgG2, IgG3, and IgG4 sequences that correspond to any given position in SEQ ID NO: 1 can be readily determined.

[0241] In one embodiment, the modified CH3 domain protein that specifically binds to the transferrin receptor binds to the apical domain of the transferrin receptor at an epitope that includes position 208 of the full-length human transferrin receptor sequence (SEQ ID NO: 235), which corresponds to position 11 of the human transferrin receptor apical domain sequence set forth in SEQ ID NO: 107. SEQ ID NO: 107 corresponds to amino acids 198-378 of the human transferrin receptor-1 monoprotein sequence P02786 (SEQ ID NO: 235). In some embodiments, the modified CH3 domain protein binds to the apical domain of the transferrin receptor at an epitope that includes positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and / or 294 of the full-length human transferrin receptor sequence (SEQ ID NO: 235). The modified CH3 domain protein may bind to the transferrin receptor without blocking or otherwise inhibiting the binding of transferrin to the receptor. In some embodiments, the binding of transferrin to the TfR is not substantially inhibited. In some embodiments, the binding of transferrin to the TfR is inhibited by less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%). In some embodiments, binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%). Exemplary CH3 domain proteins that exhibit this binding specificity include proteins having amino acid substitutions at positions 157, 159, 160, 161, 162, 163, 186, 189, and 194, as determined with reference to amino acids 114-220 of SEQ ID NO:1.

[0242] CH3 transferrin receptor binding set (i): positions 157, 159, 160, 161, 162, 163, 186, 189, and 194 In some embodiments, the modified CH3 domain proteins described herein contain at least three or at least four, and typically five, six, seven, eight, or nine substitutions in a set of amino acid positions including 157, 159, 160, 161, 162, 163, 186, 189, and 194 (set i). Exemplary substitutions that may be introduced at these positions are shown in Tables B and C. In some embodiments, the amino acid at position 161 and / or 194 is an aromatic amino acid, e.g., Trp, Phe, or Tyr. In some embodiments, the amino acid at position 161 is Trp. In some embodiments, the amino acid at position 161 is Gly. In some embodiments, the aromatic amino acid at position 194 is Trp or Phe.

[0243] In some embodiments, a modified CH3 domain protein that specifically binds to a transferrin receptor comprises at least one position having a substitution of Leu, Tyr, Met, or Val at position 157; Leu, Thr, His, or Pro at position 159; Val, Pro, or an acidic amino acid at position 160; an aromatic amino acid, e.g., Trp or Gly (e.g., Trp), at position 161; Val, Ser, or Ala at position 162; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 186; Thr or an acidic amino acid at position 189; or Trp, Tyr, His, or Phe at position 194, with respect to SEQ ID NO:1. In some embodiments, the modified CH3 domain comprises 2, 3, 4, 5, 6, 7, or 8 positions selected from: 157 is Leu, Tyr, Met, or Val; 159 is Leu, Thr, His, or Pro; 160 is Val, Pro, or an acidic amino acid; 161 is Trp or Gly; 162 is Val, Ser, or Ala; 186 is Glu, Ala, Ser, Leu, Thr, or Pro; 189 is Thr or an acidic amino acid; and 194 is Trp, Tyr, His, or Phe. In some embodiments, the modified CH3 domain comprises Leu or Met at position 157, Leu, His, or Pro at position 159, Val at position 160, Trp or Gly at position 161, Val or Ala at position 162, Pro at position 186, Thr at position 189, and / or Trp at position 194. In some embodiments, the modified CH3 domain may comprise conservative substitutions of particular amino acids at one or more positions within a set, such as amino acids within the same charge class, hydrophobic class, side chain ring structure class (e.g., aromatic amino acids), or size class, and / or polar or non-polar class. Thus, for example, Ile may be present at position 157, 159, and / or 186. In some embodiments, the acidic amino acid at one, both, or each of positions 160, 186, and 189 is Glu.In other embodiments, the acidic amino acid at one, two, or each of positions 160, 186, and 189 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 157, 159, 160, 161, 162, 186, 189, and 194 have an amino acid substitution as defined in this paragraph.

[0244] In some embodiments, a CH3 domain protein having modifications of set (i) comprises a native Asn at position 163. In some embodiments, the modified CH3 domain protein comprises a Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 163. In some embodiments, the modified CH3 domain protein further comprises one, two, three, or four substitutions at positions including positions 153, 164, 165, and 188. In some embodiments, Trp, Tyr, Leu, or Gln may be present at position 153. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 164. In some embodiments, Gln, Phe, or His may be present at position 165. In some embodiments, Glu may be present at position 188.

[0245] In certain embodiments, the modified CH3 domain protein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions selected from Trp, Leu, or Glu at position 153; Tyr or Phe at position 157; Thr at position 159; Glu at position 160; Trp at position 161; Ser, Ala, Val, or Asn at position 162; Ser or Asn at position 163; Thr or Ser at position 186; Glu or Ser at position 188; Glu at position 189; and / or Phe at position 194. In some embodiments, the modified CH3 domain protein includes all eleven positions: Trp, Leu, or Glu at position 153; Tyr or Phe at position 157; Thr at position 159; Glu at position 160; Trp at position 161; Ser, Ala, Val, or Asn at position 162; Ser or Asn at position 163; Thr or Ser at position 186; Glu or Ser at position 188; Glu at position 189; and / or Phe at position 194.

[0246] In certain embodiments, the modified CH3 domain protein comprises Leu or Met at position 157, Leu, His, or Pro at position 159, Val at position 160, Trp at position 161, Val or Ala at position 162, Pro at position 186, Thr at position 189, and / or Trp at position 194. In some embodiments, the modified CH3 domain protein further comprises Ser, Thr, Gln, or Phe at position 164. In some embodiments, the modified CH3 domain protein further comprises Trp, Tyr, Leu, or Gln at position 153 and / or Gln, Phe, or His at position 165. In some embodiments, Trp is present at position 153 and / or Gln is present at position 165. In some embodiments, the modified CH3 domain protein does not have a Trp at position 153.

[0247] In other embodiments, the modified CH3 domain protein comprises a Tyr at position 157, a Thr at position 159, a Glu or Val at position 160, a Trp at position 161, a Ser at position 162, a Ser or Thr at position 186, a Glu at position 189, and / or a Phe at position 194. In some embodiments, the modified CH3 domain protein comprises a native Asn at position 163. In certain embodiments, the modified CH3 domain protein further comprises a Trp, Tyr, Leu, or Gln at position 153, and / or a Glu at position 188. In some embodiments, the modified CH3 domain protein further comprises a Trp at position 153 and / or a Glu at position 188.

[0248] In some embodiments, the modified CH3 domain includes one or more substitutions of Trp at position 153, Thr at position 159, Trp at position 161, Val at position 162, Ser or Thr at position 186, Glu at position 188, and / or Phe at position 194.

[0249] In a further embodiment, the modified CH3 domain further comprises one, two or three positions selected from the following: 187 is Lys, Arg, Gly or Pro; 197 is Ser, Thr, Glu or Lys; and 199 is Ser, Trp or Gly.

[0250] In some embodiments, a modified CH3 domain protein that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 114-220 of any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, such a modified CH3 domain protein comprises amino acids 157-163 and / or amino acids 186-194 of any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, such a modified CH3 domain protein comprises amino acids 153 to 163 and / or amino acids 186 to 194 of any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746. In some embodiments, such a modified CH3 domain protein comprises amino acids 153 to 163 and / or amino acids 186 to 199 of any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 (e.g., 11 to 16) residues at positions corresponding to 153, 157, 159, 160, 161, 162, 163, 164, 165, 186, 187, 188, 189, 194, 197, and 199, as determined based on SEQ ID NO: 1, are not deleted or substituted in SEQ ID NOs: 4 to 29, 236 to 299, 645, 650, or 746.

[0251] In some embodiments, a modified CH3 domain protein that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 114 to 220 of SEQ ID NO: 1 or 634, except that the percent identity does not include the set of positions 157, 159, 160, 161, 162, 163, 186, 189, and 194. In some embodiments, the modified CH3 domain protein comprises amino acids 157 to 163 and / or amino acids 186 to 194 set forth in any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746.

[0252] In some embodiments, the modified CH3 domain protein has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. 9% identity thereto, provided that at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions corresponding to positions 153, 157, 159, 160, 161, 162, 163, 164, 165, 186, 187, 188, 189, 194, 197, and 199 of any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746 are not deleted or substituted.

[0253] In some embodiments, the modified CH3 domain protein has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746, and also has at least one of the following amino acids: Trp, Tyr, Leu, Gln, or Glu at position 153; Leu, Tyr, Met, or Val at position 157; Leu, Thr, His, or Pro at position 159; Val, Pro, or an acidic amino acid at position 160; At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions include an aromatic amino acid, e.g., Trp, at position 61; Val, Ser, or Ala, at position 162; Ser or Asn, at position 163; Ser, Thr, Gln, or Phe, at position 164; Gln, Phe, or His, at position 165; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro, at position 186; Lys, Arg, Gly, or Pro, at position 187; Glu or Ser, at position 188; Thr or an acidic amino acid, at position 189; Trp, Tyr, His, or Phe, at position 194; Ser, Thr, Glu, or Lys, at position 197; and Ser, Trp, or Gly, at position 199.

[0254] In some embodiments, the modified CH3 domain proteins described herein comprise one or more substitutions at a set of amino acid positions including positions 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199, where the substitutions and positions are determined with reference to the sequence of SEQ ID NO: 13. In some embodiments, the modified CH3 domain comprises Glu, Leu, Ser, Val, Trp, or Tyr at position 153, an aromatic amino acid (e.g., Tyr, Phe, or Trp), Met, Pro, or Val at position 157, Thr, Asn, or Val at position 159, Glu, Ile, Pro, or Val at position 160, an aliphatic amino acid (e.g., Ala, Ile, or Val), Ser, or Thr at position 163, Ser, Asn, Arg, or Thr at position 164, Thr, His, or Ser at position 186, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188, Glu or Arg at position 189, Phe, His, Lys, Tyr, or Trp at position 194, Ser, Thr, or Trp at position 197, and Ser, Cys, Pro, Met, or Trp at position 199. The modified CH3 domain protein comprises the sequence GQPREPQVYTLPPS-RDELTKNQVSLTCLVKGFYPSDIAVX. 1 WES 2 GX 3 X 4 WX 5 X 6 YKTTPPVLDSDGSFFLYS-KLTVX 7 KX 8 X 9 WQQGX 10 VFX 11 CX 12 VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:556), in which 1 is E, L, S, V, W, or Y, and X 2 is an aromatic amino acid (e.g., Y, F, or W), M, P, or V, and X 3 is T, N, or V, and X 4 is E, I, P, or V, and X 5is an aliphatic amino acid (e.g., A, I, or V), S, or T, and X 6 is S, N, R, or T, and X 7 is T, H, or S, and X 8 is E, S, D, G, T, P, Q, or R, and X 9 is E or R, and X 10 is F, H, K, Y, or W, and X 11 is S, T, or W, and X 12 is S, C, P, M, or W. In certain embodiments, the modified CH3 domain protein has the sequence X 1 WES 2 GX 3 X 4 WX 5 X 6 (SEQ ID NO: 554), in which 1 is E, L, S, V, W, or Y, and X 2 is an aromatic amino acid (e.g., Y, F, or W), M, P, or V, and X 3 is T, N, or V, and X 4 is E, I, P, or V, and X 5 is an aliphatic amino acid (e.g., A, I, or V), S, or T, and X 6 is S, N, R, or T. In certain embodiments, the modified CH3 domain protein has the sequence X 1 KX 2 X 3 WQQGX 4 VFX 5 CX 6 (SEQ ID NO: 555), in which 1 is T, H, or S, and X 2 is E, S, D, G, T, P, Q, or R, and X 3 is E or R, and X 4 is F, H, K, Y, or W, and X 5 is S, T, or W, and X 6 is S, C, P, M, or W.

[0255] In some embodiments, the modified CH3 domain protein comprises Glu, Leu, or Trp at position 153, an aromatic amino acid at position 157, a Thr at position 159, Glu at position 160, an aliphatic amino acid or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe, His, Tyr, or Trp at position 194, Ser at position 197, and Ser at position 199, where the substitutions and positions are determined with reference to the sequence of SEQ ID NO: 13. In certain embodiments, the aromatic amino acid at position 157 is Tyr or Phe and the aliphatic amino acid at position 162 is Ala or Val. In some embodiments, the modified CH3 domain protein comprises Glu, Leu, or Trp at position 153, Tyr or Phe at position 157, Thr at position 159, Glu at position 160, Ala, Val, or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe at position 194, Ser at position 197, and Ser at position 199, where the substitutions and positions are determined with reference to the sequence of SEQ ID NO:13.

[0256] In some embodiments, the modified CH3 domain protein described herein comprises only one substitution at the set of amino acid positions comprising: 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199, the substitutions and positions being determined with reference to the sequence of SEQ ID NO: 238. In some embodiments, the modified CH3 domain protein comprises Glu, Leu, Ser, Val, Trp, or Tyr at position 153. The modified CH3 domain protein may comprise Glu at position 153. The modified CH3 domain protein may comprise Leu at position 153. The modified CH3 domain protein may comprise Ser at position 153. The modified CH3 domain protein may comprise Val at position 153. The modified CH3 domain protein may comprise Trp at position 153. The modified CH3 domain protein may comprise Tyr at position 153. In some embodiments, the modified CH3 domain protein comprises Tyr, Phe, Trp, Met, Pro, or Val at position 157. The modified CH3 domain protein may comprise Tyr at position 157. The modified CH3 domain protein may comprise Phe at position 157. The modified CH3 domain protein may comprise Trp at position 157. The modified CH3 domain protein may comprise Met at position 157. The modified CH3 domain protein may comprise Pro at position 157. The modified CH3 domain protein may comprise Val at position 157. In some embodiments, the modified CH3 domain protein comprises Thr, Asn, or Val at position 159. The modified CH3 domain protein may comprise Thr at position 159. The modified CH3 domain protein may comprise Asn at position 159. The modified CH3 domain protein may comprise Val at position 159. In some embodiments, the modified CH3 domain protein comprises Glu, Ile, Pro, or Val at position 160. The modified CH3 domain protein may comprise a Glu at position 160. The modified CH3 domain protein may comprise an Ile at position 160. The modified CH3 domain protein may comprise a Pro at position 160. The modified CH3 domain protein may comprise a Val at position 160.In some embodiments, the modified CH3 domain protein comprises Ala, Ile, Val, Ser, or Thr at position 162. The modified CH3 domain protein may comprise Ala at position 162. The modified CH3 domain protein may comprise Ile at position 162. The modified CH3 domain protein may comprise Val at position 162. The modified CH3 domain protein may comprise Ser at position 162. The modified CH3 domain protein may comprise Thr at position 162. In some embodiments, the modified CH3 domain protein comprises Ser, Asn, Arg, or Thr at position 163. The modified CH3 domain protein may comprise Ser at position 163. The modified CH3 domain protein may comprise Asn at position 163. The modified CH3 domain protein may comprise Arg at position 163. The modified CH3 domain protein may comprise Thr at position 163. In some embodiments, the modified CH3 domain protein comprises Thr, His, or Ser at position 186. The modified CH3 domain protein may include a Thr at position 186. The modified CH3 domain protein may include a His at position 186. The modified CH3 domain protein may include a Ser at position 186. In some embodiments, the modified CH3 domain protein includes a Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188. The modified CH3 domain protein may include a Glu at position 188. The modified CH3 domain protein may include a Ser at position 188. The modified CH3 domain protein may include an Asp at position 188. The modified CH3 domain protein may include a Gly at position 188. The modified CH3 domain protein may include a Thr at position 188. The modified CH3 domain protein may include a Pro at position 188. The modified CH3 domain protein may include a Glu at position 188. The modified CH3 domain protein may include an Arg at position 188. In some embodiments, the modified CH3 domain protein comprises Glu or Arg at position 189. The modified CH3 domain protein may comprise Glu at position 189. The modified CH3 domain protein may comprise Arg at position 189.In some embodiments, the modified CH3 domain protein comprises Phe, His, Lys, Tyr, or Trp at position 194. The modified CH3 domain protein may comprise Phe at position 194. The modified CH3 domain protein may comprise His at position 194. The modified CH3 domain protein may comprise Lys at position 194. The modified CH3 domain protein may comprise Tyr at position 194. The modified CH3 domain protein may comprise Trp at position 194. In some embodiments, the modified CH3 domain protein comprises Ser, Thr, or Trp at position 197. The modified CH3 domain protein may comprise Ser at position 197. The modified CH3 domain protein may comprise Thr at position 197. The modified CH3 domain protein may comprise Trp at position 197. In some embodiments, the modified CH3 domain protein comprises Ser, Cys, Pro, Met, or Trp at position 199. The modified CH3 domain protein may comprise Ser at position 199. The modified CH3 domain protein may comprise Cys at position 199. The modified CH3 domain protein may comprise Pro at position 199. The modified CH3 domain protein may comprise Met at position 199. The modified CH3 domain protein may comprise Trp at position 199.

[0257] In some embodiments, the modified CH3 domain proteins described herein comprise one or more substitutions at a set of amino acid positions including 153, 157, 159, 160, 162, 163, 164, 186, 189, and 194, wherein the substitutions and positions are determined with reference to the sequence of SEQ ID NO:9. In some embodiments, the modified CH3 domain comprises Glu or Trp at position 153, Val, Trp, Leu, or Tyr at position 157, Leu, Pro, Phe, Thr, or His at position 159, Pro, Val, or Glu at position 160, Ala, Ser, Val, or Gly at position 162, Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr, or Glu at position 163, Thr, Phe, Gln, Val, or Tyr at position 164, Leu, Ser, Glu, Ala, or Pro at position 186, Glu, Asp, Thr, or Asn at position 189, and Trp, Tyr, Phe, or His at position 194.

[0258] In some embodiments, the modified CH3 domain protein comprises Glu or Trp at position 153, Trp, Leu, or Tyr at position 157, Thr or His at position 159, Val at position 160, Ala, Ser, or Val at position 162, Val, Asn, or Thr at position 163, Gln or Tyr at position 164, Pro at position 186, Thr or Asn at position 189, and Trp, Tyr, Phe, or His at position 194, where the substitutions and positions are determined with reference to the sequence of SEQ ID NO:9.

[0259] In a further embodiment, the transferrin receptor binding protein comprises amino acids 157 to 194, amino acids 153 to 194, or amino acids 153 to 199 of any one of SEQ ID NOs: 4 to 29, 236 to 299, and 422 to 435. In a further embodiment, the protein comprises an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 157 to 194 of any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746, or amino acids 153 to 194 or 153 to 199 of any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746.

[0260] In some embodiments, the protein comprises any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645-650, and 746. In further embodiments, the protein comprises any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645-650, and 746 that does not include the first three amino acids of the amino terminal "PCP". In further embodiments, the protein may have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746 determined not to include the first three amino acids of the amino terminal "PCP".

[0261] CH3 transferrin receptor binding set (ii): positions 118, 119, 120, 122, 210, 211, 212, and 213 In some embodiments, the modified CH3 domain proteins described herein comprise at least three or at least four, and typically five, six, seven, or eight substitutions in a set of amino acid positions including positions 118, 119, 120, 122, 210, 211, 212, and 213 (set ii). Exemplary substitutions that may be introduced at these positions are shown in Table A. In some embodiments, the modified CH3 domain proteins comprise a Gly at position 210, a Phe at position 211, and / or an Asp at position 213. In some embodiments, a Glu is present at position 213. In certain embodiments, the modified CH3 domain protein comprises at least one substitution at position 118: Phe or Ile, Asp, Glu, Gly, Ala, or Lys, at position 119, Tyr, Met, Leu, Ile, or Asp, at position 120, Thr or Ala, at position 122, Gly, at position 210, Phe, at position 211, His, Tyr, Ser, or Phe, at position 212, or Asp, at position 213. In some embodiments, 2, 3, 4, 5, 6, 7, or all 8 of positions 118, 119, 120, 122, 210, 211, 212, and 213 have a substitution as defined in this paragraph. In some embodiments, the modified CH3 domain may include conservative substitutions of particular amino acids at one or more positions within a set, e.g., amino acids within the same charge class, hydrophobic class, side chain ring structure class (e.g., aromatic amino acids), or size class, and / or polar or non-polar class.

[0262] In some embodiments, the modified CH3 domain protein has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 114 to 220 of SEQ ID NO: 1 or 634, except that the percent identity does not include the set of positions 118, 119, 120, 122, 210, 211, 212, and 213.

[0263] Exemplary Proteins Containing Modified CH2 or CH3 Domains The modified constant (e.g., CH3) domains described herein may be attached to other domains of an Fc polypeptide. In some embodiments, the modified CH3 domains described herein are attached to a CH2 domain, which may be a naturally occurring CH2 domain or a variant CH2 domain, typically at the C-terminus of the CH2 domain. In some embodiments, the modified CH2 domains described herein are attached to a CH3 domain, which may be a naturally occurring CH3 domain or a variant CH3 domain, typically at the N-terminus of the CH3 domain. In some embodiments, the modified CH3 domain attached to the CH2 domain, or the protein comprising the modified CH2 domain attached to the CH3 domain, further comprises a partial or complete hinge region of an antibody, thus forming the modified CH2 or CH3 domain as part of an Fc polypeptide with a partial or complete hinge region. The hinge region may be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, such as an IgG1 hinge region, e.g., the amino acid sequence of human IgG1 hinge EPKSCDKTHTCPPCP (SEQ ID NO: 234), or portions thereof set forth in SEQ ID NOs: 232-233. In further embodiments, the protein can be in an Fc format that includes a hinge or partial hinge region, but is further linked to other portions, e.g., a Fab fragment or portion thereof, to generate a transferrin receptor binding Fab-Fc fusion. In some embodiments, the transferrin receptor binding Fab-Fc fusion includes a modified CH2 or CH3 domain, a hinge region, and a Fab fragment or portion thereof. The Fab fragment may be targeted or non-targeted. In certain embodiments, the Fab fragment is targeted. In certain embodiments, the Fab fragment is non-targeted. In certain embodiments, the Fab fragment does not specifically bind to transferrin via its heavy or light chain variable region. In certain embodiments, the TfR binding protein described herein does not include a Fab fragment or portion thereof.In some embodiments, the TfR binding proteins described herein do not comprise an antigen binding portion or a variable domain portion of a Fab fragment. In some embodiments, the TfR binding proteins described herein do not comprise an antibody antigen binding domain or an antibody variable domain.

[0264] In some embodiments, an Fc polypeptide comprising a modified CH2 or CH3 domain as described herein, or a Fab-Fc fusion comprising a modified CH2 or CH3 domain as described herein, is a subunit of a dimer. Thus, in certain embodiments, a protein as described herein may comprise an Fc polypeptide dimer or a Fab-Fc dimer fusion comprising a first Fc polypeptide comprising a modified constant domain and a second Fc polypeptide capable of dimerizing to the first Fc polypeptide. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer comprises a single polypeptide that binds to the transferrin receptor, i.e., is monovalent with respect to transferrin receptor binding. In some embodiments, the dimer comprises a second polypeptide that binds to the transferrin receptor. The second polypeptide may comprise the same modified CH2 / CH3 domain present in the Fc or Fab-Fc fusion to provide a bivalent binding homodimer, or a second modified CH2 / CH3 domain as described herein may be used to provide a second transferrin receptor binding site. In some embodiments, the dimer comprises a first subunit that includes a modified CH2 or CH3 domain and a second subunit that includes a CH2 and a CH3 domain, neither of which binds to the transferrin receptor.

[0265] The transferrin receptor binding proteins described herein can have a wide range of binding affinities, for example, based on the format of the protein. For example, in some embodiments, proteins containing modified CH3 domains have affinities for transferrin receptor binding ranging from 1 pM to 10 μM. In some embodiments, affinity can be measured in a monovalent format. In other embodiments, affinity can be measured in a bivalent format, for example, as a dimer, including a polypeptide-Fab fusion protein.

[0266] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), equilibrium exclusion binding assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, equilibrium exclusion binding assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0267] Further mutations in the Fc polypeptide As described herein, the constant domain modified to bind to TfR can be comprised within an Fc polypeptide or a Fab-Fc fusion. In certain embodiments, the polypeptide can also include additional mutations that, for example, increase serum stability, modulate effector function, affect glycosylation, reduce human immunogenicity, and / or provide knob and hole heterodimerization of the polypeptide. In certain embodiments, the polypeptides described herein can be further modified to remove the C-terminal Lys residue (i.e., the Lys residue at position 220, numbered with reference to SEQ ID NO:1).

[0268] In certain embodiments, an Fc polypeptide modified to bind to TfR may dimerize with a second Fc polypeptide. Thus, in certain embodiments, the proteins described herein may comprise an Fc dimer comprising a modified CH2 or CH3 domain in a first polypeptide and a second Fc polypeptide. Thus, in some aspects, the proteins described herein comprise two Fc polypeptides, where one or both Fc polypeptides each comprise an independently selected modification (e.g., a modification or mutation described herein).

[0269] In some embodiments, the polypeptides described herein have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0270] In some embodiments, the polypeptides present in the Fc dimer contain knob and hole mutations to promote heterodimer formation. In general, this method involves introducing a protrusion ("knob") at the junction of one polypeptide and a corresponding hole ("hole") at the junction of the other polypeptide. The protrusion is constructed by replacing a small amino acid side chain at the junction of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary hole of identical or similar size to the protrusion is created at the junction of the second polypeptide by replacing a large amino acid side chain with a smaller one (e.g., alanine or threonine). Such additional mutations are at locations within the polypeptide that have no adverse effect on binding of the modified CH2 or CH3 domain to the transferrin receptor.

[0271] In one exemplary embodiment of the knobs and holes approach to dimerization, the position corresponding to position 139 of SEQ ID NO:1 of the first Fc polypeptide subunit to be dimerized has a tryptophan instead of the native threonine, and the second Fc polypeptide subunit of the dimer has a valine instead of a tyrosine at the position corresponding to position 180 of SEQ ID NO:1. The second subunit of the Fc polypeptide may further comprise a substitution in which the native threonine at the position corresponding to position 139 of SEQ ID NO:1 is replaced with a serine, and the native leucine at the position corresponding to position 141 of SEQ ID NO:1 is replaced with an alanine.

[0272] The polypeptides described herein can also be engineered to include other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the CH3-CH3 junction, either naturally charged or hydrophobic patch modifications.

[0273] In some embodiments, modifications to enhance serum half-life may be introduced. For example, in some embodiments, the Fc polypeptide comprises a CH2 domain that contains Tyr at position 25 of SEQ ID NO:1, Thr at position 27 of SEQ ID NO:1, and Glu at position 29 of SEQ ID NO:1.

[0274] In some embodiments, mutations, e.g., substitutions, are introduced at one or more of positions 17-30, 52-57, 80-90, 156-163, and 201-208, as determined with reference to SEQ ID NO:1. In some embodiments, one or more mutations are introduced at positions 24, 25, 27, 28, 29, 80, 81, 82, 84, 85, 87, 158, 159, 160, 162, 201, 206, 207, or 209, as determined with reference to SEQ ID NO:1. In some embodiments, mutations are introduced at one or two of positions 201 and 207, as determined with reference to SEQ ID NO:1 (e.g., M201L and N207S). In some embodiments, the polypeptides described herein include the mutations N207S or N207A, with or without M201L. In some embodiments, the polypeptides described herein include substitutions at one, two, or all three of positions T80, E153, and N207, numbered with respect to SEQ ID NO:1 (e.g., T80Q and N207A, or T80A, E153A, and N207A). In some embodiments, the polypeptides described herein include substitutions at positions T23 and M201, numbered with respect to SEQ ID NO:1 (e.g., T23Q and M201L).

[0275] In some embodiments, the C-terminal Lys residue is removed or absent in an Fc polypeptide described herein, or in a protein comprising an Fc polypeptide described herein (i.e., the Lys residue at position 220, numbered with reference to SEQ ID NO: 1, is removed or absent). For example, in certain embodiments, the C-terminal Lys residue may be removed or absent from any one of SEQ ID NOs: 1, 4-29, 236-299, 302, 361-372, 397-408, 421-435, 491-497, 521-518, 557-561, 623-633, 655, 702-731, 743-744, 801-810, and 812-818. Exemplary truncated Fc polypeptide sequences, or proteins comprising such truncated Fc polypeptide sequences, include, for example, SEQ ID NOs: 634-654, 733-742, and 745-746.

[0276] Fc effector function In some embodiments, the first Fc polypeptide comprising a modified CH2 or CH3 domain and / or the second Fc polypeptide has effector function, i.e., the ability to induce a specific biological function upon binding to an Fc receptor expressed on an effector cell that mediates the effector function. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells.

[0277] Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions may vary depending on the antibody class. For example, native human IgG1 and IgG3 antibodies can trigger ADCC and CDC activities when bound to the appropriate Fc receptors present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 can trigger ADCP functions when bound to the appropriate Fc receptors present on immune cells.

[0278] In some embodiments, the polypeptides described herein may include additional modifications that reduce or eliminate effector function. Alternatively, in some embodiments, the first and / or second polypeptides comprising modified CH2 or CH3 domains described herein may include additional modifications that enhance effector function.

[0279] Exemplary Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions corresponding to positions 7 and 8 of SEQ ID NO:1. In some embodiments, the substitutions in the modified CH2 domain include Ala at positions 7 and 8 of SEQ ID NO:1. In some embodiments, the substitutions in the modified CH2 domain include Ala at positions 7 and 8 of SEQ ID NO:1 and Gly at position 102. In some embodiments, the substitutions in the modified CH2 domain include Ala at positions 7 and 8 of SEQ ID NO:1 and Ser at position 102.

[0280] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, one or more substitutions at positions 238, 265, 269, 270, 297, 327, and 329 (corresponding to positions 11, 38, 42, 43, 70, 100, and 102 numbered with reference to the EU numbering scheme, SEQ ID NO: 1). Exemplary substitutions (as numbered by the EU numbering scheme) include position 329, which may include a mutation in which proline is replaced with glycine, alanine, serine, or arginine, or an amino acid residue of sufficient size to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp87 and Trp110 of FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may also be present, for example, L234A and L235A in human IgG1 Fc polypeptides; L234A, L235A, and P329G in human IgG1 Fc polypeptides; L234A, L235A, and P329S in human IgG1 Fc polypeptides; S228P and L235E in human IgG4 Fc polypeptides; L234A and G237A in human IgG1 Fc polypeptides; L234A, L235A, and G237A in human IgG1 Fc polypeptides; V234A and G237A in human IgG2 Fc polypeptides; L235A, G237A, and E318A in human IgG4 Fc polypeptides; and S228P and L236E in human IgG4 Fc polypeptides. In some embodiments, the polypeptides described herein may have one or more amino acid substitutions that modulate ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc polypeptide according to the EU numbering scheme.

[0281] In some embodiments, the polypeptides described herein may have one or more amino acid substitutions that increase or decrease ADCC, or may have mutations that alter C1q binding and / or CDC.

[0282] Exemplary Polypeptides Containing Additional Mutations The polypeptides described herein, such as polypeptides comprising a modified CH3 domain (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) and / or second polypeptides, may comprise a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a hole mutation (e.g., T139R, numbered with reference to SEQ ID NO:1), a nucleotide mutation (e.g., T139S, numbered with reference to SEQ ID NO:1), or a nucleotide mutation (e.g., T139T, numbered with reference to SEQ ID NO:1). 1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and / or a mutation that increases serum stability (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0283] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) are The polypeptide may have a knob mutation (e.g., T139W numbered with reference to SEQ ID NO: 1), and may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746 may be modified to have a knob mutation. In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) may have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:1 or 634. In some embodiments, a polypeptide having the sequence of SEQ ID NO:1 or 634 may be modified to have a knob mutation.

[0284] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., T139R, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., T139S, numbered with reference to SEQ ID NO:1), or a mutation that modulates effector function (e.g., T139T, numbered with reference to SEQ ID NO:1). The polypeptide may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746, numbered as references, L7A, L8A, and / or P102G or P102S (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S), and any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746 may be modified to have a knob mutation and a mutation that modulates effector function. In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:1 or 634. In some embodiments, a polypeptide having the sequence of SEQ ID NO:1 or 634 can be modified to have a knob mutation and a mutation that modulates effector function.

[0285] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T139W, as numbered with reference to SEQ ID NO:1), a mutation that enhances serum stability (e.g., , (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO: 1, or (ii) N207S, with or without M201L, numbered with reference to SEQ ID NO: 1), and may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, a polypeptide having a sequence of any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746 may be modified to have a knob mutation and a mutation that enhances serum stability. In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) may have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a sequence of SEQ ID NO:1 or 634. In some embodiments, a polypeptide having a sequence of SEQ ID NO:1 or 634 may be modified to have a knob mutation and a serum stability enhancing mutation.

[0286] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T139W, as numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, as numbered with reference to SEQ ID NO:1 (e.g., L7A, L8A, and / or P102S, as numbered with reference to SEQ ID NO:1), or a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, as numbered with reference to SEQ ID NO:1). and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S), mutations that increase serum stability (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L, numbered with reference to SEQ ID NO:1), and may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746 may be modified to have a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability.In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) comprises a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), a mutation that enhances serum stability ( For example, it may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:1 or 634, as well as (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S, with or without M201L, numbered with reference to SEQ ID NO:1. In some embodiments, a polypeptide having a sequence of SEQ ID NO:1 or 634 may be modified to have knob mutations, mutations that modulate effector function, and mutations that increase serum stability.

[0287] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) contain a hole mutation (e.g., , T139S, L141A, and Y180V numbered based on SEQ ID NO: 1), and any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746 may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746 may be modified to have a hole mutation. In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) may have a hole mutation (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:1 or 634. In some embodiments, a polypeptide having the sequence of SEQ ID NO:1 or 634 may be modified to have a hole mutation.

[0288] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise any of the following mutations: hole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., For example, it may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of L7A, L8A, and / or P102G or (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S) numbered based on SEQ ID NO: 1, and any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4 to 29, 236 to 299, 422 to 435, 645, 650, and 746 may be modified to have a hole mutation and a mutation that modulates effector function. In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) may have a hole mutation (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S), numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:1 or 634.In some embodiments, a polypeptide having the sequence of SEQ ID NO: 1 or 634 can be modified to have a hole mutation and a mutation that modulates effector function.

[0289] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) contain whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), which enhance serum stability. The polypeptide may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746, including mutations (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO: 1, or (ii) N207S, with or without M201L, numbered with reference to SEQ ID NO: 1), and the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, a polypeptide having any one of the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746 may be modified to have a whole mutation and a mutation that enhances serum stability. In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) may have a whole mutation (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a sequence of SEQ ID NO:1 or 634. In some embodiments, a polypeptide having a sequence of SEQ ID NO:1 or 634 may be modified to have a whole mutation and a serum stability enhancing mutation.

[0290] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise or are modified by any of the following mutations: hole mutations (e.g., T139S, L141A, and Y180V, as numbered with reference to SEQ ID NO:1), effector function modulating mutations (e.g., L7A, L8A, and / or P102G or P102S, as numbered with reference to SEQ ID NO:1), or mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, as numbered with reference to SEQ ID NO:1). For example, the antibody may have a mutation that enhances serum stability (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L, numbered with reference to SEQ ID NO:1), and may have at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequences of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4-29, 236-299, 422-435, 645, 650, and 746 may be modified to have a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability.In some embodiments, a polypeptide described herein (e.g., a second Fc polypeptide) may comprise any of the following mutations: whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), mutations that enhance serum stability, and mutations that enhance cellular function (e.g., cellular function ... In some embodiments, the polypeptide having the sequence of SEQ ID NO:1 or 634 may have mutations that enhance affinity (e.g., (i) M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1, or (ii) N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:1 or 634. In some embodiments, a polypeptide having the sequence of SEQ ID NO:1 or 634 may be modified to have hole mutations, mutations that modulate effector function, and mutations that enhance serum stability.

[0291] Clone CH3C.35.23.2 In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:361 or 646. In some embodiments, clone CH3C.35.23.2 with a knob mutation has the sequence of SEQ ID NO:361. In some embodiments, clone CH3C.35.23.2 with a knob mutation has the sequence of SEQ ID NO:646.

[0292] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequences of SEQ ID NOs: 362, 363, 632, and 647-649 (e.g., 362 or 363). In some embodiments, clone CH3C.35.23.2 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO: 362, 363, or 632. In some embodiments, clone CH3C.35.23.2 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO: 647-649. In some embodiments, the N-terminus of clone CH3C.35.23.2 having a knob mutation and a mutation that modulates effector function comprises a hinge sequence or a portion of a hinge sequence (see, e.g., SEQ ID NO: 804 or 741). In some embodiments, the N-terminus of clone CH3C.35.23.2 having a knob mutation and a mutation that modulates effector function is further joined to a CH1 region (see, e.g., SEQ ID NO: 743-745).

[0293] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 364. In some embodiments, clone CH3C.35.23.2 with the knob mutation and the serum stability enhancing mutation has the sequence of SEQ ID NO:364.

[0294] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 492. In some embodiments, clone CH3C.35.23.2 with a knob mutation and a serum stability enhancing mutation has the sequence of SEQ ID NO:492.

[0295] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:365 or 366. In some embodiments, clone CH3C.35.23.2, which has knob mutations, mutations that modulate effector function, and mutations that increase serum stability, has the sequence of SEQ ID NO: 365 or 366.

[0296] In some embodiments, clone CH3C.35.23.2 may have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), a mutation that increases serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:493 or 494. In some embodiments, clone CH3C.35.23.2, which has knob mutations, mutations that modulate effector function, and mutations that increase serum stability, has the sequence of SEQ ID NO: 493 or 494.

[0297] In some embodiments, clone CH3C.35.23.2 can have hole mutations (e.g., T139S, L141A, and Y180V numbered with reference to SEQ ID NO:1) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 367. In some embodiments, clone CH3C.35.23.2 with hole mutations has the sequence of SEQ ID NO:367.

[0298] In some embodiments, clone CH3C.35.23.2 can have hole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:368 or 369. In some embodiments, clone CH3C.35.23.2 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:368 or 369.

[0299] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), serum stability enhancing mutations (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 370. In some embodiments, clone CH3C.35.23.2 with whole mutations and serum stability enhancing mutations has the sequence of SEQ ID NO:370.

[0300] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:495. In some embodiments, clone CH3C.35.23.2 with whole mutations and serum stability enhancing mutations has the sequence of SEQ ID NO:495.

[0301] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:371 or 372. In some embodiments, clone CH3C.35.23.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability, has the sequence of SEQ ID NO: 371 or 372.

[0302] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:496 or 497. In some embodiments, clone CH3C.35.23.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability, has the sequence of SEQ ID NO:496 or 497.

[0303] Clone CH3C.35.23.3 In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0304] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that enhance serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that enhance serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1).In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0305] Clone CH3C.35.23.4 In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). For example, in some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:718 or 742. In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0306] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that enhance serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that enhance serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1).In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0307] Clone CH3C.35.21.17.2 In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 397 or 651. In some embodiments, clone CH3C.35.21.17.2 with a knob mutation has the sequence of SEQ ID NO: 397. In some embodiments, clone CH3C.35.21.17.2 with a knob mutation has the sequence of SEQ ID NO: 651.

[0308] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:398, 399, 633, or 652-654 (e.g., 398 or 399). In some embodiments, clone CH3C.35.21.17.2 having knob mutations and mutations that modulate effector function has the sequence of SEQ ID NO: 398, 399, or 633. In some embodiments, clone CH3C.35.21.17.2 having knob mutations and mutations that modulate effector function has the sequence of SEQ ID NO: 652-654.

[0309] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 400. In some embodiments, clone CH3C.35.21.17.2 with knob mutations and serum stability enhancing mutations has the sequence of SEQ ID NO:400.

[0310] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 513. In some embodiments, clone CH3C.35.21.17.2 with a knob mutation and a serum stability enhancing mutation has the sequence of SEQ ID NO:513.

[0311] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:401 or 402. In some embodiments, clone CH3C.35.21.17.2, which has knob mutations, mutations that modulate effector function, and mutations that increase serum stability, has the sequence of SEQ ID NO: 401 or 402.

[0312] In some embodiments, clone CH3C.35.21.17.2 may have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), a mutation that increases serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:514 or 515. In some embodiments, clone CH3C.35.21.17.2, which has knob mutations, mutations that modulate effector function, and mutations that increase serum stability, has the sequence of SEQ ID NO:514 or 515.

[0313] In some embodiments, clone CH3C.35.21.17.2 can have hole mutations (e.g., T139S, L141A, and Y180V numbered with reference to SEQ ID NO:1) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 403. In some embodiments, clone CH3C.35.21.17.2 with hole mutations has the sequence of SEQ ID NO:403.

[0314] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:404 or 405. In some embodiments, clone CH3C.35.21.17.2, which has a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO: 404 or 405.

[0315] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), serum stability enhancing mutations (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 406. In some embodiments, clone CH3C.35.21.17.2 with whole mutations and serum stability enhancing mutations has the sequence of SEQ ID NO:406.

[0316] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), a serum stability enhancing mutation (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:516. In some embodiments, clone CH3C.35.21.17.2 with whole mutations and serum stability enhancing mutations has the sequence of SEQ ID NO:516.

[0317] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:407 or 408. In some embodiments, clone CH3C.35.21.17.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability, has the sequence of SEQ ID NO: 407 or 408.

[0318] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:517 or 518. In some embodiments, clone CH3C.35.21.17.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability, has the sequence of SEQ ID NO:517 or 518.

[0319] Clone CH3C.35.23 In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0320] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1).In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0321] Clone CH3C.35.21 In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0322] In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1).In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0323] Clone CH3C.35.23.1.1 In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1) and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T139W, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and a mutation that enhances serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0324] In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)). In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that enhance serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1) and mutations that enhance serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1). In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., M25Y, S27T, and T29E, numbered with reference to SEQ ID NO:1).In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and mutations that increase serum stability (e.g., N207S with or without M201L, numbered with reference to SEQ ID NO:1).

[0325] Fc Polypeptides In certain embodiments, an Fc polypeptide comprising a modified CH2 or CH3 domain as described herein, or a Fab-Fc fusion comprising a modified constant domain as described herein, is a subunit of a dimer. Thus, in certain embodiments, a protein as described herein may comprise an Fc dimer comprising a constant domain in a first polypeptide modified to bind to TfR and a second Fc polypeptide.

[0326] In some embodiments, the second Fc polypeptide may have hole mutations (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO: 1) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO: 557 or 635. In some embodiments, the second Fc polypeptide having hole mutations has the sequence of SEQ ID NO: 557 or 635.

[0327] In some embodiments, the second Fc polypeptide may have a whole mutation (e.g., T139S, L141A, and Y180V, numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S, numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:558, 627, 628, 636, 637, or 638. In some embodiments, the second Fc polypeptide having hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO: 558, 627, 628, 636, 637, or 638.

[0328] In some embodiments, the second Fc polypeptide can have a knob mutation (e.g., T139W numbered with reference to SEQ ID NO:1). In some embodiments, the second Fc polypeptide can have a knob mutation (e.g., T139W numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G or P102S numbered with reference to SEQ ID NO:1 (e.g., L7A and L8A; L7A, L8A, and P102G; or L7A, L8A, and P102S)), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of SEQ ID NO:655. In some embodiments, the second Fc polypeptide having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:655.

[0329] Exemplary Proteins Containing Non-Targeting Fab Fragments In some embodiments, the first and second Fc polypeptides are each linked to an NTF or portion thereof to generate a Fab-Fc dimeric fusion.

[0330] In some embodiments, the NTF or portion thereof comprises a non-binding variable region (NBVR). The NBVR comprises a light chain variable region and a heavy chain variable region and does not specifically bind to an epitope that occurs naturally in a subject. In some embodiments, the NBVR does not specifically bind to an antigen expressed in a given mammal, mammalian tissue, or mammalian cell type. The antigen can be a mammalian antigen, or an antigen found in a mammal, such as one derived from an infectious organism, such as a virus, bacteria, fungus, or parasite. The mammal can be, but is not limited to, a non-human primate, a human, or a rodent (e.g., a mouse). The NBVR can be, but is not limited to, an scFv.

[0331] Specific binding of an antibody to an antigen means an affinity of at least 106M-1. Specific binding is binding that is detectably greater in magnitude and distinguishable from non-specific binding that occurs to at least one unrelated target. Non-specific binding is often the result of van der Waals forces. Non-binding does not mean that the NBVR does not bind to the antigen with any affinity. Rather, in some embodiments, the NBVR does not exhibit specific binding to (a) any protein or epitope in a mammalian cell, mammalian tissue, or mammal, (b) a protein or epitope accessible on the surface of a mammalian cell or mammalian tissue, or (c) a protein or epitope accessible in the serum of a mammalian tissue or mammal.

[0332] In some embodiments, the NBVR is a humanized NBVR. A humanized Fab may be humanized in one or more of the light chain variable domain, the heavy chain variable domain, the light chain constant domain, and the heavy chain constant (CH1) domain. A humanized NBVR is a genetically engineered NBVR in which the CDRs of a non-human "donor" antibody are grafted onto the heavy and / or light chain variable region, light chain constant region, and / or heavy chain CH1 region sequences of a human "acceptor" antibody (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody that has at least three, four, five, or all CDRs that are completely or substantially derived from a donor antibody, and completely or substantially human antibody variable region framework sequences and / or constant region sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs that are completely or substantially derived from a donor antibody heavy chain, and heavy chain variable region framework sequences and heavy chain constant region sequences, if present, that are substantially derived from human heavy chain variable region framework sequences and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs that are completely or substantially derived from a donor antibody light chain, and light chain variable region framework sequences and light chain constant region sequences, if present, that are substantially derived from human light chain variable region framework and constant region sequences. The CDRs of a humanized antibody are substantially derived from the corresponding CDRs in a non-human antibody when at least 85%, 90%, 95% or 100% of the corresponding residues (any conventional definition, preferably those defined by Kabat) between each CDR are identical. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues defined by Kabat are identical.

[0333] In some embodiments, the NBVR is a chimeric NBVR. A chimeric NBVR comprises a non-human light and / or heavy chain variable region and a human heavy chain (CH1) and / or light chain constant region.

[0334] In some embodiments, the NBVR is a veneered NBVR. A veneered NBVR comprises a partially humanized light and / or heavy chain variable region and a human heavy chain (CH1) and / or light chain constant region.

[0335] Exemplary NBVRs include NBVR1 or NBVR2. Unless otherwise clear from the context, reference to NBVR1 or NBVR2 should be understood as referring to any of the murine, chimeric, veneered, humanized, and modified forms of NBVR1 or NBVR2.

[0336] Exemplary NTFs include NBVR1 or NBVR2. Unless otherwise clear from the context, reference to NBVR1 or NBVR2 should be understood as referring to any of the murine, chimeric, veneered, humanized, and modified forms of NBVR1 or NBVR2.

[0337] The sequences of the light and heavy chain variable regions of NBVR1 are set forth in SEQ ID NOs: 832 and 837, respectively. The sequences of the light and heavy chain variable regions of NBVR1 are set forth in SEQ ID NOs: 833 and 838, respectively.

[0338] In some embodiments, the NBVR comprises the CDR sequences of NBVR1. The light chain CDRs (L1, L2, and L3) of NBVR1 are set forth in SEQ ID NOs: 819, 821, and 823, respectively. The heavy chain CDRs (H1, H2, and H3) of NBVR1 are set forth in SEQ ID NOs: 825, 827, and 829, respectively. In some embodiments, the NBVR comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H3 sequences of NBVR1, and a CDR-H2 sequence comprising SEQ ID NO: 869.

[0339] In some embodiments, the NBVR comprises a light chain comprising the amino acid sequence of SEQ ID NO: 833 or 835, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 838, 839, 840, 841, 844, 845, 846, or 847.

[0340] In some embodiments, the NBVR comprises a light chain comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 832, 833, or 835, and a heavy chain comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 837, 838, 839, 840, 841, 844, 845, 846, or 847, and contains the CDR sequences of NBVR1 and maintains the non-binding properties of NBVR1.

[0341] In some embodiments, NBVRs comprise light and heavy chain variable regions that differ from the NBVR1 light and heavy chain variable regions by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are NBVRs having 1, 2, 3, 4, 5, or 6 CDRs, as defined by any conventional definition (preferably Kabat), that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of NBVR1 or NBVR2.

[0342] The sequences of the light and heavy chain variable regions of NBVR2 are set forth in SEQ ID NOs: 851 and 853, respectively. The sequences of the light and heavy chains of NBVR2 are set forth in SEQ ID NOs: 851 and 854, respectively.

[0343] In some embodiments, the NBVR comprises the CDR sequences of NBVR2. The light chain CDRs (L1, L2, and L3) of NBVR2 are set forth in SEQ ID NOs: 820, 822, and 824, respectively. The heavy chain CDRs (H1, H2, and H3) of NBVR2 are set forth in SEQ ID NOs: 826, 828, and 829, respectively. In some embodiments, the NBVR comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H3 sequences of NBVR2, and a CDR-H2 sequence comprising SEQ ID NO: 869.

[0344] In some embodiments, the NBVR comprises a light chain comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 850, 851, or 852, and a heavy chain comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 853, 854, 855, 856, 857, 859, 860, 861, or 862, and contains the CDR sequences of NBVR2 and maintains the non-binding properties of NBVR2.

[0345] In some embodiments, NBVRs comprise light and heavy chain variable regions that differ from the NBVR2 light and heavy chain variable regions by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are NBVRs having 1, 2, 3, 4, 5, or 6 CDRs, defined by any conventional definition (preferably Kabat), that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of NBVR1 or NBVR2.

[0346] In some embodiments, an NBVR comprises light and heavy chain variable regions having some or all (e.g., 3, 4, 5, and 6) CDRs that are completely or substantially derived from NBVR1 or NBVR2. Such NBVRs can comprise a heavy chain variable region having at least two, and usually all three, CDRs that are completely or substantially derived from the heavy chain variable region of NBVR1 or NBVR2, and / or a light chain variable region having at least two, and usually all three, CDRs that are completely or substantially derived from the light chain variable region of NBVR1 or NBVR2. When a CDR contains no more than 4, 3, 2, or 1 substitution, insertion, or deletion, the CDR is substantially derived from the corresponding NBVR1 or NBVR2 CDR, except that CDR-H2 (as defined by Kabat) can contain no more than 6, 5, 4, 3, 2, or 1 substitution, insertion, or deletion. Such antibodies may have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the described NBVR1 or NBVR2 light and heavy chain amino acid sequences, retain their functional properties, and / or differ from NBVR1 or NBVR2. In some embodiments, the NBVR does not exhibit specific binding to (a) any protein or epitope that occurs naturally in a mammalian cell, mammalian tissue, or mammal, (b) any protein or epitope that is accessible on the surface on a native mammalian cell or mammalian tissue, or (c) any protein or epitope that is accessible in the serum of a native mammalian tissue or mammal.

[0347] In some embodiments, a nucleic acid encoding an NBVR light chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 832, 833, 835, 850, 851, or 852. In some embodiments, a nucleic acid encoding an NBVR heavy chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 837, 838, 839, 840, 841, 844, 845, 846, 847, 853, 854, 855, 856, 857, 859, 860, 861, or 862.

[0348] In some embodiments, a nucleic acid encoding an NBVR light chain comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 864 or 866. In some embodiments, a nucleic acid encoding an NBVR heavy chain comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 865 or 867.

[0349] Cells are described that contain nucleic acids encoding the heavy and light chains of any of the described NBVRs. In some embodiments, the cells contain a nucleic acid encoding an NBVR light chain comprising the amino acid sequence of SEQ ID NO: 832, 833, or 835, and a nucleic acid encoding an NBVR heavy chain comprising the amino acid sequence of SEQ ID NO: 837, 838, 839, 840, 841, 844, 845, 846, or 847. In some embodiments, the cells contain a nucleic acid encoding an NBVR light chain comprising the amino acid sequence of SEQ ID NO: 850, 851, or 852, and a nucleic acid encoding an NBVR heavy chain comprising the amino acid sequence of SEQ ID NO: 853, 854, 855, 856, 857, 859, 860, 861, or 862. The cells can be bacterial cells, yeast cells, insect cells, or mammalian cells.

[0350] Protein variants engineered for conjugation to "L" As described herein, the TfR binding protein may be linked to the oligonucleotide(s) via a linking group "L". In one embodiment, the protein includes one or more amino acid residues (e.g., amino acid residues present at accessible sites in the protein) that can be used to link the protein to L. For example, in one embodiment, the protein includes one or more cysteine ​​residues (e.g., cysteine ​​residues present at accessible sites in the protein). In certain embodiments, the protein is linked to L via a cysteine ​​residue of the protein (e.g., via a sulfur atom of the cysteine ​​residue). In other embodiments, the protein includes one or more glutamine residues. In certain embodiments, the protein is linked to L via a glutamine residue (e.g., via an amide bond in the side chain of the glutamine residue).

[0351] In other aspects, it may be desirable to generate engineered proteins with one or more modification sites. These modification sites may be used to facilitate attachment of P to each L. For example, P may be attached to each L at the modification site. In other embodiments, the modification site allows for attachment of L to an amino acid residue located near the modification site (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the modification site, within 2 or 3 amino acids of the modification site, etc.). In certain embodiments, such modification sites are substituted residues that occur at accessible sites of the protein. In certain embodiments, the proteins described herein include one or more modification sites (e.g., one or more amino acid substitutions, such as cysteine, alanine, or glycine substitutions). In certain embodiments, the proteins include at least or exactly 1, 2, 3, 4, 5, 6, 7, or 8 modification sites. In certain embodiments, the proteins include 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 modification sites. In certain embodiments, the protein comprises between two and four modification sites.

[0352] In certain embodiments, the modification site in the protein (P) is an amino acid substitution or insertion. In certain embodiments, the protein (P) is or comprises an Fc dimer, e.g., where at least one of the Fc polypeptides is modified to bind to TfR. The modification site in the protein (P) can be in the Fc polypeptide that binds to TfR and / or in the Fc polypeptide that dimerizes with the Fc polypeptide that binds to TfR. In certain embodiments, the Fc polypeptide(s) can be part of a Fab-Fc fusion or a Fab-Fc dimer fusion, and the modification site can be in the Fab-Fc polypeptide that binds to TfR and / or in the Fab-Fc polypeptide that dimerizes with the Fab-Fc polypeptide that binds to TfR.

[0353] In certain embodiments, the modification site is in the CL domain. In certain embodiments, the modification site is in the CH1 domain. In certain embodiments, the modification site is in the CH2 domain. In certain embodiments, the modification site is in the CH3 domain.

[0354] In certain embodiments, the modification site is an amino acid substitution. In certain embodiments, the modification site is a cysteine, glycine, or alanine substitution.

[0355] In certain embodiments, the modification site is a cysteine ​​substitution. By substituting these residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the protein, which can be used to conjugate the protein (P) to the oligonucleotide (X) via the linking group (L) to generate the conjugates described herein. In certain embodiments, the protein comprises an Fc polypeptide or an Fc polypeptide dimer and comprises a cysteine ​​substitution selected from the group consisting of S239C, S442C, A330C, and T289C, the positions and substitutions being according to EU numbering. In other embodiments, the Fc polypeptide is attached to the CH1 domain and comprises an A114C substitution. In other embodiments, the protein comprises a Fab-Fc fusion, and the light chain comprises a K149C substitution.

[0356] In other aspects, the modification site is an alanine or glycine substitution. Such modified amino acids may facilitate enzymatic conjugation of L to proteins (e.g., using bacterial transglutaminase (BTG)) at nearby amino acids such as glutamine residues. For example, in certain embodiments, the alanine / glycine substitution is N297A or N297G, where the positions and substitutions are according to EU numbering. These substitutions remove glycosylation at position 297 and prevent enzymatic conjugation of the linker to the protein at position Q295 (i.e., the linker is attached to the protein via an amide bond of the glutamine side chain). Thus, in certain embodiments, the modification site is N297A or N297G, and the protein (P) is attached to L at Q295 (e.g., by enzymatic conjugation).

[0357] Thus, certain embodiments include a) one or more cysteine, alanine, or glycine substitutions (e.g., S239C, S442C, A330C, K149C (light chain), T289C, N297A, and / or N297G according to EU numbering, and / or A114C according to Kabat numbering); b) a modified constant domain (e.g., a modified CH2 or CH3 domain) that specifically binds to a transferrin receptor as described herein (e.g., a modified CH3 domain that includes 5, 6, 7, 8, or 9 substitutions at a set of amino acid positions including 157, 159, 160, 161, 162, 163, 186, 189, and 194, wherein the substitutions and positions are determined with reference to SEQ ID NO:1); and an Fc polypeptide, an Fc dimer (e.g., including an Fc polypeptide (a "first Fc polypeptide") and a second Fc polypeptide), or a Fab-Fc fusion (e.g., including an Fc dimer). In certain embodiments, the Fc polypeptide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NOs: 4-29, 236-299, 361-372, 397-408, 422-435, 491-497, 512-518, 632-633, 645-654, 702-708, 710-716, 718, 738-740, and 742. In certain embodiments, the N-terminus of the Fc polypeptide comprises a portion of the hinge region (e.g., DKTHTCP (SEQ ID NO: 232) or DKTHTCPPCP (SEQ ID NO: 233)). Thus, in some embodiments, the Fc polypeptide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 804 and 741. In certain embodiments, the N-terminus of the Fc polypeptide is linked to a CH1 domain sequence via a hinge region. Thus, in some embodiments, the protein comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 709, 717, and 743-745.In certain embodiments, the Fc polypeptide has a tryptophan at a position corresponding to position 139 of SEQ ID NO: 1. In certain embodiments, the second polypeptide or second Fab-Fc fusion may or may not comprise a modified constant domain. In certain embodiments, the second polypeptide or second Fab-Fc fusion may or may not comprise one or more modified sites (e.g., S239C, S442C, A330C, K149C (light chain), T289C, N297A, and / or N297G according to EU numbering, and / or A114C according to Kabat numbering).

[0358] Certain embodiments also provide a protein that is or comprises an Fc polypeptide dimer or a Fab-Fc dimer fusion thereof comprising a first and a second Fc polypeptide, wherein the first Fc polypeptide comprises a modified constant domain (e.g., a modified CH2 or CH3 domain) that specifically binds to the transferrin receptor, the second Fc polypeptide is capable of dimerizing with the first Fc polypeptide, and the first and / or second Fc polypeptide comprises one or more modification sites, or the first and / or second Fab-Fc fusion comprises one or more modification sites (e.g., one or more amino acid substitutions, such as cysteine ​​substitutions). In certain embodiments, the first Fc polypeptide comprises a modified CH3 domain that specifically binds to the transferrin receptor, and the modified CH3 domain comprises 5, 6, 7, 8, or 9 substitutions at the set of amino acid positions comprising: 157, 159, 160, 161, 162, 163, 186, 189, and 194. In certain embodiments, the first and second Fc polypeptides are each linked to a non-targeting Fab fragment or portion thereof to generate a Fab-Fc dimeric fusion. In certain embodiments, the first and second Fc polypeptides (or the first and second Fab-Fc fusions) of the dimer comprise substitutions that promote heterodimerization. For example, in certain embodiments, the first polypeptide or the first Fab-Fc fusion has a tryptophan instead of the native threonine at the position corresponding to position 139 of SEQ ID NO:1, and the second Fc polypeptide or the second Fab-Fc fusion has a valine at position 180 of SEQ ID NO:1, a serine at the position corresponding to position 139 of SEQ ID NO:1, and an alanine at the position corresponding to position 141 of SEQ ID NO:1.

[0359] In certain embodiments, the first Fc polypeptide or Fab-Fc fusion comprises one or more amino acid substitutions (e.g., one or more cysteine ​​substitutions). In certain embodiments, the first Fc polypeptide or Fab-Fc fusion comprises one or more substitutions selected from the group consisting of S239C, S442C, A330C, K149C (light chain), T289C, N297A and N297G according to EU numbering, and A114C according to Kabat numbering. In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises S239C. In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises S442C. In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises A330C. In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises T289C. In certain embodiments, the first Fc polypeptide or first Fab-Fc fusion comprises N297A. In certain embodiments, the first Fc polypeptide or first Fab-Fc fusion comprises N297G. In certain embodiments, the first Fc polypeptide or first Fab-Fc fusion comprises S239C and A330C. In certain embodiments, the first Fab-Fc fusion comprises K149C on the light chain. In certain embodiments, the first Fab-Fc fusion comprises A114C. In certain embodiments, the first Fc polypeptide or first Fab-Fc fusion comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NOs: 4-29, 236-299, 361-372, 397-408, 422-435, 491-497, 512-518, 632-633, 645-654, 702-708, 709-717, 718, 738-740, 741-746, and 804.

[0360] In certain embodiments, the second Fc polypeptide or Fab-Fc fusion comprises one or more amino acid substitutions (e.g., one or more cysteine ​​substitutions). In certain embodiments, the second Fc polypeptide or Fab-Fc fusion comprises one or more substitutions selected from the group consisting of S239C, S442C, A330C, K149C (light chain), T289C, N297A and N297G according to EU numbering, and A114C according to Kabat numbering. In certain embodiments, the second Fc polypeptide or second Fab-Fc fusion comprises S239C. In certain embodiments, the second Fc polypeptide or second Fab-Fc fusion comprises S442C. In certain embodiments, the second Fc polypeptide or second Fab-Fc fusion comprises A330C. In certain embodiments, the second Fc polypeptide or second Fab-Fc fusion comprises T289C. In certain embodiments, the second Fc polypeptide or the second Fab-Fc fusion comprises N297A. In certain embodiments, the second Fc polypeptide or the second Fab-Fc fusion polypeptide comprises N297G. In certain embodiments, the second Fc polypeptide or the second Fab-Fc fusion comprises S239C and A330C. In certain embodiments, the second Fab-Fc fusion comprises K149C on the light chain. In certain embodiments, the second Fab-Fc fusion comprises A114C. In certain embodiments, the second Fc polypeptide or the second Fab-Fc fusion comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NOs: 557-561, 627-631, 635-644, 719-723, 724-731, 733-737, and 810.

[0361] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide (or the first and second Fab-Fc fusions) each comprise one or more amino acid substitutions (e.g., one or more cysteine ​​substitutions). In certain embodiments, the one or more substitutions are S239C, S442C, A330C, T289C, N297A, and / or N297G according to EU numbering, and A114C according to Kabat numbering. In certain embodiments, the light chain comprises a K149C substitution. In certain embodiments, the one or more substitutions are S239C, S442C, A330C, K149C (light chain), A114C, and / or T289C. In certain embodiments, the one or more substitutions are S239C, S442C, A114C, and / or T289C. In certain embodiments, the one or more substitutions are N297A and / or N297G. In certain embodiments, the first and second Fc polypeptides (or the first and second Fab-Fc fusions) each comprise one or more amino acid substitutions (e.g., one cysteine ​​substitution). In certain embodiments, the first and second Fc polypeptides each comprise a cysteine ​​substitution at S239C, or the first and second Fab-Fc fusions each comprise a cysteine ​​substitution at S239C. In certain embodiments, the first and second Fc polypeptides each comprise two amino acid substitutions (e.g., two cysteine ​​substitutions), or the first and second Fab-Fc fusions each comprise two amino acid substitutions (e.g., cysteine ​​substitutions). In certain embodiments, the first and second Fc polypeptides each comprise a cysteine ​​substitution at S239C and A330C, or the first and second Fab-Fc fusions each comprise a cysteine ​​substitution at S239C and A330C.

[0362] In certain embodiments, the first Fc polypeptide or first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 279, 361, 362, 363, 743-744, or 804, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ala at position 162, Asn at position 163, Thr at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with respect to SEQ ID NO: 1, and the second Fc polypeptide or second Fab-Fc fusion comprises the sequence of any one of SEQ ID NOs: 559-561, 719-731, or 810. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 362 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 559. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 362 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 560. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 362 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 561. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 363 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 726. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 744 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 731. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 363 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 725. In certain embodiments, the N-terminus of the first and second Fc polypeptides comprises a portion of the hinge region (e.g., DKTHTCP (SEQ ID NO: 232) or DKTHTCPPCP (SEQ ID NO: 233)).Thus, in a particular embodiment, the first polypeptide comprises the sequence of SEQ ID NO:804 and the second polypeptide comprises the sequence of SEQ ID NO:810.

[0363] In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 645-648, 741, and 745, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ala at position 162, Asn at position 163, Thr at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered based on SEQ ID NO: 1, and the second Fc polypeptide or the second Fab-Fc fusion comprises a sequence of any one of SEQ ID NOs: 639-641 and 733-737. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO:647 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO:639. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO:647 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO:640. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO:647 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO:641. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO:648 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO:735. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO:745 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO:737. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 648 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 734. In certain embodiments, the N-terminus of the first and second Fc polypeptides comprises a portion of the hinge region (e.g., DKTHTCP (SEQ ID NO: 232) or DKTHTCPPCP (SEQ ID NO: 233)).Thus, in a particular embodiment, the first polypeptide comprises the sequence of SEQ ID NO:741 and the second polypeptide comprises the sequence of SEQ ID NO:736.

[0364] In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 702-717, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ala at position 162, Asn at position 163, Thr at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO: 1, and the second Fc polypeptide or the second Fab-Fc fusion comprises the sequence of any one of SEQ ID NOs: 557-561, 627, 719-731, and 810. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 702 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 559. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 708 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 722. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 713 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 726. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 713 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 627.

[0365] In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 738-741, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ala at position 162, Asn at position 163, Thr at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO: 1, and the second Fc polypeptide or the second Fab-Fc fusion comprises any one of the sequences of SEQ ID NOs: 635-637, 639-641, and 733-737. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 738 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 639. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 739 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 733. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 740 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 735. In certain embodiments, the first polypeptide or first Fab-Fc fusion comprises the sequence of SEQ ID NO: 740 and the second polypeptide or second Fab-Fc fusion comprises the sequence of SEQ ID NO: 637.

[0366] In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 281 or 718, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser at position 162, Asn at position 163, Ser at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO: 1, and the second Fc polypeptide or the second Fab-Fc fusion comprises the sequence of any one of SEQ ID NOs: 559-561, 719-731, or 810. In a specific embodiment, the first polypeptide or the first Fab-Fc fusion comprises the sequence of SEQ ID NO:718 and the second polypeptide or the second Fab-Fc fusion comprises the sequence of SEQ ID NO:559.

[0367] In certain embodiments, the first Fc polypeptide or the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 742 or 746, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser at position 162, Asn at position 163, Ser at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO: 1, and the second Fc polypeptide or the second Fab-Fc fusion comprises the sequence of any one of SEQ ID NOs: 639-641 and 733-737. In a specific embodiment, the first polypeptide or the first Fab-Fc fusion comprises the sequence of SEQ ID NO:742 and the second polypeptide or the second Fab-Fc fusion comprises the sequence of SEQ ID NO:639.

[0368] In certain embodiments, the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 279, 361, 362, 363, 743-744, or 804, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ala at position 162, Asn at position 163, Thr at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered relative to SEQ ID NO: 1, and the second Fab-Fc fusion comprises the sequence of any one of SEQ ID NOs: 557-558, and 627. In certain embodiments, the first and Fab-Fc fusions each further comprise a CL comprising the sequence of SEQ ID NO: 732.

[0369] In certain embodiments, the first Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 645-648, 741, and 745, the polypeptide comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ala at position 162, Asn at position 163, Thr at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered relative to SEQ ID NO: 1, and the second Fab-Fc fusion comprises the sequence of any one of SEQ ID NOs: 635-637. In certain embodiments, the first and Fab-Fc fusions each further comprise a CL comprising the sequence of SEQ ID NO: 732.

[0370] Fc polypeptides or Fab-Fc fusions, or dimers thereof, described herein, containing one or more modification sites (e.g., cysteine ​​substitutions) (P) may be used in the conjugates described herein. Thus, certain embodiments also provide a conjugate of formula I: P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide (e.g., selected from an ASO and an RNAi agent); each L is independently a linking group as described herein; P is a protein comprising a first Fc polypeptide as described herein, or a dimer thereof, or a Fab-Fc fusion as described herein, or a dimer thereof, and comprising one or more modification sites (e.g., cysteine ​​substitutions), which modification sites facilitate binding of P to each L; each y is independently 1 or greater (e.g., 1, 2, 3, or 4); n is 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0371] In certain embodiments, one or more oligonucleotides are bound to the linking group (L). In certain embodiments, two or more oligonucleotides are bound to the linking group (L). In certain embodiments, one oligonucleotide is bound to the linking group (L). In certain embodiments, two oligonucleotides are bound to the linking group (L).

[0372] In embodiments, useful values ​​of y include integers from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0373] In embodiments, useful values ​​of n include integers from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0374] X.How to use The conjugates described herein may be used for a variety of purposes, including therapeutic applications.

[0375] In some embodiments, the conjugates are used to deliver oligonucleotides (e.g., ASOs or RNAi agents) to target cell types that express the transferrin receptor. In some embodiments, the conjugates can be used to transport the oligonucleotides (e.g., ASOs or RNAi agents) across the endothelium, e.g., the blood-brain barrier, and be taken up into the brain.

[0376] For example, certain embodiments provide a method of transcytosis of an oligonucleotide (e.g., an ASO or an RNAi agent) across the endothelium, comprising contacting the endothelium (e.g., the blood-brain barrier (BBB)) with a conjugate described herein. Accordingly, certain embodiments provide a method of transporting an oligonucleotide through the BBB in a subject in need of oligonucleotide transport, comprising administering to the subject a conjugate described herein. In certain embodiments, a conjugate described herein is provided for use in transporting an oligonucleotide through the BBB in a subject in need of oligonucleotide transport. In some embodiments, provided herein is a method of delivering an oligonucleotide to the CNS. In some embodiments, provided herein is a method of delivering an oligonucleotide to deep brain regions (e.g., the cortex, brainstem, hippocampus, striatum, cerebellum, thalamus, caudate putamen, and substantia nigra). In some embodiments, provided herein is a method of delivering an oligonucleotide to deep brain regions and the spinal cord (e.g., cervical spinal cord, lumbar spinal cord). In some embodiments, provided herein is a method of delivering an oligonucleotide to the CNS and muscle (e.g., the heart and skeleton). In some embodiments, provided herein are methods for delivering oligonucleotides to the CNS, peripheral nerves (e.g., retina, sciatic nerve), muscles (e.g., quadriceps), and other peripheral organs (e.g., heart, diaphragm, spleen, intestine, lungs, liver, kidneys).

[0377] Certain embodiments also provide a method of modulating expression of a target gene or sequence in a subject in need thereof comprising administering to the subject an effective amount of a conjugate as described herein. In some embodiments, a conjugate as described herein is provided for use in modulating expression of a target gene.

[0378] In certain embodiments, the target gene or sequence is expressed in cells in the brain of a subject.In certain embodiments, the target gene or sequence is expressed in cells that express TfR.In certain embodiments, the target gene or sequence is expressed in muscle cells, such as skeletal muscle cells or cardiac muscle cells.

[0379] In certain embodiments, the regulation of target gene expression is gene knockdown or gene knockout.Thus, in certain embodiments, the expression of target gene or sequence is inhibited or reduced, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, compared with the expression in control (e.g., the subject that does not receive the conjugate).

[0380] The conjugates described herein are administered to subjects in a therapeutically effective amount or dose.However, the dosage may vary according to several factors, including the selected administration route, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight, and the prescribing physician's judgment.Dosage may be increased or decreased over time according to the needs of an individual patient.

[0381] In various embodiments, the conjugates described herein are administered parenterally. In some embodiments, the conjugates are administered intravenously. Intravenous administration can be performed, for example, by infusion over about 10 to about 30 minutes, or over at least 1 hour, 2 hours, or 3 hours. In some embodiments, the conjugates are administered as an intravenous bolus. A combination of infusion and bolus administration can also be used.

[0382] In some parenteral embodiments, the conjugate is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the conjugate is administered intradermally or intramuscularly. In some embodiments, the conjugate is administered intrathecally, such as by epidural administration, or intracerebroventricularly.

[0383] In other embodiments, the conjugates can be administered orally, pulmonary, intranasally, intraocularly, or topically. Pulmonary administration can be employed, for example, by use of an inhaler or nebulizer and a formulation which includes an aerosolizing agent.

[0384] XI. Pharmaceutical Compositions and Kits In other aspects, pharmaceutical compositions and kits are provided that include the conjugates described herein.

[0385] Pharmaceutical Compositions Guidance for preparing formulations for the uses described herein can be found in many handbooks on pharmaceutical preparations and formulations known to those skilled in the art.

[0386] In some embodiments, the pharmaceutical composition comprises a conjugate as described herein and further comprises one or more pharma- ceutically acceptable carriers and / or excipients. In certain embodiments, the composition comprises a plurality of conjugates as described herein, which may be identical or different (e.g., a mixture of different conjugates). In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 1:1 to about 4:1. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 1:1 to about 2:1. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 1.23. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 2:1 to about 3:1. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 2.5.

[0387] As used herein, the term pharma- ceutically acceptable carrier includes any solvent, dispersion medium, or coating agent that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the active agent. A variety of pharma- ceutically acceptable excipients are well known in the art. In some embodiments, the carrier is suitable for intravenous, intrathecal, intracerebroventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. The pharma- ceutically acceptable carrier can include one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the conjugate. Physiologically acceptable compounds can include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agent, or excipients or other stabilizers and / or buffers. Other pharma- ceutically acceptable carriers and their formulations are also available in the art.

[0388] The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The following methods and excipients are merely illustrative and in no way limiting.

[0389] For oral administration, the conjugates described herein can be formulated by combining with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, and the like, to be taken orally by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the conjugates with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules to obtain tablets or dragee cores, after adding suitable excipients, if desired. Suitable excipients include fillers such as sugars, including, for example, lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

[0390] As disclosed above, the conjugates described herein can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the conjugates can be formulated into preparations by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvents, for example, vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, and, if necessary, with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, the conjugates can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, and physiological saline buffer. The formulations for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of a preservative. The compositions can take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0391] Typically, pharmaceutical compositions for use in in vivo administration are sterile, which can be achieved by methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0392] The dosage and desired drug concentration of the pharmaceutical compositions described herein may vary depending on the specific use envisaged. The determination of the appropriate dosage or route of administration is well within the skill of the art. Suitable dosages are also described above.

[0393] kit In some embodiments, a kit is provided that includes the conjugates described herein. In some embodiments, the kit is for use in modulating expression of a target gene or sequence (e.g., expression of a target gene in the brain or central nervous system (CNS)). In some embodiments, the kit is for use in modulating expression of a target gene.

[0394] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a conjugate as described herein and further comprises one or more additional therapeutic agents. In some embodiments, the kit further comprises instructions including instructions (i.e., protocols) for carrying out the methods described herein (e.g., instructions for using the kit to administer a composition across the blood-brain barrier). Instructions typically include, but are not limited to, written or printed material. Any medium capable of storing such instructions and transmitting them to an end user is contemplated herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses to internet sites providing such instructional materials.

[0395] The subject matter described herein includes the following non-limiting embodiments.

[0396] 1. (a) a first Fc polypeptide comprising a modified constant domain that specifically binds to the transferrin receptor; (b) a second Fc polypeptide capable of dimerizing with the first Fc polypeptide of (a); and or a Fab-Fc dimer fusion thereof, The Fc polypeptide dimer or Fab-Fc dimer fusion thereof, wherein the first and / or the second Fc polypeptide comprises one or more cysteine ​​substitutions or the first and / or the second Fab-Fc fusion comprises one or more cysteine ​​substitutions.

[0397] 2. The Fc polypeptide dimer of embodiment 1, wherein said Fab-Fc dimer fusion is a non-targeted Fab-Fc dimer fusion.

[0398] 3. An Fc polypeptide dimer according to embodiment 1 or 2, wherein said cysteine ​​substitution(s) is selected from the group consisting of S239C, S442C, A330C, K149C, A118C, and T289C.

[0399] 4. The Fc polypeptide dimer of any one of embodiments 1 to 3, wherein the first and second Fc polypeptides each comprise a cysteine ​​substitution, or the first and second Fab-Fc fusions each comprise a cysteine ​​substitution.

[0400] 5. The Fc polypeptide dimer of embodiment 4, wherein said first and second Fc polypeptides each comprise a cysteine ​​substitution at S239C or said first and second Fab-Fc fusions each comprise a cysteine ​​substitution at S239C.

[0401] 6. The Fc polypeptide dimer of any one of embodiments 1 to 5, wherein the first and second Fc polypeptides each comprise two cysteine ​​substitutions, or the first and second Fab-Fc fusions each comprise two cysteine ​​substitutions.

[0402] 7. The Fc polypeptide dimer of embodiment 6, wherein said first and second Fc polypeptides comprise cysteine ​​substitutions at S239C and A330C, respectively, or said first and second Fab-Fc fusions comprise cysteine ​​substitutions at S239C and A330C, respectively.

[0403] 8. The Fc polypeptide dimer of any one of embodiments 1 to 3, wherein said second Fc polypeptide comprises a cysteine ​​substitution or said second Fab-Fc fusion comprises a cysteine ​​substitution.

[0404] 9. The Fc polypeptide dimer of embodiment 8, wherein said cysteine ​​substitution is S239C.

[0405] 10. (a) a first Fc polypeptide comprising a modified constant domain that specifically binds to the transferrin receptor; (b) a second Fc polypeptide capable of dimerizing with the first Fc polypeptide of (a); and An Fc polypeptide dimer comprising: The Fc polypeptide dimer, wherein the first and / or the second Fc polypeptide comprises one or more substitutions selected from the group consisting of N297A and N297G.

[0406] 11. The Fc polypeptide dimer of embodiment 10, wherein said first and said second Fc polypeptides are each linked to a non-targeting Fab fragment or portion thereof to generate a Fab-Fc dimeric fusion.

[0407] 12. The modified constant domain has, as numbered with respect to SEQ ID NO:1, at position 153, Trp, Tyr, Leu, Gln, or Glu; at position 157, Leu, Tyr, Met, Val, Phe, or Trp; at position 159, Leu, Thr, His, Pro, or Phe; at position 160, Val, Pro, or an acidic amino acid; at position 161, Trp; at position 162, Val, Ser, Ala, or Gly; at position 163, Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Thr, or Glu; at position 164, Ser, Thr, Gln, Phe, Tyr, or Val; and at position 165, Gln, Phe, or Hi. 12. The Fc polypeptide dimer of any one of embodiments 1 to 11, wherein the Fc polypeptide dimer is a modified CH3 domain comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions selected from: 186 is Glu, Ala, Ser, Leu, Thr, Pro, or Asp; 187 is Lys, Arg, Gly, or Pro; 188 is Glu, or Ser; 189 is Thr, Asn, or an acidic amino acid; 194 is Trp, Tyr, His, or Phe; 197 is Ser, Thr, Glu, Lys, or Trp; and 199 is Ser, Trp, Gly, Cys, Pro, or Met.

[0408] 13. The first Fc polypeptide or Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 279, 281, 361-366, 491-494, 702-718, 632, 645-649, 738-746, 804; The second Fc polypeptide or Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 557 to 561, 627 to 631, 635 to 644, 719 to 723, 724 to 731, 733 to 737, and 810; An Fc polypeptide dimer according to any one of embodiments 1 to 12.

[0409] 14. The Fc polypeptide dimer of embodiment 13, wherein the first Fc polypeptide or Fab-Fc fusion comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser or Ala at position 162, Asn at position 163, Thr or Ser at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO:1.

[0410] 15. the first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362, and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 702, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 708, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 722; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 718, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 560; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362 and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 561; or The first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 804, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 810. An Fc polypeptide dimer according to embodiment 13.

[0411] 16. A conjugate of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein that contains 1) a modified constant domain that specifically binds to the transferrin receptor, and 2) one or more modification sites that facilitate binding of P to each L; each y is independently at least 1; The conjugate, wherein n is at least 1.

[0412] 17. The conjugate of embodiment 16, wherein said protein (P) comprises an Fc polypeptide comprising said modified constant domain.

[0413] 18. The conjugate of embodiment 16, wherein the protein comprises an Fc polypeptide dimer comprising a first Fc polypeptide comprising the modified constant domain and a second Fc polypeptide capable of dimerizing to the first Fc polypeptide.

[0414] 19. The conjugate of embodiment 17 or 18, wherein said Fc polypeptide is linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc fusion, or wherein said first and second Fc polypeptides of said Fc polypeptide dimer are each linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc dimer fusion.

[0415] 20. The conjugate according to any one of embodiments 16 to 19, wherein the modification site is an amino acid substitution or insertion.

[0416] 21. The conjugate according to embodiment 20, wherein the modification site is a cysteine ​​substitution.

[0417] 22. The conjugate of embodiment 21, wherein said cysteine ​​substitutions are selected from the group consisting of S239C, S442C, A330C, K149C, A118C, and T289C according to EU numbering.

[0418] 23. The linking group L comprises a group of the formula: [ka] 23. The conjugate of any one of embodiments 16 to 22, wherein P is attached to the sulfur atom of the modification site at the position marked with an *.

[0419] 24. The conjugate of embodiment 20, wherein the modification site is an alanine or glycine substitution.

[0420] 25. The conjugate of embodiment 24, wherein said alanine or glycine substitution is N297A or N297G according to EU numbering.

[0421] 6. The conjugate according to any one of embodiments 16 to 20 and 24 to 25, which is prepared by conjugation of L to P with Q295 by enzymatic conjugation.

[0422] 27. The conjugate of embodiment 26, wherein the enzymatic conjugation uses bacterial transglutaminase (BTG).

[0423] 28. The modified constant domain has, numbered with respect to SEQ ID NO:1, at position 153, Trp, Tyr, Leu, Gln, or Glu; at position 157, Leu, Tyr, Met, Val, Phe, or Trp; at position 159, Leu, Thr, His, Pro, or Phe; at position 160, Val, Pro, or an acidic amino acid; at position 161, Trp; at position 162, Val, Ser, Ala, or Gly; at position 163, Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Thr, or Glu; at position 164, Ser, Thr, Gln, Phe, Tyr, or Val; at position 165, Gln, Phe, or is His, at position 186 is Glu, Ala, Ser, Leu, Thr, Pro, or Asp, at position 187 is Lys, Arg, Gly, or Pro, at position 188 is Glu, or Ser, at position 189 is Thr, Asn, or an acidic amino acid, at position 194 is Trp, Tyr, His, or Phe, at position 197 is Ser, Thr, Glu, Lys, or Trp, and at position 199 is Ser, Trp, Gly, Cys, Pro, or Met.

[0424] 29. The first Fc polypeptide or Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 279, 281, 361-366, 491-494, 702-718, 632, 645-649, 738-746, 804; The conjugate of any one of embodiments 16 to 28, wherein the second Fc polypeptide or Fab-Fc fusion comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 557 to 561, 627 to 631, 635 to 644, 719 to 723, 724 to 731, 733 to 737, and 810.

[0425] 30. The conjugate of embodiment 29, wherein the first Fc polypeptide or Fab-Fc fusion comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser or Ala at position 162, Asn at position 163, Thr or Ser at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO:1.

[0426] 31. The first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362, and the second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 702, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 708, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 722; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 718, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 559; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362, and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 560; said first Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 362 and said second Fc polypeptide or Fab-Fc fusion comprises the amino acid sequence of SEQ ID NO: 561; or The first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 804, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 810. 30. The conjugate of embodiment 29.

[0427] 32. A conjugate according to any one of embodiments 16 to 31, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or an siRNA.

[0428] 33. The conjugate of embodiment 32, wherein each oligonucleotide is independently an ASO.

[0429] 34. The ASO has the formula 5'-X a -Y a -Z a -3' modification pattern, wherein X a and Z a each of the gap regions Y a 34. The conjugate of embodiment 33, wherein

[0430] 35. The conjugate of embodiment 33 or 34, wherein each L is linked to the 5' end of the ASO.

[0431] 36. The conjugate of embodiment 33 or 34, wherein each L is linked to the 3' end of the ASO.

[0432] 37.L is a compound of the formula -(CH 2 ) 6 -NH-C 6 37. The conjugate of any one of embodiments 16 to 36, comprising an amine group.

[0433] 38. A conjugate according to any one of embodiments 16 to 37, wherein P binds to the transferrin receptor with an affinity of between 3 nM and 600 nM, or between 50 nM and 250 nM.

[0434] 39. The conjugate of embodiment 38, wherein P binds to the transferrin receptor with an affinity of about 100 nM.

[0435] 40. A conjugate according to any one of embodiments 16 to 18 and 20 to 39, wherein P does not comprise a Fab fragment or a portion thereof.

[0436] 41. A conjugate of formula I comprising two or more oligonucleotides, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1; The conjugate, wherein n is 2 or more.

[0437] 42. The conjugate of embodiment 41, wherein n is 2 or 4.

[0438] 43. The conjugate of embodiment 41 or 42, wherein said protein (P) comprises an Fc polypeptide comprising said modified constant domain.

[0439] 44. The conjugate of any one of embodiments 41 to 43, wherein the protein (P) comprises an Fc polypeptide dimer comprising a first Fc polypeptide comprising the modified constant domain and a second Fc polypeptide capable of dimerizing to the first Fc polypeptide.

[0440] 45. The conjugate of embodiment 43 or 44, wherein the Fc polypeptide is linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc fusion, or the first and second Fc polypeptides of the Fc polypeptide dimer are each linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc dimer fusion.

[0441] 46. ​​A conjugate according to any one of embodiments 41 to 45, wherein the protein (P) comprises one or more modification sites facilitating binding of P to each L.

[0442] 47. The conjugate according to embodiment 46, wherein the modification site is an amino acid substitution or insertion.

[0443] 48. The conjugate according to embodiment 47, wherein the modification site is a cysteine ​​substitution.

[0444] 49. The conjugate of embodiment 48, wherein said cysteine ​​substitutions are selected from the group consisting of S239C, S442C, A330C, K149C, A118C, and T289C according to EU numbering.

[0445] 50. A conjugate according to any one of embodiments 44 to 49, wherein n is 2 and the first Fc polypeptide and the second Fc polypeptide are each linked to L via a cysteine ​​residue at position 239.

[0446] 51. A conjugate according to any one of embodiments 44 to 49, wherein n is 4 and the first Fc polypeptide and the second Fc polypeptide are linked to two L's via cysteine ​​residues at positions 239 and 330, respectively.

[0447] 52. A conjugate according to any one of embodiments 41 to 51, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or an siRNA.

[0448] 53. A conjugate of formula I comprising two or more oligonucleotides, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein containing a modified constant domain that specifically binds to the transferrin receptor, each y is independently at least 1, and at least one y is 2 or greater; The conjugate, wherein n is at least 1.

[0449] 54. The conjugate of embodiment 53, wherein y is 2.

[0450] 55. A conjugate according to any one of embodiments 53-54, wherein at least one L is separately attached to two oligonucleotides.

[0451] 56. The conjugate of any one of embodiments 53 to 55, wherein y is 2, at least one L is linked to the 5' end of a first oligonucleotide, and a second oligonucleotide is linked to the 3' end of said first oligonucleotide.

[0452] 57. The conjugate according to embodiment 56, wherein the first and second oligonucleotides are linked by a nucleic acid linker or a non-oligonucleotide cleavable linker.

[0453] 58. A conjugate according to any one of embodiments 53 to 57, wherein said protein (P) comprises an Fc polypeptide comprising said modified constant domain.

[0454] 59. The conjugate of any one of embodiments 53 to 57, wherein the protein (P) comprises an Fc polypeptide dimer comprising a first Fc polypeptide comprising the modified constant domain and a second Fc polypeptide capable of dimerizing to the first Fc polypeptide.

[0455] 60. The conjugate of embodiment 58 or 59, wherein the Fc polypeptide is linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc fusion, or the first and second Fc polypeptides of the Fc polypeptide dimer are each linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc dimer fusion.

[0456] 61. The conjugate according to any one of embodiments 53-60, wherein P further comprises one or more modification sites facilitating attachment of P to each L.

[0457] 62. The conjugate according to embodiment 61, wherein the modification site is an amino acid substitution or insertion.

[0458] 63. The conjugate according to embodiment 62, wherein the modification site is a cysteine ​​substitution.

[0459] 64. The conjugate of embodiment 63, wherein said cysteine ​​substitutions are selected from the group consisting of S239C, S442C, A330C, K149C, A118C, and T289C according to EU numbering.

[0460] 65. A conjugate according to any one of embodiments 53 to 64, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or an siRNA.

[0461] 66. A low affinity transferrin receptor binding conjugate of formula I, P-(L-(X) y ) n (I) During the ceremony, each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising a modified constant domain that specifically binds to the transferrin receptor with an affinity of about 3 nM to about 600 nM; each y is independently at least 1; The conjugate, wherein n is at least 1.

[0462] 67. The conjugate of embodiment 66, wherein P optionally binds to the transferrin receptor with an affinity of about 3 nM to about 600 nM or about 40 nM to about 1200 nM.

[0463] 68. The conjugate of embodiment 67, wherein P binds to the transferrin receptor with an affinity of about 50 nM to 250 nM.

[0464] 69. The conjugate of embodiment 68, wherein P binds to the transferrin receptor with an affinity of about 100 nM.

[0465] 70. A conjugate according to any one of embodiments 66 to 69, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or an siRNA.

[0466] 71. A conjugate according to any one of embodiments 66 to 70, wherein said protein (P) comprises an Fc polypeptide comprising said modified constant domain.

[0467] 72. The conjugate of any one of embodiments 66 to 70, wherein the protein (P) comprises an Fc polypeptide dimer comprising a first Fc polypeptide comprising the modified constant domain and a second Fc polypeptide capable of dimerizing to the first Fc polypeptide.

[0468] 73. The conjugate of embodiment 71 or 72, wherein the Fc polypeptide is linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc fusion, or the first and second Fc polypeptides of the Fc polypeptide dimer are each linked to a non-targeting Fab fragment or portion thereof to form a Fab-Fc dim...

Claims

**Claim 1** A conjugate of formula I, wherein: P-(L-(X)y)n (I) wherein each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising an engineered binding site that specifically binds to the transferrin receptor (TfR) with an affinity of about 3 nM to about 600 nM; each y is independently at least 1; n is at least 1, conjugate. **Claim 2** The conjugate according to claim 1, wherein the engineered binding site binds to TfR with an affinity of about 500 nM to about 3 nM, about 400 nM to about 20 nM, about 300 nM to about 30 nM, about 200 nM to about 40 nM, about 150 nM to about 50 nM, or about 130 nM to about 80 nM. **Claim 3** (a) P comprises a modified Fc polypeptide that specifically binds to TfR; (b) P comprises a modified Fc polypeptide, and the engineered binding site comprises a modified CH3 constant domain that specifically binds to TfR; or (c) P comprises an Fc polypeptide dimer comprising a first modified Fc polypeptide and a second Fc polypeptide, wherein the first modified Fc polypeptide comprises a modified CH3 constant domain that specifically binds to TfR, conjugate according to claim 1. **Claim 4** (a) The modified Fc polypeptide is linked to an untargeted Fab fragment or a part thereof to form a Fab-Fc dimer fusion; (b) The first modified Fc polypeptide is linked to a first Fab fragment or a part thereof to form a first Fab-Fc fusion, and / or the second Fc polypeptide is linked to a second Fab fragment or a part thereof to form a second Fab-Fc dimer fusion; or (c) The first modified Fc polypeptide is linked to a first untargeted Fab fragment or a part thereof to form a first untargeted Fab-Fc fusion, and / or the second Fc polypeptide is linked to a second untargeted Fab fragment or a part thereof to form a second untargeted Fab-Fc dimer fusion, conjugate according to claim 3. **Claim 5** The conjugate according to claim 1, wherein P comprises one or more modification sites that facilitate the binding of P to each L. **Claim 6** The conjugate according to claim 5, wherein the one or more modification sites comprise one or more amino acid substitutions. **Claim 7** The one or more substitutions are (a) one or more cysteine substitutions, or (b) An N297A substitution and / or an N297G substitution according to EU numbering The conjugate according to claim 6, comprising the same. **Claim 8**: The conjugate according to claim 7, wherein the one or more cysteine substitutions are selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering, and A114C according to Kabat numbering. **Claim 9**: The conjugate according to claim 1, wherein at least one L is attached to the 5'-end of the first oligonucleotide, and the second oligonucleotide is linked to the 3'-end of the first oligonucleotide. **Claim 10**: The conjugate according to claim 1, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or siRNA. **Claim 11**: The conjugate according to claim 1, wherein the linking group L comprises a moiety having the following structure, [Chemical Formula 30] wherein * indicates the point of attachment to the sulfur atom of the modified site in P. **Claim 12**: (a) y is an integer from 1 to 4, and n is an integer from 1 to 6; (b) y is 1; (c) n is 1; or (d) y is 1 and n is 1, The conjugate according to claim 1. **Claim 13**: (a) A first Fc polypeptide comprising a modified CH3 constant domain that specifically binds to the transferrin receptor with an affinity of about 3 nM to about 600 nM, (b) A second Fc polypeptide dimerized to the first Fc polypeptide, An Fc polypeptide dimer, or a Fab-Fc dimer fusion thereof, comprising one or more cysteine substitutions. **Claim 14**: (i) The first Fc polypeptide is fused to a first Fab to form a first Fab-Fc fusion, and / or the second Fc polypeptide is fused to a second Fab to form a second Fab-Fc fusion; or (ii) The first Fc polypeptide is fused to a first non-targeting Fab to form a first non-targeting Fab-Fc fusion, and / or the second Fc polypeptide is fused to a second non-targeting Fab to form a second non-targeting Fab-Fc fusion. The Fc polypeptide dimer, or a Fab-Fc dimer fusion thereof, according to claim 13. **Claim 15**: (i) The first Fc polypeptide and / or the second Fc polypeptide comprises one or more cysteine substitutions selected from the group consisting of S239C, S442C, A330C, and T289C according to EU numbering. (ii) The first Fab-Fc fusion and / or the second Fab-Fc fusion, or the first non-targeted Fab-Fc fusion and / or the second non-targeted Fab-Fc fusion, comprises one or more cysteine substitutions selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering and substitutions according to Kabat numbering, A114C, or (iii) The first Fc polypeptide and / or the second Fc polypeptide comprises S239C substitution and A330C substitution according to EU numbering. The Fc polypeptide dimer according to claim 14, or its Fab-Fc dimer fusion. **Claim 16**: The Fc polypeptide dimer according to claim 13, or its Fab-Fc dimer fusion, linked to one or more oligonucleotides. **Claim 17**: A conjugate of formula I, wherein P-(L-(X)y)n (I) wherein each X is independently an oligonucleotide, each L is independently a linking group, P is a protein comprising (a) a modified CH3 constant domain that specifically binds to the transferrin receptor and (b) one or more modification sites that facilitate the binding of P to each L, each y is independently at least 1, and n is at least 1, the conjugate. **Claim 18**: P comprises an Fc polypeptide, an Fc polypeptide dimer, a Fab-Fc dimer fusion, or a non-targeted Fab-Fc dimer fusion, and the one or more modification sites are (a) one or more cysteine substitutions selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering and A114C according to Kabat numbering, and / or (b) N297A substitution and / or N297G substitution according to EU numbering The conjugate according to claim 17. **Claim 19**: Each L is independently of the following formula, wherein A is (C1-C15) alkylene, 【Chemical 37】 P comprises a Fab-Fc dimer fusion, and the modified CH3 constant domain specifically binds to TfR with an affinity of about 100 nM. The conjugate according to claim 18. ​

20. A conjugate of formula I, wherein: P-(L-(X)y)n (I) wherein each X is independently an oligonucleotide; each L is independently a linking group; P is a protein comprising a modified CH3 constant domain that specifically binds to the transferrin receptor; each y is independently at least 1; n is 2 or more, conjugate.

21. (a) n is 2 or 4; (b) n is 2 or 4 and y is 1; (c) at least one of y is 2 or more; or (d) y is 2, the conjugate according to Claim 20.

22. P comprises an Fc polypeptide, an Fc polypeptide dimer, a Fab-Fc dimer fusion, or a non-targeted Fab-Fc dimer fusion, (i) the Fc polypeptide dimer comprises a first Fc polypeptide and a second Fc polypeptide, and the first Fc polypeptide comprises the modified CH3 constant domain; (ii) the Fab-Fc dimer fusion comprises a first Fc polypeptide linked to a first Fab fragment or a portion thereof to form a first Fab-Fc fusion, and a second Fc polypeptide linked to a second Fab fragment or a portion thereof to form a second Fab-Fc fusion, and the first Fc polypeptide comprises the modified CH3 constant domain; or (iii) the Fab-Fc dimer fusion comprises a first Fc polypeptide linked to a first non-targeted Fab fragment or a portion thereof to form a first non-targeted Fab-Fc fusion, and a second Fc polypeptide linked to a second non-targeted Fab fragment or a portion thereof to form a second non-targeted Fab-Fc fusion, and the first Fc polypeptide comprises the modified CH3 constant domain, the conjugate according to Claim 20.

23. P comprises one or more modification sites that facilitate the binding of P to each L, the one or more modification sites are (i) one or more cysteine substitutions selected from the group consisting of S239C, S442C, A330C, K149C, and T289C according to EU numbering and A114C according to Kabat numbering; and / or (ii) N297A substitution and / or N297G substitution according to EU numbering the conjugate according to Claim 20.

24. A conjugate of formula I, wherein: P-(L-(X)y)n (I) wherein Each X is independently an oligonucleotide, Each L is independently a linking group, P is a protein comprising a modified CH3 constant domain that specifically binds to the transferrin receptor and does not contain a Fab fragment or a portion thereof, Each y is independently 1 or more, n is 1 or more, a conjugate. **Claim 25**: P comprises an Fc polypeptide or an Fc polypeptide dimer, The Fc polypeptide dimer comprises a first Fc polypeptide and a second Fc polypeptide, and the first Fc polypeptide comprises the modified CH3 constant domain. The conjugate according to claim 24. **Claim 26**: P further comprises one or more modification sites that facilitate binding to each L of P, and the one or more modification sites are (i) one or more cysteine substitutions selected from the group consisting of S239C, S442C, A330C, and T289C according to EU numbering, and / or (ii) N297A substitution and / or N297G substitution according to EU numbering The conjugate according to claim 24. **Claim 27**: The modified CH3 constant domain has Trp, Tyr, Leu, Gln, or Glu at position 153 numbered based on SEQ ID NO: 1, Leu, Tyr, Met, Val, Phe, or Trp at position 157, Leu, Thr, His, Pro, or Phe at position 159, Val, Pro, or an acidic amino acid at position 160, Trp at position 161, Val, Ser, Ala, or Gly at position 162, Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Thr, or Glu at position 163, Ser, Thr, Gln, Phe, Tyr, or Val at position 164, Gln, Phe, or His at position 165, Glu, Ala, Ser, Leu, Thr, Pro, or Asp at position 186, Lys, Arg, Gly, or Pro at position 187, Glu or Ser at position 188, Thr, Asn, or an acidic amino acid at position 189, Trp, Tyr, His, or Phe at position 194, Ser, Thr, Glu, Lys, or Trp at position 197, and Ser, Trp, Gly, Cys, Pro, or Met at position 199, and comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions selected from these positions, the conjugate according to any one of claims 3 to 12 and 17 to 26, or the Fc polypeptide dimer according to any one of claims 13 to 16. **Claim 28**: The first Fc polypeptide or Fab-Fc dimer fusion has a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences of SEQ ID NOs: 279, 281, 361 - 366, 491 - 494, 702 - 718, 632, 645 - 649, 738 - 746, 804, The second Fc polypeptide or Fab-Fc dimer fusion is a conjugate according to any one of claims 3 to 12 and 17 to 26, or an Fc polypeptide dimer according to any one of claims 13 to 16, comprising a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences of SEQ ID NOs: 557-561, 627-631, 635-644, 719-723, 724-731, 733-737, and 810. **Claim 29** The conjugate or Fc polypeptide dimer according to claim 27, wherein the first Fc polypeptide or Fab-Fc dimer fusion comprises Glu at position 153, Tyr at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser or Ala at position 162, Asn at position 163, Thr or Ser at position 186, Glu at position 188, Glu at position 189, and Phe at position 194, numbered with reference to SEQ ID NO:

1. **Claim 30** The first Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO: 362, and the second Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO:

559. The first Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO: 702, and the second Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO:

559. The first Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO: 708, and the second Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO:

722. The first Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO: 718, and the second Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO:

559. The first Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO: 362, and the second Fc polypeptide or Fab-Fc dimer fusion comprises the amino acid sequence of SEQ ID NO:

560. The first Fc polypeptide or Fab-Fc dimer fusion contains the amino acid sequence of SEQ ID NO: 362, and the second Fc polypeptide or Fab-Fc dimer fusion contains the amino acid sequence of SEQ ID NO: 561, or The conjugate or Fc polypeptide dimer according to claim 27, wherein the first Fc polypeptide contains the amino acid sequence of SEQ ID NO: 804 and the second Fc polypeptide contains the amino acid sequence of SEQ ID NO:

810. **Claim 31** Each of the non-targeted Fab or a part thereof contains a heavy chain variable region including CDR-H1, CDR-H2, and CDR-H3, and a light chain variable region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 contains SEQ ID NO: 825 or 826, CDR-H2 contains SEQ ID NO: 827, 828, or 869, CDR-H3 contains SEQ ID NO: 829, CDR-L1 contains SEQ ID NO: 819 or 820, CDR-L2 contains SEQ ID NO: 821 or 822, CDR-L3 contains SEQ ID NO: 823 or 824, The conjugate according to any one of claims 4 to 12, 18, 22 to 23, or the Fab-Fc dimer fusion according to any one of claims 14 to 16. **Claim 32** (a) The heavy chain variable region contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 837, and the light chain variable region contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 832, or (b) The heavy chain variable region contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 853, and the light chain variable region contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 851, The conjugate or Fab-Fc dimer fusion according to claim 31.

33. The Fab-Fc dimer fusion body includes a heavy chain and a light chain, (a) the heavy chain includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of the amino acid sequences of SEQ ID NOs: 838, 839, 840, 841, 844, 845, 846, 847, 854, 855, 856, 857, 859, 860, 861, and 862, (b) the light chain includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of the amino acid sequences of SEQ ID NOs: 833, 835, 850, and 852, The Fab-Fc dimer fusion body or conjugate according to claim 31, including a heavy chain and a light chain.

34. Each L is 【Chemical Formula 38-1】 【Chemical 38-2】 a linking group independently selected from the group consisting of wherein each A is independently (C1-C15) alkylene, each D is -(CH2-CH2-O)m-, each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; the conjugate according to any one of claims 3 to 12, 17 to 18, 20 to 22, or the Fc polypeptide dimer according to any one of claims 13 to 16.

35. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 12 and 17 to 26, or the Fc polypeptide dimer according to claim 16, and a pharmaceutically acceptable excipient.

36. The ratio of oligonucleotide to protein is (a) about 1:1 to about 2:1, (b) about 1.23, (c) about 2:1 to about 3:1, or (d) about 2.5; the conjugate according to any one of claims 1 to 12 and 17 to 26, or the Fc polypeptide dimer according to claim 16.

37. (a) Targeting the delivery of the oligonucleotide to the muscle tissue, brain tissue, and / or central nervous system (CNS) of a patient, (b) transporting the oligonucleotide through the blood-brain barrier, (c) delivering the oligonucleotide across the brain region, (d) delivering the oligonucleotide to the deep brain region, (e) delivering the oligonucleotide to the spinal cord, (f) regulating gene expression in a brain cell or a plurality of brain cells, (g) modifying a nerve cell so as to reduce target gene expression in a neuron, or (h) generating a nerve cell in which target gene expression is reduced The pharmaceutical composition according to claim 35 for use in (i).

38. (i) The deep brain region includes the cortex, brainstem, hippocampus, striatum, cerebellum, thalamus, caudate putamen, substantia nigra, (ii) The spinal cord is the cervical spinal cord and / or the lumbar spinal cord, (iii) The plurality of brain cells are distributed throughout the brain region or the entire CNS, (iv) The brain cell or plurality of brain cells includes one or more cell types selected from the group consisting of neurons, excitatory neurons, inhibitory neurons, endothelial cells, oligodendrocytes, astrocytes, and microglia, (v) The oligonucleotide is distributed throughout the brain region or the entire central nervous system, or (vi) The oligonucleotide regulates gene expression throughout the brain region or the entire central nervous system, The pharmaceutical composition according to claim 37 for use in (i).

39. The pharmaceutical composition according to claim 37, wherein the oligonucleotide reduces the gene expression of the target gene by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to the expression when the conjugate, the Fc polypeptide, or the pharmaceutical composition is not administered.

40. A composition for use in regulating gene expression in a brain cell or a plurality of brain cells, comprising a conjugate, The conjugate is (a) a protein that specifically binds to TfR, (b) an oligonucleotide, wherein the oligonucleotide is conjugated to the protein via a linker having the following structure 【Chemical Formula 39】 and * represents a binding point to the sulfur atom of the modification site in P, wherein the protein binds to TfR with low affinity, the conjugate is transported through the BBB, and in the cell, the oligonucleotide regulates the expression of the target gene. A composition.

41. A method for making an oligonucleotide delivery vehicle, (a) providing a protein comprising an Fc polypeptide having a modified constant domain that specifically binds to TfR with an affinity of about 3 nM to about 600 nM, (b) providing an oligonucleotide, and (c) conjugating the oligonucleotide to the protein A method comprising.

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