Improved linkers and methods for the synthesis of Anti-transferrin receptor antibody conjugates

Abstract

The present disclosure relates to improved linkers for conjugating oligonucleotidebased agents (e.g., RNAi agents) to an antibody or antibody fragment (e.g., anti-transferrin receptor (TfR1) antibodies or fragments).

Description

[0001] IMPROVED LINKERS AND METHODS FOR THE SYNTHESIS OF ANTI-TRANSFERRIN RECEPTOR ANTIBODY CONJUGATES

[0002] RELATED APPLICATIONS

[0003]

[0001] This application claims the benefit of priority7to United States Provisional Patent Application Serial No. 63 / 624,417, filed on January 24, 2024, United States Provisional Patent Application Serial No. 63 / 573,151. filed on April 2, 2024, United States Provisional Patent Application Serial No. 63 / 662,243, filed on June 20, 2024, United States Provisional Patent Application Serial No. 63 / 662,272, filed on June 20, 2024, and United States Provisional Patent Application Serial No. 63 / 718,062 filed on November 8, 2024, the contents of each of which are incorporated herein by reference in their entirety.

[0004] FIELD OF THE INVENTION

[0005]

[0002] The present disclosure relates to improved linkers for conjugating oligonucleotide- based agents (e.g., RNAi agents) to an antibody or antibody fragment (e.g.. anti-transferrin receptor (TfRl) antibodies or fragments).

[0006] SEQUENCE LISTING

[0007]

[0003] This application contains a Sequence Listing (in compliance with Standard ST26), which has been submitted in xml format and is hereby incorporated by reference in its entirety7. The xml sequence listing file is named 30745-WO_SeqListing.xml, created January 22, 2025, and is 990 kb in size.

[0008] BACKGROUND

[0009]

[0004] Directing therapeutic payloads to specific tissues and cells of interest in a subject in vivo continues to be a great challenge in the field of medicine. This is particularly true for oligonucleotide-based therapeutics, such as antisense oligonucleotides (ASOs) and RNA interference (RNAi) agents (typically comprised of small (or short) interfering RNA that employ chemically modified nucleotides), which have shown great promise and potential to revolutionize the field of medicine and provide potent therapeutic treatment options for previously undruggable diseases; provided, of course, that the therapeutic oligonucleotide can reach the desired cells and tissues in vivo. Indeed, obtaining suitable delivery of oligonucleotide-based therapeutics remains the most pressing challenge to overcome in discovering and identifying viable therapeutics.

[0005] Several other interfering RNA deliver^' methods are being tested / developed for in vivo use. For example. siRNAs can be deliverediLnaked?’ in saline solution; complexed with poly cations, cationic lipids / lipid transfection reagents, or cationic peptides; as components of defined molecular conjugates (e.g., cholesterol-modified siRNA, TAT-DRBD / siRNA complexes); as components of liposomes; and as components of nanoparticles. These approaches have shown varying degrees of success. Another approach is conjugating a siRNA with an antibody or antibody fragment. However, there is a need for new and improved methods and linkers for conjugating siRNAs and other oligonucleotide-based agents to antibodies and antibody fragments to provide targeted in vivo delivery of RNAi molecules, which can enhance the therapeutic potential of RNAi.

[0010] SUMMARY

[0011]

[0006] Provided herein are novel linkers for conjugating oligonucleotide-based agents (e.g., RNAi agents) to an antibody or antibody fragment (e.g., anti-transferrin receptor (TfRl) antibodies or fragments).

[0012]

[0007] In some aspects, provided here in are compounds of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0013] Fab is an antibody or antibody fragment;

[0014] R1is -ORla, -C(O)NHRla’ or -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl;

[0015] Z is a bivalent or trivalent aryl group, or a bivalent or trivalent heteroaryl group;

[0016] Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted ary lene, or substituted or unsubstituted heteroarylene;

[0017] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0018] L1is absent or a linker comprising a PEG chain;

[0019] L2is absent or a linker;

[0020] R2comprises an oligonucleotide-based agent; m is 0 or 1 ; and n is 0 or 1 as valency permits.

[0021]

[0008] In another aspect, provided herein is a compound comprising an antibody or antibody fragment; and a structure selected from the group consisting of

[0022] L2is absent or a linker.

[0023] R2comprises an oligonucleotide-based agent, and represents an attachment point to the antibody or antibody fragment.

[0024]

[0009] In certain embodiments, the compounds provided herein are of the formula: wherein L2is absent or a linker, R2comprises an oligonucleotide-based agent, and * represents an attachment point to the antibody or antibody fragment.

[0025]

[0010] In certain embodiments, the compounds provided herein are of the formula: wherein R2comprises an oligonucleotide-based agent, and ' represents an attachment point to the antibody or antibody fragment. [OH] In certain embodiments, the compounds provided herein are of the formula: , wherein L2is absent or a linker, R2comprises an oligonucleotide-based agent, and5represents an attachment point to the antibody or antibody fragment.

[0026]

[0012] In certain embodiments, the compounds provided herein are of the formula: , wherein R2comprises an oligonucleotide- based agent, and5represents an attachment point to the antibody or antibody fragment.

[0027]

[0013] In some aspects, provided herein are compounds of Formula (II): or a pharmaceutically acceptable salt thereof, wherein Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroar l group, a branched alkyl chain, or a branched heteroalkyl chain; L1is absent or a linker comprising a PEG chain; L2is absent or a linker; R2comprises an oligonucleotide-based agent; p is 0 or 1; and X is a leaving group.

[0028]

[0014] In another aspect, provided herein are compounds of Formula (III): or a pharmaceutically acceptable salt thereof, wherein Z1is absent, a bivalent or trivalent ary l group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L1is absent or a linker comprising a PEG chain; L2is absent or a linker; R3is a reactive moiety; p is 0 or 1; and X is a leaving group.

[0015] In certain embodiments, provided herein is a compound of the formula: a pharmaceutically acceptable salt thereof, wherein L2is absent or a linker, R2comprises an oligonucleotide-based agent, and X is a leaving group.

[0029]

[0016] In certain embodiments, provided herein is a compound of the formula:

[0030]

[0017] In some aspects, also provided herein are methods of synthesizing compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein Fab is an antibody or antibody fragment; R1is -ORla, -C(O)NHRUor -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Cs alkyl, or substituted or unsubstituted aryl; Z is a bivalent or trivalent aryl group, or a bivalent or trivalent heteroaryl group; Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted ary lene, or substituted or unsubstituted heteroarylene; Z1is absent, a bivalent or trivalent ary l group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L1is absent or a linker comprising a PEG chain; L2is absent or a linker; R2comprises an oligonucleotide-based agent; m is 0 or 1; and n is 0 or 1 as valency permits; wherein the method comprises reacting a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroary 1 group, a branched alkyl chain, or a branched heteroalkyl chain; L1is absent or a linker comprising a PEG chain; L2is absent or a linker; R2comprises an oligonucleotide-based agent; p is 0 or 1; and X is a leaving group; with an antibody comprising a cysteine residue.

[0031] DEFINITIONS

[0032]

[0018] For convenience, certain terms employed herein, in the specification, examples, and claims are collected herein. The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3.sup.rd Edition or a dictionary known to those of skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

[0033]

[0019] As used herein, all percentages are percentages by weight, unless stated otherwise.

[0020] As used herein and unless otherwise indicated, the terms “a” and "an" are taken to mean "one", “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0034]

[0021] The term “receptor” as used herein is intended to encompass the entire receptor or ligand-binding portions thereof. These portions of the receptor particularly include those regions sufficient for specific binding of the ligand to occur, including those regions capable of being recognized by an antibody or antibody fragments.

[0035]

[0022] An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody”, e.g., anti-TfRl antibody, encompasses not only intact (e g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., nanobody), single domain antibodies (e.g., a VH only antibody), multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0023] An antibody, e.g., anti-TfRl antibody, includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0036]

[0024] The term “Fab” as used herein refers to the fragment antigen-binding (Fab) region that binds to the TfRl antigen, and includes 6 complementary' determining regions (CDRs), wherein 3 CDRs are on the variable light chain (VL), and 3 CDRs are on the variable heavy chain (VH).

[0037]

[0025] The term “ligand” is defined as any molecule or atom that binds to a receiving molecule for example binding to proteins, nucleotides, or fragments, binding domains, or complements thereof.

[0038]

[0026] The term “protein” as used herein includes peptides, polypeptides, consensus molecules, fusion proteins, purified naturally occurring proteins, artificially synthesized proteins, recombinant proteins, antibodies, antibody fragments, and analogs, derivatives or combinations thereof.

[0039]

[0027] The term “antigen binding fragment” (e.g., Fab) is further intended to encompass fully humanized and chimeric antibody fragments comprising portions from more than one species, bifunctional antibody fragments, etc.

[0040]

[0028] The term “antibody” used herein can refer to the fragment antigen-binding region (Fab) or an antibody including the Fc portion of an antibody, depending on the context. Examples of TfRl antibodies are described herein.

[0029] The term “conjugate'’ is defined as consisting of two or more molecules; or two or more entities that are coupled together. Preferably, the two molecules or entities are conjugated by non-specific or specific protein-protein interaction, by covalent bonding, by non-covalent bonding or by coordinating chemical bonding. In the context of the present invention, the first molecule may be an interfering RNA molecule (i.e., an RNAi agent), whereas the second molecule may be a ligand for a receptor on a target cell as defined herein. In some embodiments, the term conjugate includes TfRl Fab coupled to an oligonucleotide- based agent, such as an siRNA, which may also include a linker and / or peptide or protein for release of the oligonucleotide-based agent from the endosome as described herein.

[0041]

[0030] As used herein, the terms “interfering RNA'’ and “interfering RNA molecule'’ refer to all RNA or RNA-like molecules that can interact with RISC and participate in RISC- mediated changes in gene expression.

[0042]

[0031] The term “siRNA” as used herein refers to a double-stranded interfering RNA unless otherwise noted.

[0043]

[0032] As used herein, the term “therapeutically effective amount” refers to the amount of interfering RNA or a pharmaceutical composition comprising an interfering RNA determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary7skill in the art and using methods as described herein.

[0033] The phrase “attenuating expression” with reference to a gene or an mRNA as used herein means administering or expressing an amount of interfering RNA (e.g., an siRNA) to reduce translation of a target mRNA into protein, either through mRNA cleavage or through direct inhibition of translation.

[0044]

[0034] The terms “inhibit,” “silencing,” and “attenuating” as used herein refer to a measurable reduction in expression of a target mRNA or the corresponding protein as compared with the expression of the target mRNA or the corresponding protein in the absence of an interfering RNA of the invention.

[0045]

[0035] The term “knockdown” refers to the reduction in expression of the target mRNA or the corresponding protein.

[0046]

[0036] The phrases “target sequence” and “target mRNA” as used herein refer to the mRNA or the portion of the mRNA sequence that can be recognized by an interfering RNA used in a method of the invention, whereby the interfering RNA can silence gene expression as discussed herein.

[0047]

[0037] As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F. Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0048]

[0038] The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990. modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al.. Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0049]

[0039] As used herein, the terms “oligonucleotide” and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodified.

[0050]

[0040] As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”) means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double stranded RNAs (dsRNA). micro RNAs (miRNAs). short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0051]

[0041] As used herein, the terms “silence,” “reduce,” “inhibit,” "down-regulate." or "knockdow n" when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.

[0052]

[0042] As used herein, the terms “sequence” and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature.

[0053]

[0043] As used herein, a “base,” “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.

[0054]

[0044] As used herein, the term “nucleotide” has the same meaning as commonly understood in the art, and thus refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate intemucleoside linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and / or base moieties, which are also referred to as nucleotide analogs or modified nucleotides herein. Herein, a single nucleotide can be referred to as a monomer or unit.

[0055]

[0045] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleobase or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the ability' of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or otherwise suitable in vivo or in vitro conditions)) and form a duplex or double helical structure under certain standard conditions with an oligonucleotide that includes the second nucleotide sequence.

[0046] The person of ordinary skill in the art would be able to select the set of conditions most appropriate for a hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0056]

[0047] Unless stated otherwise, use of the symbol as used herein means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein.

[0057]

[0048] As used herein, the term '‘isomers” refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not minor images of one another are termed '‘diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a “chiral center.”

[0058]

[0049] As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are present and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.

[0059]

[0050] As used in a claim herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0060]

[0051] The person of ordinary skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art. Correspondingly, compounds described herein with labile protons or basic atoms should also be understood to represent salt forms of the corresponding compound.

[0061]

[0052] Compounds described herein may be in a free acid, free base, or salt form. Pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.

[0062]

[0053] As used herein, the term “linked'’ or “conjugated’" when referring to the connection between two compounds or molecules means that two compounds or molecules are joined by a covalent bond. Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.

[0063]

[0054] As used herein, the term “including” is used to herein mean, and is used interchangeably with, the phrase “including but not limited to.” The term “or” is used herein to mean, and is used interchangeably with, the term “and / or,” unless the context clearly indicates otherwise.

[0064]

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0065]

[0056] Where a value is explicitly recited, it is to be understood that values which are about the same uantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each sub-combination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0066]

[0057] Other objects, features, aspects, and advantages of the invention will be apparent from the following detailed description, accompanying figures, and from the claims.

[0067] BRIEF DESCRIPTION OF THE DRAWINGS

[0068]

[0058] These and other features, aspects, and advantages of the present disclosure may be better understood when the following detailed description is read with reference to the accompanying drawings.

[0069]

[0059] FIG. 1 shows relative androgen receptor (AR) mRNA expression knockdown after the delivery of select TfRl -targeting Fabs conjugated to siRNA with the antisense strand complementary to androgen receptor mRNA relative to control siRNA in different brain regions.

[0070]

[0060] FIG. 2 shows relative androgen receptor mRNA expression knockdown after the delivery of select TfRl -targeting Fabs conjugated to siRNA with the antisense strand complementary to androgen receptor mRNA relative to control siRNA in different brain regions.

[0071]

[0061] FIG. 3 shows relative androgen receptor mRNA expression knockdown after the delivery of select TfRl -targeting Fabs conjugated to siRNA with the antisense strand complementary to androgen receptor mRNA relative to control siRNA in different brain regions.

[0072]

[0062] FIG. 4 shows binding affinities of select TfRl -targeting Fabs in KD normalized to Fab0002.

[0073]

[0063] FIG. 5 shows the knockdown of MAPT protein in various tissues according to the description in Example 10.

[0074] DETAILED DESCRIPTION

[0075]

[0064] The present disclosure relates to improved linkers for conjugating oligonucleotide- based agents (e.g.. RNAi agents) to an antibody or antibody fragment (e.g.. anti-transferrin receptor (TfRl) antibodies or fragments).

[0076]

[0065] In certain aspects, provided herein is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0077] Fab is an antibody or antibody fragment;

[0078] R1is -ORla, -C(O)NHRla>or -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Cs alkyl, or substituted or unsubstituted aryl;

[0079] Z is a bivalent or trivalent ary l group, or a bivalent or trivalent hcteroaryl group;

[0080] Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0081] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0082] L1is absent or a linker comprising a PEG chain;

[0083] L2is absent or a linker;

[0084] R2comprises an oligonucleotide-based agent; m is 0 or 1; and n is 0 or 1 as valency permits.

[0085]

[0066] In certain embodiments, m is 0. In certain embodiments, m is 1.

[0086]

[0067] In certain embodiments, n is 0. In certain embodiments, n is i.

[0087]

[0068] In certain embodiments, n is 0 and m is 1. In certain embodiments, n is 1 and m is 0.

[0088]

[0069] In certain embodiments, the compounds of Formula (I) are of Formula (I-a): or a pharmaceutically acceptable salt thereof, wherein:

[0089] Fab is an antibody or antibody fragment; each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0090] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0091] L1is absent or a linker comprising a PEG chain;

[0092] L2is absent or a linker; and R2comprises an oligonucleotide-based agent.

[0093]

[0070] In certain embodiments, the compounds of Formula (I) are of Formula (I-b): or a pharmaceutically acceptable salt thereof, wherein:

[0094] Fab is an antibody or antibody fragment;

[0095] R1is -ORla, -C(O)NHRlaor -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl;

[0096] Z is a bivalent or trivalent aryl group, or a bivalent or trivalent heteroaryl group;

[0097] Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene:

[0098] Y1is substituted or unsubstituted ary lene, or substituted or unsubstituted heteroarylene;

[0099] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0100] L1is absent or a linker comprising a PEG chain;

[0101] L2is absent or a linker; and

[0102] R2comprises an oligonucleotide-based agent.

[0103]

[0071] In some embodiments, the compounds of Formula (I) as disclosed herein contain the substituent R1. In certain embodiments, R1is -ORla, wherein Rlais substituted or unsubstituted C i-Ce alky l, or substituted or unsubstituted aryl . In certain embodiments, R1is -C(O)NHRla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl. In certain embodiments, R1is -NHC(O)Rla. wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl. In certain embodiments, R1is -C(O)NHRla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl. In certain embodiments, Rlais substituted or unsubstituted Ci-Ce alkyl. In certain embodiments, Rlais substituted or unsubstituted methyl. In certain embodiments, Rlais unsubstituted methyl.

[0104]

[0072] In some embodiments, the compounds of Formula (I) as disclosed herein contain the substituent Y. In certain embodiments, Y is substituted or unsubstituted arylene. In certain embodiments, Y is substituted or unsubstituted hcteroarylene. In certain embodiments, Y is of the formula:

[0073] In some embodiments, the compounds of Formula (I) as disclosed herein contain the substituent Z. In certain embodiments. Z is a bivalent aryl group. In certain embodiments, Z is a bivalent phenyl group. In certain embodiments, Z is of the formula: in certain embodiments, Z is a trivalent aryl group. In certain embodiments, Z is a bivalent heteroaryl group. In certain embodiments, Z is a trivalent heteroaryl group.

[0105]

[0074] Further disclosed herein is a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein:

[0106] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0107] L1is absent or a linker comprising a PEG chain;

[0108] L2is absent or a linker:

[0109] R2comprises an oligonucleotide-based agent; p is 0 or 1; and

[0110] X is a leaving group.

[0111]

[0075] Also disclosed herein is a compound of Formula (III): or a pharmaceutically acceptable salt thereof, wherein:

[0112] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0113] L1is absent or a linker comprising a PEG chain;

[0114] L2is absent or a linker;

[0115] R3is a reactive moiety; p is 0 or 1; and

[0116] X is a leaving group.

[0117]

[0076] In some embodiments, the compounds of Formula (III) as disclosed herein contain the substituent X. In certain embodiments, X is a sulfonate leaving group. In certain embodiments, X is -SChMe.

[0077] In some embodiments, the compounds of Formula (III) as disclosed herein contain the substituent R3. In certain embodiments, R3is an activated ester. In certain embodiments,

[0118] R3is of the formula: certain embodiments, R3is an azide.

[0119]

[0078] In some embodiments, the compounds of Formula (I), Formula (II), and Formula (HI) as disclosed herein contain the substituent Y1. In certain embodiments, at least one instance of Y1is substituted or unsubstituted arylene. In certain embodiments, at least one instance of Y1is substituted or unsubstituted heteroarylene. In certain embodiments, two instances of Y1are both substituted or unsubstituted ary lene. In certain embodiments, two instances of Y1are both substituted or unsubstituted heteroarylene. In certain embodiments, two instances of Y1are the same. In certain embodiments, two instances of Y1are different. In certain embodiments, one or two instances of Y1are a substituted or unsubstituted benzothiazole or a substituted or unsubstituted oxadiazole. In certain embodiments, one or

[0120] N-N two instances of Y1are of the formula: . In certain embodiments, one or two instances of Y1are of the formula:

[0121]

[0079] In some embodiments, the compounds of Formula (I). Formula (II), and Formula (III) as disclosed herein contain the substituent L1. In certain embodiments, L1is absent. In certain embodiments, L1is a linker comprising a PEG chain. In certain embodiments, L1comprises 1-10 PEG units. In certain embodiments, L1comprises 3-6 PEG units. In certain embodiments, L1is of the formula: In certain embodiments, L1is of the formula: In certain embodiments, L1is of the formula: In certain embodiments. L1is of the formula: In certain embodiments, L1is of the o formula: H . In certain embodiments, L1is of the formula: In certain embodiments. L1is of the formula: . In certain embodiments, L1is of the

[0122] ° . In certain embodiments, L1is of the formula: certain embodiments, L1is of the formula:

[0123] In certain embodiments, L1is of the formula:

[0124]

[0080] In some embodiments, the compounds of Formula (I), Formula (II), and Formula

[0125] (III) as disclosed herein contain the substituent L2. In certain embodiments, L2is absent. In certain embodiments, L2is a linker. In certain embodiments, L2is of the formula:

[0126]

[0081] In some embodiments, the compounds of Formula (I), Formula (II), and Formula (III) as disclosed herein contain the substituent Z1. In certain embodiments, Z1is absent. In certain embodiments, Z1is a bivalent ary l group. In certain embodiments, Z1is a bivalent phenyl group. In certain embodiments, Z1is of the formula: . In certain embodiments, Z1is a trivalent aryl group. In certain embodiments, Z1is a trivalent phenyl group. In certain embodiments, Z1is of the formula: certain embodiments,

[0127] Z1is a bivalent heteroaryl group. In certain embodiments, Z1is a trivalent heteroaryl group. In certain embodiments, Z1is a branched alkyl chain. In certain embodiments, Z1is a branched heteroalkyl chain. In certain embodiments, Z1is of the formula:

[0128]

[0082] In some embodiments, the compounds of Formula (I) and Formula (II) as disclosed herein contain the substituent R2. In certain embodiments, R2comprises an RNAi agent. In certain embodiments, R2comprises a sense strand containing one inverted abasic residue. In certain embodiments, R2comprises a sense strand containing two inverted abasic residues. In certain embodiments, R2comprises a sense strand conjugated to a Fab at the 5’ terminal end of the sense strand.

[0083] In certain embodiments, provided herein is a compound comprising an antibody or

[0129] or a pharmaceutically acceptable salt thereof, wherein:

[0130] L2is absent or a linker,

[0131] R2comprises an oligonucleotide-based agent, and represents attachment point to the antibody or antibody fragment.

[0132]

[0084] In another aspect, provided herein are compounds of the formula:

[0133] or a pharmaceutically acceptable salt thereof, wherein:

[0134] L2is absent or a linker,

[0135] R2comprises an oligonucleotide-based agent, and

[0136] X is a leaving group.

[0137]

[0085] In some aspects, provided herein are compounds of the formula:

[0138]

[0139] or a salt thereof.

[0086] Further provided herein are methods of synthesizing a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0140] Fab is an antibody or antibody fragment;

[0141] R1is -ORla, -C(O)NHRla’ or -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted ary l;

[0142] Z is a bivalent or trivalent aryl group, or a bivalent or trivalent heteroaryl group;

[0143] Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0144] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0145] L1is absent or a linker comprising a PEG chain;

[0146] L2is absent or a linker;

[0147] R2comprises an oligonucleotide-based agent; m is 0 or 1; and n is 0 or 1 as valency permits; comprising, reacting a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein:

[0148] Z1is absent, a bivalent or trivalent ary l group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0149] L1is absent or a linker comprising a PEG chain;

[0150] L2is absent or a linker;

[0151] R2comprises an oligonucleotide-based agent; p is 0 or 1; and

[0152] X is a leaving group; with an antibody comprising a cysteine residue. Modified Nucleotides

[0153]

[0087] Modified nucleotides, when used in various oligonucleotide constructs, can preserve activity of the compound in cells while at the same time increasing the serum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans upon administering of the oligonucleotide construct.

[0154]

[0088] In some embodiments, an RNAi agent contains one or more modified nucleotides. As used herein, a ‘"modified nucleotide’7is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, inverted nucleotides, modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3 '-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3'-methoxy (2' intemucleoside linked) nucleotides, 2'-F- Arabino nucleotides. 5'-Methyl, 2'-fluoro nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides. 2'- modified nucleotides (z.e., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (also referred to as 2'-methoxy nucleotides), 2'-fluoro nucleotides (also referred to herein as 2'-deoxy-2'-fluoro nucleotides). 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-(2- methoxylethyl)) nucleotides (also referred to as 2'-M0E), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary' for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in a single RNAi agent or even in a single nucleotide thereof. Modification at one nucleotide is independent of modification at another nucleotide. Various modified nucleotides are well known and described in the art.

[0155]

[0089] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenme, 5-propynyluracil, or 5-propynylcytosine). 5-methylcytosine (5-me- C), 5 -hydroxymethyl cytosine, inosine (hypoxanthine), xanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil). 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, and 3 -deazaadenine.

[0156]

[0090] In some embodiments, the 5' and / or 3' end of the antisense strand can include abasic residues (Ab), which can also be referred to as an “abasic site” or “abasic nucleotide.” An abasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the sugar moiety. In some embodiments, an abasic residue can be placed internally in a nucleotide sequence. In some embodiments, Ab or AbAb can be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb. or Ab are added to the 3' end of the sense strand. In some embodiments, an abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.

[0157]

[0091] In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e.. 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. As used herein, an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is an unmodified ribonucleotide. Chemical structures for certain modified nucleotides are set forth in Table A herein.

[0158] Modified Intemucleoside Linkages

[0159]

[0092] In some embodiments, one or more nucleotides of an RNAi agent are linked by nonstandard linkages or backbones (i.e.. modified intemucleoside linkages or modified backbones). Modified intemucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a low er case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, diphosphorothioates. alkyl phosphonates (e.g., methyl phosphonates or 3 '-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates. or thionophosphoramidates), thionoalkyl- phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0160]

[0093] In some embodiments, a sense strand of an RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of an RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.

[0161]

[0094] In some embodiments, an RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate intemucleoside linkage is at the 5’ end of the sense strand nucleotide sequence, and another phosphorothioate linkage is at the 3’ end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate intemucleoside linkage are located at the 5’ end of the sense strand, and another phosphorothioate linkage is at the 3’ end of the sense strand. In some embodiments, the sense strand does not include any phosphorothioate intemucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5’ and 3‘ ends and the optionally present inverted abasic residue terminal caps.

[0162] Capping Residues or Moieties

[0163]

[0095] In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a '‘cap,” a “terminal cap." or a “capping residue. As used herein, a “capping residue” is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of a nucleotide sequence of an RNAi agent disclosed herein. A capping residue can provide the RNAi agent, in some instances, with certain beneficial properties, such as, for example, protection against exonuclease degradation. In some embodiments, inverted abasic residues (invAb) (also referred to in the art as “inverted abasic sites”) are added as capping residues (see Table A). (See, e.g., F. Czaudema, Nucleic Acids Res.. 2003, 31(1 1), 2705-16; U.S. Patent No. 5.998,203). Capping residues are generally known in the art, and include, for example, inverted abasic residues as well as carbon chains such as a terminal C3H7 (propyl), CeHi3 (hexyl), or C12H25 (dodecyl) groups. In some embodiments, a capping residue is present at either the 5' terminal end, the 3' terminal end, or both the 5' and 3' terminal ends of the sense strand. In some embodiments, the 5’ end and / or the 3' end of the sense strand may include more than one inverted abasic deoxyribose moiety as a capping residue.

[0164]

[0096] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between the linker and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.

[0165]

[0097] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)s)), or other intemucleoside linkages. In some embodiments, the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent. In some embodiments, an inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence, or the 3' end of the antisense strand sequence, may include an inverted abasic residue. Chemical structures for inverted abasic deoxyribose residues are shown in Table A below.

[0166]

[0098] The following notations are used herein to indicate modified nucleotides, linkers, and linking groups:

[0167] A adenosine-3 '-phosphate C = cytidine-3 '-phosphate

[0168] G = guanosine-3 '-phosphate

[0169] U = uridine-3 '-phosphate

[0170] I = inosine-3 '-phosphate a = 2'-O-methyladenosine-3 '-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3 '-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate i = 2'-O-methylinosine-3 ’-phosphate is = 2'-O-methylinosine-3'-phosphorothioate t = 2'-O-methyl-5-methyluridine-3 '-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3 '-phosphate us = 2'-O-methyluridine-3'-phosphorothioate

[0171] Af = 2'-fluoroadenosine-3'-phosphate

[0172] Afs = 2'-fluoroadenosine-3'-phosporothioate

[0173] Cf = 2'-fluorocytidine-3 '-phosphate

[0174] Cfs = 2'-fluorocytidine-3'-phosphorothioate

[0175] Gf = 2'-fluoroguanosine-3'-phosphate

[0176] Gfs = 2'-fluoroguanosine-3'-phosphorothioate

[0177] Tf = 2'-fluoro-5'-methyluridine-3'-phosphate

[0178] Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate

[0179] Uf = 2'-fluorouridine-3'-phosphate

[0180] Ufs = 2'-fluorouridine-3'-phosphorothioate a_2N = see Table A a_2Ns = see Table A

[0181] (invAb) = inverted abasic deoxy ribonucleotide-5 '- phosphate, see Table A

[0182] (invAb)s = inverted abasic deoxy ribonucleotide-5'- phosphorothioate. see Table A s = phosphorothioate linkage p = terminal phosphate (as synthesized) (Alk-SS-C6) = see Table A (C6-SS-C6) = see Table A (C6-SS-Alk-Me) = see Table A (NH2-C6) = see Table A

[0183] L20 = see Table A

[0184] L-1026 = see Table A

[0185] L-1274 = see Table A

[0186] L-1288 = see Table A

[0187] L-1166 = see Table A

[0188] L-1275 = see Table A

[0189] L-1100 = see Table A

[0190] L-1045 = see Table A

[0191] L-1176 = see Table A

[0192] L-1064 = see Table A

[0193] L-1063 = see Table A

[0194]

[0099] As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence (such as, for example, by a phosphorothioate linkage “s”), when present in an oligonucleotide, the nucleotide monomers are mutually linked by 5’-3’- phosphodiester bonds. As the person of ordinary skill in the art would clearly understand, the inclusion of a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides. Further, the person of ordinary skill in the art would readily understand that the terminal nucleotide at the 3’ end of a given oligonucleotide sequence would typically have a hydroxyl (-OH) group at the respective 3’ position of the given monomer instead of a phosphate moiety ex vivo. Moreover, as the person of ordinary skill w ould readily understand and appreciate, while the phosphorothioate chemical structures depicted herein typically show the anion on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers (e.g., where the sulfur atom has a double-bond and the anion is on an oxygen atom). Unless expressly indicated otherwise herein, such understandings of the person of ordinary skill in the art are used when describing the RNAi agents described herein.

[0195]

[0100] Certain examples of linking groups used with RNAi agents disclosed herein are included in the chemical structures provided below in Table A. Each sense strand and / or antisense strand can have any linking groups listed herein, as well as other linking groups, conjugated to the 5' and / or 3' end of the sequence.

[0196]

[0101] In some embodiments, the synthesized RNAi agent is prepared or provided as a salt, mixed salt, a free-acid, or a free base. In some embodiments, an RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, a RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms that are well known in the art are within the scope of the inventions disclosed herein.

[0197] Anti-TfRl Fabs and Anti-TfRl Antibodies

[0198]

[0102] The present disclosure provides antibodies binding to TfRl . for example, human TfRl. In some embodiments, the anti-TIRl antibodies disclosed herein are capable of binding to TfRl expressed on a cell surface, for example, on brain endothelial cells. As such, the antibodies disclosed herein may be used for facilitating delivery' of therapeutic or diagnostic agents conjugated to the antibody to cells and tissues outside of the liver. In some instances, the anti-TIRl antibodies provided herein may also be used for detecting the presence of the TfRl receptor, either in vitro or in vivo. As used herein, the term “anti-TIRl antibody” refers to any antibody capable of binding to a TfRl polypeptide (e.g., a TfRl polypeptide expressed on cell surface such as on brain endothelial cells), which can be of a suitable source, for example, human or a non-human mammal (e.g.. mouse, rat. rabbit, primate such as monkey, etc.).

[0199]

[0103] A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The Vn and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”). interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology’ known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17: 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0200]

[0104] The anti-Tf l antibody described herein may be a full-length antibody, which contains two heavy chains and two light chains, each including a variable domain and a constant domain. Alternatively, the anti-TfRl antibody can be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed within the term "‘antigen-binding fragment7’ of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0201]

[0105] In some embodiments, the anti-TfRl antibody provided here is a TfRl fragment antigen-binding region (Fab) that binds with specificity to the TfRl. In some examples, the TfRl -specific Fab binds to TfRl on brain endothelial cells. In some embodiments, the TfRl - specific Fab are capable of recognizing and binding with specificity to one or more epitopes on the TfRl.

[0202]

[0106] The anti-TfRl antibody discloses herein such as the TfRl -specific Fab may bind competitively to TfRl with transferrin (Tl), the physiological TfRl ligand. Upon binding of the Fab to TfRl on a cell surface, transfer of the Fab and the attached interfering RNA enters the cell through constitutive clathrin-mediated endocytosis.

[0203]

[0107] The antibodies described herein can be of a suitable origin, for example, murine, rat, or human. Such antibodies are non-naturally occurring, i.e.. would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries). Any of the antibodies described herein, e.g., anti-TfRl antibody, can be either monoclonal or polyclonal. A “monoclonal antibody’’ refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

[0204]

[0108] In some embodiments, the anti-TfRl antibodies are human antibodies, which may be isolated from a human antibody library or generated in transgenic mice. For example, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XenomouseTM from Amgen, Inc. (Fremont, Calif.) and HuMAb- MouseTM and TC MouseTM from Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, the antibody library display technology, such as phage, yeast display, mammalian cell display, or mRNA displaytechnology as known in the art can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0205]

[0109] In other embodiments, the anti-TfRl antibodies may be humanized antibodies. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat. or rabbit having the desired specificity-, affinity, and capacity. In some instances, one or more Fv framework region (FR) residues of the human immunoglobulin are replaced by- corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In some instances, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation. Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). [HO] In some embodiments, the anti-TfRl antibodies described herein specifically bind to the corresponding target antigen (here TfRl) or an epitope thereof. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (TfRl) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that “specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (i.e.., only baseline binding activity can be detected in a conventional method).

[0206] [Hl] In some embodiments, an anti-TfRl antibody as described herein has a suitable binding affinity for the target antigen (e.g., TfRl) or antigenic epitopes thereof. As used herein, “binding affinity” refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The anti-TfRl antibody described herein may have a binding affinity (KD) of at least 2 nM, InM, 0.5 nM, 0. 1 nM, or lower for TfRl. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical value KD) for binding the first antigen than the I< \ (or numerical value KD) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g, a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g, the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5. 10. 15. 20, 37.5, 50, 70, 80, 90, 100. 500, 1000. 10,000 or 105fold. In some embodiments, any of the anti-TfRl antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0207]

[0112] Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration. ELISA, surface plasmon resonance, or spectroscopy (e.g. using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v / v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free] / (Kd+ [Free]).

[0208]

[0113] It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g, determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

[0209] Exemplary Anti-TfRl Antibodies

[0210]

[0114] In some embodiments, the anti-TfRl antibodies provided herein are humanized antibodies derived from a mouse monoclonal antibody clone. Briefly, the heavy chain and light chain complementarity determining domains of the mouse parent are grafted to human consensus VH and VL frameworks to produce a parent humanized antibody. A humanized library was generated using Kunkel mutagenesis to introduce variations at certain framework regions positions and CDR-FR junction positions was constructed and screened for clones exhibiting high binding affinity to human TfRl. mablG derived from the screening showed the highest binding affinities and thus was selected for further affinity maturation. See Examples below. Exemplary anti-TfRl antibodies derived from affinity maturation, comprising variations in certain CDRs relative to the mablG parent, exhibited further enhanced binding affinity.

[0211]

[0115] Also within the scope of the present disclosure are functional variants of any of the exemplary anti-TfRl antibodies as disclosed herein. Such functional variants are substantially similar to the exemplary antibody, both structurally and functionally. A functional variant comprises substantially the same VH and VL CDRS as the exemplary7antibody. For example, it may comprise only up to 8 (e.g. 8, 7, 6, 5, 4, 3, 2. or 1) amino acid residue variations in the total CDR regions of the antibody and binds the same epitope of TfRl with substantially similar affinity (e.g., having a KD value in the same order). In some instances, the functional variants may have the same heavy chain CDR3 as the exemplary7antibody, and optionally the same light chain CDR3 as the exemplary7antibody. Alternatively or in addition, the functional variants may have the same heavy chain CDR2 as the exemplary antibody. Such an anti-TfRl antibody may comprise a VH fragment having CDR amino acid residue variations in only the heavy chain CDR1 as compared with the VH of the exemplary' antibody. In some examples, the anti-TfRl antibody may further comprise a VL fragment having the same VL CDR3, and optionally same VL CDR1 or VL CDR2 as the exemplary antibody.

[0212]

[0116] Alternatively or in addition, the amino acid residue variations can be conservative amino acid residue substitutions.

[0213]

[0117] In some embodiments, the anti-TfRl antibody may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRs of an exemplary antibody described herein. Alternatively7or in addition, the anti-TfRl antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as an exemplary antibody described herein. As used herein, “individually"’ means that one CDR of an antibody shares the indicated sequence identity7relative to the corresponding CDR of the exemplary' antibody. “Collectively” means that three V11 or VL CDRS of an antiody in combination share the indicated sequence identity relative the corresponding three VH or VL CDRS of the exemplary7antibody in combination.

[0214]

[0118] In some embodiments, the heavy chain of any of the anti-TfRl antibodies as described herein may further comprise a heavy chain constant region (CH) or a portion thereof e.g., CHI, CH2, CH3, or a combination thereof). The heavy chain constant region can of any suitable origin, e.g., human, mouse, rat, or rabbit. Alternatively or in addition, the light chain of the anti-TfRl antibody may further comprise a light chain constant region (CL), which can be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. Antibody heavy' and light chain constant regions are well known in the art, e.g., those provided in the IMGT database (www.imgt.org) or at vvww.v'base2.org / vbstat.php., both of which are incorporated byreference herein.

[0119] In some instances, the anti-TfRl antibodies are in Fab format, which comprises a heavy chain comprising any of the VH regions disclosed herein and a CHI fragment and a light chain comprising any of the VL regions disclosed herein and a light chain constant region (LC). The CHI fragment may be of any immunoglobin (Ig) heavy chain constant region (e.g., a human Ig heavy chain constant region). In some instances, the CHI may be of an IgG (e.g., IgGl. IgG2, or IgG4) heavy chain. The LC may be of human original in some instances. It can be a kappa chain. Alternatively, it can be a lambda chain.

[0215]

[0120] In some embodiments, the anti-TfRl antibody disclosed herein may be a single chain antibody (scFv). A scFv antibody may comprise a VH fragment and a VL fragment, which may be linked via a flexible peptide linker. In some instances, the scFv antibody may be in the VH-> VL orientation (from N-terminus to C-terminus). In other instances, the scFv antibody may be in the VL- VH orientation (from N-terminus to C-terminus).

[0216]

[0121] A Fab library with mutated CDRs generated by Kunkel mutagenesis was sorted for TfRl affinity as described in Example 2.

[0217] Preparation of Anti-TfRl Antibodies

[0218]

[0122] Antibodies capable of binding TfRl as described herein can be made by any method known in the art, for example, via recombinant technology. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory. New York.

[0219]

[0123] If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma cell line or isolated from an antibody library) may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. In an alternative, the polynucleotide sequence may be used for genetic manipulation to, e.g., humanize the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is from a non-human source and is to be used in clinical trials and treatments in humans. Alternatively or in addition to, it may be desirable to genetically manipulate the antibody sequence to obtain greater affinity and / or specificity to the target antigen and greater efficacy in enhancing the activity of TfRl. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

[0124] Alternatively, antibodies capable of binding to the target antigens as described herein (a TfRl molecule) may be isolated from a suitable antibody library via routine practice. Antibody libraries can be used to identify proteins that bind to a target antigen (e.g, human TfRl such as cell surface TfRl) via routine screening processes. In the selection process, the polypeptide component is probed with the target antigen or a fragment thereof and, if the polypeptide component binds to the target, the antibody library member is identified, typically by retention on a support. Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the polypeptide component and purification of the polypeptide component for detailed characterization.

[0220]

[0125] There are a number of routine methods known in the art to identify and isolate antibodies capable of binding to the target ant gens described herein, including phage display, yeast display, ribosomal display, or mammalian display technology.

[0221]

[0126] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g, conventional recombinant technology. In one example, DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g.. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Once isolated, the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., PCT Publication No. WO 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81 :6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.

[0222]

[0127] Methods for constructing humanized antibodies are also well known in the art. See, e.g, Queen et al.. Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). In one example, variable regions of VH and VL of a parent non-human antibody are subjected to three- dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.

[0223]

[0128] The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

[0224]

[0129] A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques descnbed for the production of single chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage-display, yeast-display, mammalian cell-display, or mRNA-display scFv library and scFv clones specific to TfRl can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that enhance CD 19 activity.

[0225]

[0130] Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y.. 1999. In an additional example, epitope mapping can be used to determine the sequence, to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may. for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries).

[0226]

[0131] Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of TfRl have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein (such as another member of the tumor necrosis factor receptor family). By assessing binding of the antibody to the mutant TfRl , the importance of the particular antigen fragment to antibody binding can be assessed.

[0227]

[0132] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

[0228]

[0133] In some examples, an anti-TfRl antibody (e.g., in Fab format) is prepared by recombinant technology as exemplified below.

[0229]

[0134] Nucleic acids encoding the heavy and light chain of an anti-TfRl antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter. Alternatively, the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter. When necessary, an internal ribosomal entry site (IRES) can be inserted between the heavy chain and light chain encoding sequences.

[0230]

[0135] In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.

[0231]

[0136] Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors / promoter would depend on the type of host cells for use in producing the antibodies.

[0232]

[0137] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR. HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

[0233]

[0138] Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell. 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard. H.. Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al.. Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK.506 dimer, VP 16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0234]

[0139] Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987): Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR- mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Y ao et al.. Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al.. Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

[0235]

[0140] Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability7; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0236]

[0141] Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

[0237]

[0142] One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g.. from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.

[0238]

[0143] In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti-TfRl antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g.. a dhfr- CHO cell) by a conventional method, e.g.. calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.

[0239]

[0144] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti-TfRl antibody and the other encoding the light chain of the anti-TfRl antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g, calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cells, the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.

[0240]

[0145] Standard molecular biology7techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

[0241]

[0146] Any of the nucleic acids encoding the heavy chain, the light chain, or both of an anti- TfRl antibody as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure. Also within the scope of the present disclosure are methods for producing the anti-TfRl antibodies via expressing such in host cells and harvesting the antibody thus produced from the host cells and / or the culture supernatant. Linking Groups, and Delivery Vehicles

[0242]

[0147] In some embodiments, an RNAi agent as disclosed herein contains or is conjugated to one or more non-nucleotide groups including, but not limited to, a Fab, a linking group, a delivery polymer, or a delivery vehicle. The Fab can enhance targeting, delivery, or attachment of the RNAi agent. The non-nucleotide group can be covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, a RNAi agent contains a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of a RNAi agent sense strand. A non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0243]

[0148] In some embodiments, a non-nucleotide group (e.g., a Fab) enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.

[0244]

[0149] The RNAi agents described herein can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine), at the 5'-terminus and / or the 3'- terminus.

[0245]

[0150] For example, in some embodiments, the RNAi agents disclosed herein are synthesized having an NH2-C6 group (represented as (NH2-C6) in the modified sequences herein) at the

[0246] 5 '-terminus of the sense strand of the RNAi agent. The terminal amino group subsequently can be reacted to form a conjugate with, for example, a group that includes a Fab. In some embodiments, the RNAi agents disclosed herein are synthesized having one or more alkyne groups at the 5 '-terminus of the sense strand of the RNAi agent. The terminal alkyne group(s) can subsequently be reacted to form a conjugate with, for example, a group that includes a Fab.

[0247]

[0151] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the oligonucleotide-based agent to a Fab, pharmacokinetic modulator, delivery polymer, or deliver}' vehicle. The linking group can be linked to the 3' and / or the 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5' end of an RNAi agent sense strand. Examples of linking groups, include, but are not limited to: C6-SS-C6, C6-SS- MeC5, 6-SS-6. reactive groups such as primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and / or PEG groups.

[0248]

[0152] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a Fab) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility7and / or length to the linkage. Spacers include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.

[0249]

[0153] Examples of certain modified nucleotides and linking groups are provided in Table A.

[0250] Table A. Structures Representing Various Modified Nucleotides, Linkers, and Linking Groups

[0251]

[0252]

[0253]

[0154] Alternatively, other linking groups known in the art may be used. In many instances, linking groups can be commercially acquired or alternatively, are incorporated into commercially available nucleotide phosphorami dites.

[0254]

[0155] In some embodiments, a delivery vehicle may be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.

[0255]

[0156] In some embodiments, the RNAi agents described herein can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art for nucleic acid delivery. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholestery l and cholesteryl derivatives), encapsulating in nanoparticles, liposomes, micelles, conjugating to polymers or DPCs (see, for example WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169, and WO 2012 / 083185, WO 2013 / 032829. WO 2013 / 158141, each of which is incorporated herein by reference), by iontophoresis, or by incorporation into other delivery vehicles or systems available in the art such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors.

[0157] Additional embodiments of the present disclosure are provided below:

[0256] Embodiment 1. A compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0257] Fab is an antibody or antibody fragment;

[0258] R1is -OR1'1, -C(O)NHRlaor -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl;

[0259] Z is a bivalent or bivalent aryl group, or a bivalent or bivalent heteroaryl group;

[0260] Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0261] Z1is absent, a bivalent or bivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0262] L1is absent or a linker comprising a PEG chain;

[0263] L2is absent or a linker:

[0264] R2comprises an oligonucleotide-based agent; m is 0 or 1; and n is 0 or 1 as valency permits.

[0265] Embodiment 2. The compound of embodiment 1. wherein m is 0.

[0266] Embodiment 3. The compound of embodiment 1, wherein m is 1.

[0267] Embodiment 4. The compound of any one of embodiments 1-3, wherein n is 0.

[0268] Embodiment 5. The compound of any one of embodiments 1-3, wherein n is 1.

[0269] Embodiment 6. The compound of embodiment 1. wherein n is 0 and m is 1. Embodiment 7. The compound of any one of embodiments 1, 3, 4, and 6 wherein the compound is of Formula (I-a): or a pharmaceutically acceptable salt thereof.

[0270] Embodiment 8. The compound of embodiment 1, wherein n is 1 and m is 0.

[0271] Embodiment 9. The compound of any one of embodiments 1, 2, 5. and 8 wherein the compound is of Formula (I-b): or a pharmaceutically acceptable salt thereof.

[0272] Embodiment 10. The compound of embodiment 9. wherein Y is substituted or unsubstituted heteroarylene.

[0273] Embodiment 11. The compound of embodiment 9 or 10, wherein Y is of the formula:

[0274] Embodiment 12. The compound of any one of embodiments 9-11, wherein Z is a bivalent ary l group.

[0275] Embodiment 13. The compound of any one of embodiments 9-12, wherein Z is a bivalent phenyl group.

[0276] Embodiment 14. The compound of any one of embodiments 9-13. wherein Z is of the formula: Embodiment 15. The compound of any one of embodiments 9-14. wherein R1is -

[0277] C(O)NHRla.

[0278] Embodiment 16. The compound of any one of embodiments 1-15, wherein Rlais substituted or unsubstituted Ci-Ce alkyl.

[0279] Embodiment 17. The compound of any one of embodiments 1-16, wherein Rlais substituted or unsubstituted methyl.

[0280] Embodiment 18. The compound of any one of embodiments 1-17. wherein Rlais unsubstituted methyl.

[0281] Embodiment 19. A compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein:

[0282] Z1is absent, a bivalent or bivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0283] L1is absent or a linker comprising a PEG chain;

[0284] L2is absent or a linker;

[0285] R2comprises an oligonucleotide-based agent; p is 0 or 1; and

[0286] X is a leaving group.

[0287] Embodiment 20. A compound of Formula (III): or a pharmaceutically acceptable salt thereof, wherein:

[0288] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L1is absent or a linker comprising a PEG chain;

[0289] L2is absent or a linker:

[0290] R3is a reactive moiety: p is 0 or 1; and

[0291] X is a leaving group.

[0292] Embodiment 21. The compound of embodiment 19 or 20, wherein X is a sulfonate leaving group.

[0293] Embodiment 22. The compound of any one of embodiments 19-21, wherein X is -

[0294] SChMe.

[0295] Embodiment 23. The compound of any one of embodiments 20-22, wherein R3is an activated ester.

[0296] Embodiment 24. The compound of any one of embodiments 20-23. wherein R3is of the formula:

[0297] Embodiment 25. The compound of any one of embodiments 20-22, wherein R3is an azide.

[0298] Embodiment 26. The compound of any one of embodiments 1-25. wherein Y1is a substituted or unsubstituted heteroarylene.

[0299] Embodiment 27. The compound of any one of embodiments 1-26, wherein Y1is a substituted or unsubstituted benzothiazole or a substituted or unsubstituted oxadiazole.

[0300] Embodiment 28. The compound of any one of embodiments 1-27, wherein Y1is of the formula:

[0301] Embodiment 29. The compound of any one of embodiments 1-28, wherein L1is a linker comprising a PEG chain.

[0302] Embodiment 30. The compound of any one of embodiments 1-29, wherein L1comprises

[0303] 1-10 PEG units.

[0304] Embodiment 31. The compound of any one of embodiments 1-30. wherein L1is of the formula: Embodiment 32. The compound of any one of embodiments 1-28, wherein L1is absent.

[0305] Embodiment 33. The compound of any one of embodiments 1-32, wherein L2is of the formula:

[0306] Embodiment 34. The compound of any one of embodiments 1-32, wherein L2is absent.

[0307] Embodiment 35. The compound of any one of embodiments 1-34. wherein Z1is a bivalent aryl group, or a trivalent and group.

[0308] Embodiment 36. The compound of any one of embodiments 1-35, wherein Z1is a bivalent phenyl group, or a trivalent phenyl group.

[0309] Embodiment 37. The compound of any one of embodiments 1-36, wherein Z1is of the formula:

[0310] Embodiment 38. The compound of any one of embodiments 1-34, wherein Z1comprises a branched alkyl or heteroalkyd chain.

[0311] Embodiment 39. The compound of any one of embodiments 1-34, or 38 wherein Z1comprises a branched heteroalky l chain.

[0312] Embodiment 40. The compound of any one of embodiments 1-34. or 38-39 wherein Z1is of the formula:

[0313] Embodiment 41. The compound of any one of embodiments 1-34. wherein Z1is absent.

[0314] Embodiment 42. A compound comprising an antibody or antibody fragment; and a

[0315]

[0316]

[0317] L2is absent or a linker,

[0318] R2comprises an oligonucleotide-based agent, and represents an attachment point to the antibody or antibody fragment.

[0319]

[0320]

[0321] or a pharmaceutically acceptable salt thereof, wherein: L2is absent or a linker,

[0322] R2comprises an oligonucleotide-based agent, and

[0323] X is a leaving group.

[0324] Embodiment 44. The compound of embodiment 43, wherein X is a sulfonate leaving group.

[0325] Embodiment 45. The compound of embodiment 43 or 44, wherein X is -SChMe.

[0326] Embodiment 46. The compound of any one of embodiments 42-45, wherein L2is of the formula:

[0327] Embodiment 47. The compound of any one of embodiments 42-45, wherein L2is absent.

[0328] Embodiment 48. The compound of any one of embodiments 1-19. or 26-47, wherein R2comprises an RNAi agent.

[0329] Embodiment 49. The compound of any one of embodiments 1-19, or 26-48, wherein R2comprises a sense strand containing one or two inverted abasic residues. Embodiment 50. The compound of any one of embodiments 1-19, or 26-49, wherein R2comprises a sense strand containing two inverted abasic residues.

[0330] Embodiment 51. The compound of any one of embodiments 1-19, or 26-50, wherein R2comprises a sense strand containing one or two inverted abasic residues.

[0331] Embodiment 52. The compound of embodiment 50 or 51, wherein the Fab is conjugated to the 5’ terminal end of the sense strand.

[0332]

[0333] « A represents a point of connection of the structure to an antibody or antibody fragment, and represents a point of connection of the structure to a moiety comprising an RNAi agent.

[0334] or a salt thereof.

[0335] Embodiment 55. A method of synthesizing a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0336] Fab is an antibody or antibody fragment; R1is -0Rla, -C(O)NHRla’ or -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl;

[0337] Z is a bivalent or trivalent aryl group, or a bivalent or trivalent heteroaryl group:

[0338] Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0339] Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0340] L1is absent or a linker comprising a PEG chain;

[0341] L2is absent or a linker;

[0342] R2comprises an oligonucleotide-based agent; m is 0 or 1 ; and n is 0 or 1 as valency permits; comprising, reacting a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein:

[0343] Z1is absent, a bivalent or tnvalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;

[0344] L1is absent or a linker comprising a PEG chain;

[0345] L2is absent or a linker;

[0346] R2comprises an oligonucleotide-based agent; p is 0 or 1; and

[0347] X is a leaving group; with an antibody comprising a cysteine residue.

[0348]

[0158] The above provided embodiments and items are now illustrated with the following, non-limiting examples. EXAMPLES

[0349] Example 1. Anti-Transferrin antibody fragments (Fabs)

[0350]

[0159] Antibodies and antibody fragments for combination with linkers described herein may be produced by methods known in the art. Some example anti-transferrin Fabs are described below'.

[0351]

[0160] The CDR3 is the same sequence in the 15 Fab clones VL (QHFWGTPLT; SEQ ID NO: 13) and VH (GTRAYHY; SEQ ID NO: 24).

[0352] Table 1. Light Chain Complementarity Determining Regions (CDRs)

[0353] Table 2. Heavy Chain Complementarity Determining Regions (CDRs)

[0354]

[0355]

[0161] Table 3 shows exemplary light chain framework regions used in the exemplary anti- TfRl antibodies provided herein. An exemplary' CL sequence for use in the Fab light chain is also provided in Table 3.

[0356] Table 3. Exemplary Light Chain Framework Regions and Constant Region

[0357]

[0162] Table 4 shows exemplary heavy chain framework regions used in the exemplary anti- TfRl antibodies provided herein. An exemplary CHI sequence for use in the Fab heavy chain is also provided in Table 4.

[0358] Table 4. Exemplary Heavy Chain Framework Regions and Constant Region Fragment

[0359]

[0163] Table 5 below lists VH and VL sequences of exemplary' anti-TfRl antibodies provided herein and their heavy and light chain sequences when in Fab format. Table 5. Sequences of Exemplary Anti-TfRl Fab Antibodies

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] Example 2. Synthesis of antibody-siRNA linkers

[0366]

[0164] The synthesis of various Fab linkers used throughout the present application are provided below.

[0367] Synthesis of 2.3.5.6-tetrafluorophenyl 16-((3,5-bis(5-(methylsulfonyl)-l,3>4-oxadiazol-2- yl)phenyl)amino)-16-oxo-4,7,10,13-tetraoxahexadecanoate (i.e., L-1026-p)

[0368]

[0165] Compound 6 (2.35 g. 7.31 mmol; prepared according to Sarbisheh et al. Bioconjugate Chemistry 2020 31 (12), 2789-2806), EDC-HC1 (2.38 g, 12.43 mmol), and K-Oxyma (2.50 g, 13.9 mmol) were combined as solids and slurried in DMF (190 mL) under N2 at ambient temperature. Compound 7 (1.1.92 g, 5.48 mmol) was added as a solution in DMF (10 mL). After 5 m, triethylamine (4.5 mL, 32.2 mmol) was added dropwise at ambient temperature. The reaction mixture was heated at 50°C for 2 days. The reaction mixture was concentrated under reduced pressure to a red oil which was slurried in DCM (250 mL) and washed with sat. aq. sodium bicarbonate (200 mL). The layers were separated, and the aqueous layer was further extracted with DCM (100 mL). The combined organic phase was washed with water (200 mL) and brine (200 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated. The residue was purified by normal phase SiCh chromatography with a gradient of ethyl acetate in DCM (0-100%). Yield of compound 8: 1.77 g (49%), partially contaminated with compound 6. Calculated mw for compound 8: 653.77 g / mol, found m / z (ESI, positive mode): 654.83.

[0369]

[0166] Compound 8 (1.77 g. 2.71 mmol) was dissolved in TFA:DCM [1 :1] (18 mL) and stirred at ambient temperature for 1 hour. The reaction mixture was concentrated under reduced pressure then coevaporated with toluene (3 x 30 mL). The residue was purified by normal phase SiCh chromatography with a gradient of DCM containing 0.1% formic acid and methanol (0-7%). Yield of compound 9: 1.30 g (80%). Calculated mw for compound 9: 597.66 g / mol, found m / z (ESI. positive mode): 598.79.

[0370]

[0167] Compound 9 (1.30 g, 2.18 mmol) was dissolved in DCM (50 mL) and cooled to 0°C. A 100 mg / mL solution of zw-CPBA solution was prepared by dissolving 10.38 g zw-CPBA (77 wt%) in 80 mL DCM and dry ing with sodium sulfate until clear. To the solution of compound 9 was added 58 mL m-CPBA (5.85 g, 26.1 mmol) dropwise at 0°C. The reaction mixture was warmed to ambient temperature and allowed to proceed overnight. The reaction mixture was concentrated, slurried in DCM 0.1% formic acid (50 mL), and filtered. The filtrate was purified by normal phase SiCh chromatography with a gradient of DCM containing 0.1% formic acid and methanol (0-10%). Yield of compound 10: 1.03 g (72%). Calculated mw for compound 10: 661.65 g / mol, found m / z (ESI, positive mode): 662.65.

[0371]

[0168] To a solution of compound 10 (1.03 g. 1.56 mmol) in DCM:ACN [4: 1] (15 mL) at 0°C was added EDC (0.448 g, 2.34 mmol) followed by a solution of TFP (0.310 g, 1.87 mmol) in DCM: ACN [4:1] (5 mL). After 5 m. the reaction mixture was warmed to ambient temperature. After 1.5 h. the reaction mixture was concentrated to dryness. The crude was purified by preparative reverse phase HPLC (Phenomenex Gemini C18 50 mm x 250 mm, 10 um) using a gradient of water / acetonitrile containing 0.1% TFA. Product-containing fractions were concentrated under reduced pressure. Yield of L-1026-p: 1.10 g (87%). Calculated mw for compound L-1026-p: 809.71 g / mol, found m / z (ESI, positive mode): 810.62. NMR (400 MHZ.[D6]DMSO, 25°C): 5 = 2.64 (t. 2H), 3.00 (t, 2H), 3.49 (m. 12H), 3.74 (m. 10H), 7.92 (m, 1H), 8.34 (t, 1H), 8.68 (d, 2H), 10.67 (s, 1H).

[0372] Synthesis of 2,3,5,6-tetrafluorophenyl l-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenyl)-14-(l-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenyl)-l-oxo-5,8,ll-trioxa-

[0373] 2-azatridecan-13-yl)-l-oxo-5,8,ll,17,20,23-hexaoxa-2,14-diazahexacosan-26-oate (i.e., L-

[0374]

[0169] To a solution of compound 5 (250 mg, 0.44 mmol) in DCM (5 mL) was added DIEA (190 uL, 1.09 mmol) then Compound L20-p (400 mg, 0.96 mmol) as a si urn- in DCM (5 mL) at 0°C. After 5 m, the reaction was allowed to warm to ambient temperature. After 2.5 h, the mixture was concentrated under reduced pressure. The residue was coevaporated with ACN (5 mL x 2) then used in the next step without purification assuming 100% yield. Calculated mw for compound 6: 1072.17 g / mol, found m / z (ESI. positive mode): 1073.05.

[0375]

[0170] To a solution of compound 6 (467 mg, 0.44 mmol) in DCM (4 mL) was added EDC (100 mg. 0.52 mmol) at 0°C. After 5 min, TFP (87 mg, 0.52 mmol) was added as a solution in DCM (0.5 mL). After 5 min, the reaction was allowed to warm to ambient temperature. After 4 h, an additional portion of EDC (25 mg, 0.13 mmol) was added. After an additional 30 m, the reaction mixture was concentrated. The residue was coevaporated with ACN (2 x 5 mL).

[0376]

[0171] The crude reaction mixture was dissolved in water / ACN 0. 1% TFA (25 mL) and MeOH (5 mL). The mixture had solids which were removed by centrifugation, and the solution was filtered. The solution was purified by semipreparative reverse phase HPLC (Phenomenex Gemini C18 21.2 mm x 250 mm) using a gradient of water / acetonitrile containing 0. 1% TFA. Product containing fractions (60 mL) were diluted 1 : 1 with DCM. The pH of the mixture was adjusted with sat. aq. sodium bicarbonate until pH 7 (2 mL). The layers were separated and the organic phase was washed with water (20 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated. The residue was coevaporated with ACN (50 mL). Yield of L-1045-p: 76 mg (14% over 2 steps). Calculated mw for compound L-1045-p: 1220.22 g / mol, found m / z (ESI, positive mode): 1221.02. 'H NMR (400 MHZ,[D6]DMSO, 25°C): 5 = 3.00 (t, 2H), 3.40-3.55 (m, 40H), 3.72 (s, 6H), 3.75 (t, 2H), 7.93 (m, 1H), 8.08 (m, 4H), 8.19 (m, 4H), 8.80 (t, 2H).

[0377] Synthesis of 2,3,5,6-tetrafluorophenyl 2-(methylsulfonyl)benzo[d]thiazole-6-carboxyIate

[0378] (i.e., L-1064-p)

[0379]

[0172] To a solution of compound 1 (200 mg, 0.77 mmol) in DMF (3 mL) was added sodium methanesulfinate (technical grade 85 wt%, 162 mg, 1.16 mmol). The reaction mixture was heated at 70°C for 30 m then cooled to ambient temperature. EtOAc (10 mL) and water (10 mL) were added and the layers were separated. The aqueous phase was further extracted with EtOAc (10 mL). The combined organic phase was washed with water (3 x 10 mL) then brine (10 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated, sodium sulfate, filtered, and concentrated. Calculated mw for compound 2: 257.28 g / mol, found m / z (ESI. positive mode): 258.29.

[0380]

[0173] To a slurry of compound 2 (182 mg, 0.71 mmol) in DCM (2 rnL) was added EDC (203 mg, 1.06 mmol) followed by a solution of TFP (129 mg, 0.78 mmol) in DCM (1 mL) at ambient temperature. After 2 h, the reaction had become clear. The reaction was concentrated to dryness. The residue was adsorbed onto SiCh (3 mL) and purified by normal phase SiCh chromatography with a gradient of DCM in hexanes (0-50% DCM). Yield of L-1064: 191 mg (67%). Calculated mw for compound L-1064-p: 405.34 g / mol, found m / z (ESI, positive mode): 406.23. ’H NMR (400 MHz,[D6]DMSO, 25°C): 5 = 3.66 (s, 3H), 8.06 (m, 1H), 8.4- 8.5 (m. 2H), 9.35 (d, 1H).

[0381] Synthesis of 2,3,5,6-tetrafluorophenyl l-(2-(methylsulfonyl)benzo[d]thiazol-6-yl)-14-(l- (2-(methylsulfonyl)benzo[d]thiazol-6-yl)-l-oxo-5,8,ll-trioxa-2-azatridecan-13-yl)-l-oxo- 5,8,11,17, 20, 23-hexaoxa-2,14-diazahexacosan-26-oate (i.e., L-1100-p)

[0382]

[0174] To a solution of compound 1 (250 mg, 0.44 mmol) in DCM (5 mL) was added DIEA (190 uL, 1.09 mmol) then a solution of L-1064 (400 mg, 0.96 mmol) in DCM (5 mL) dropwise at 0°C. The reaction mixture was warmed to ambient temperature. After 1 h, EDC (168 mg, 0.87 mmol) was added to the reaction mixture. After an additional 1.5 h, the reaction mixture was concentrated to an oil. The residue was purified by normal phase SiCh chromatography with a gradient of methanol in DCM (0-10%). Yield of L-1100-p: 299 mg (57%). Calculated mw for compound L-1100-p: 1198.29 g / mol, found m / z (ESI, positive mode): 1198.70. 'H NMR (400 MHz,[D6]DMSO, 25°C): 8 = 2.99 (t, 2H), 3.10 (m, 4H), 3.44- 3.60 (m, 46H), 3.74 (t, 2H), 7.91 (m, 1H), 8.11 (m, 2H), 8.30 (m, 2H), 8.88 (m, 4H).

[0383] Synthesis of 2,3,5,6-tetrafluorophenyl l-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)-3,6,9,12-tetraoxapentadecan- 15-oate (

[0384]

[0175] A solution of compound 1 (500 mg, 2.40 mmol) in THF (6 mL) was sparged with nitrogen for 10 m. To the solution was added potassium carbonate (664 mg, 4.80 mmol). The mixture was stirred at ambient temperature for 30 m. A solution of compound 2 (2.78 g, 7.2 mmol) in THF (2 mL) was added dropwise at rt. The reaction was allowed to proceed overnight. After 24 h, LCMS showed a mixture of product and starting materials; the reaction mixture was heated at 40°C for 5 h. The reaction mixture was concentrated under reduced pressure and dissolved in ethyl acetate (30 mL) and sat. aq. sodium bicarbonate (15 mL). The organic phase was w ashed with brine, dried with sodium sulfate, filtered, and concentrated. The residue w as purified by normal phase SiCh chromatography with a gradient of ethyl acetate in DCM (0-50%). Yield of compound 3: 980 mg (80%). Calculated mw for compound 3: 512.62 g / mol, found m / z (ESI, positive mode): 513.69.

[0385]

[0176] Compound 3 (980 mg, 1.91 mmol) was dissolved in TFA:DCM [1: 1] (6 mL) at rt. After 30 m, toluene (30 mL) was added and the mixture was concentrated under reduced pressure. The residue was coevaporated with toluene (3 x 20 mL) then acetonitrile (20 mL). The product was used in the next step without further purification assuming 100% yield. Calculated mw for compound 4: 456.51 g / mol, found m / z (ESI. positive mode): 457.05.

[0386]

[0177] Compound 4 (1.30 g, 2.18 mmol) was dissolved in DCM (2 mL) and cooled to 0°C. A 100 mg / mL solution of m-CPBA solution was prepared by dissolving 1.3 g m-CPBA (77 wt%) in 10 mL DCM and drying with sodium sulfate until clear. To the solution of compound 4 was added 6.5 mL m-CPBA (654 mg, 2.92 mmol) dropwise at 0°C. The reaction mixture was warmed to ambient temperature and allowed to proceed overnight. The reaction mixture was quenched by addition of aq. sodium sulfite (10% w / w, 4 mL) with vigorous stirring. The mixture was diluted with DCM (20 mL) and water (10 mL). The organic phase was collected and w ashed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in DCM (10 mL), cooled to -20°C. and centrifuged. The supernatant was collected and the process was repeated with additional DCM (10 mL). The combined harvest was concentrated under reduced pressure. Yield: 355 mg. NMR analysis in DMSO shows 60 wt% contamination by 3-chlorobenzoic acid. Corrected yield for compound 5: 213 mg (97%). Calculated mw for compound 5: 488.51 g / mol, found m / z (ESI, positive mode): 489.57.

[0387]

[0178] To a solution of compound 5 (213 mg. 0.44 mmol) in DCM (2.2 mL) was added EDC (293 mg, 1.53 mmol) followed by a solution of TFP (217 mg, 1.31 mmol) in DCM (1 mL) at 0°C. The reaction mixture was allowed to warm to ambient temperature. After 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by normal phase S1O2 chromatography with a gradient of ethyl acetate in DCM (0-50%). Yield of L-1063-p: 121 mg (44%). Calculated mw for compound L-1063: 636.57 g / mol, found m / z (ESI, positive mode): 637.66. ‘H NMR (400 MHz,[D6]DMSO, 25°C): 5 = 3.01 (t, 2H), 3.51- 3.60 (m, 12H), 3.69 (s. 3H), 3.78 (m, 4H), 4.22 (m, 2H), 7.21 (m, 2H), 7.93 (m, 1H), 8.01 (m, 2H).

[0388] Synthesis of 2,3,5,6-tetrafluorophenyl (S)-l-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)-18-(l-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenoxy)-3,6,9,12- tetraoxapentadecan-15-amido)-15,19-dioxo-3,6,9,12,23,26-hexaoxa-16,20- diazanonacosan-29-oate

[0389]

[0179] To a solution of 3-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-L-alanine 1,1- dimethylethyl ester (CAS 2084868-72-2) (1.00 g. 2.61 mmol) in ACN (6.3 mL) was added diethylamine (2.70 mL, 26.15 mmol) at rt. After 30 m, the reaction mixture was concentrated under reduced pressure. The residue was coevaporated with ACN (2 x 20 mL) and used in the next step without further purification assuming 100% yield. Calculated mw for compound 2: 160.22 g / mol, found m / z (ESI. positive mode): 161.36.

[0390]

[0180] To a slurry of compound 2 (125 mg, 0.78 mmol) in DMF (2.5 mL) was added DIEA (408 uL, 2.34 mmol) then a solution of compound 1 (784 mg. 1.72 mmol; prepared as discussed above) in DCM (5 mL). To the turbid mixture was added TBTU (626 mg, 1.95 mmol) at ambient temperature. After 30 m, the reaction mixture was concentrated under reduced pressure (to remove DCM) and EtOAc (30 mL) was added. The mixture was washed with sat. aq. sodium bicarbonate (2 x 15 mL), sat. aq. ammonium chloride (2 x 15 mL), water (15 mL). and brine (15 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was adsorbed onto SiCh (7 mL) and purified by normal phase SiCh chromatography with a gradient of methanol in DCM (0-7%). Yield of compound 3: 492 mg (61%). Calculated mw for compound 3: 1037.21 g / mol, found m / z (ESI, positive mode): 1037.89.

[0391]

[0181] Compound 3 (490 mg, 0.47 mmol) was dissolved in TFA:DCM [1 : 1] (5 mL) and stirred at ambient temperature for 2 hours. The reaction mixture was concentrated under reduced pressure then coevaporated with toluene (3 x 30 mL). The product was used in the next step without further purification assuming 100% yield. Calculated mw for compound 4: 981.10 g / mol, found m / z (ESI, positive mode): 981.84.

[0392]

[0182] To a solution of compound 4 (291 mg, 0.30 mmol) and compound 5 (58 mg, 0.33 mmol) in DMF (4.4 mL) was added DIEA (77 uL, 0.44 mmol) then HATU (169 mg, 0.44 mmol) at ambient temperature. The reaction mixture was allowed to proceed overnight. The reaction mixture was diluted with water 0.1% TFA (5 mL) and purified by semi -preparative RP HPLC (Phenomenex Gemini C18 21.2 x 250 mm) using a gradient of water / acetonitrile containing 0.1% TFA. Product-containing fractions were concentrated under reduced pressure. Yield of compound 6: 229 mg (68%). Calculated mw for compound 6: 1140.28 g / mol, found m / z (ESI, positive mode): 1140.97.

[0393]

[0183] To a solution of compound 6 (229 mg, 0.20 mmol) in DCM (3.4 mL) was added EDC (77 mg, 0.40 mmol) then TFP (67 mg, 0.40 mmol) at 0°C. After 1 h, additional TFP (267 mg, 1.61 mmol) and EDC (77 mg, 0.40 mmol) were added. After an additional 2 h. additional EDC (96 mg, 0.50 mmol) was added. Finally, after 30 m, the reaction mixture was concentrated to 2 mL and purified by normal phase SiCh chromatography with a gradient of methanol in DCM (0-7%). Yield of compound 7: 69 mg (27%). Calculated mw for compound 7: 1288.34 g / mol, found m / z (ESI. positive mode): 1288.83.

[0394]

[0184] A solution of compound 7 (69 mg, 0.054 mmol) in DCM (2 mL) was prepared and cooled to 0°C. A 100 mg / mL solution of m-CPBA solution was prepared by dissolving 1.3 g m-CPBA (77 wt%) in 10 mL DCM and dry ing with sodium sulfate until clear. To the solution of compound 7 was added 1.4 mL m-CPBA ( l40 mg, 0.80 mmol) dropwise at 0°C. The reaction mixture was warmed to ambient temperature and allowed to proceed overnight. The reaction mixture was concentrated then suspended in water / ACN containing 0. 1% TFA (10 mL) and purified by semi-preparative RP HPLC (Phenomenex Gemini C18 21.2 x 250 mm) using a gradient of water / acetonitrile containing 0.1% TFA . Product-containing fractions were concentrated under reduced pressure. Yield of L-1176-p: 60 mg (83%). Calculated mw for compound L-1176-p: 1352.34 g / mol, found m / z (ESI, positive mode): 1352.79. 'l l NMR (400 MHZ,[D6]DMSO, 25°C): 5 = 2.99 (m, 3H), 3.15-3.32 (m, 6H), 3.37 (t, 2H). 3.42-3.68 (m, 33H), 3.68 (s, 6H), 3.73-3.77 (m, 6H), 4.20 (m, 5H), 4.28 (m, 3H), 7.17 (m, 4H), 7.73 (t, 1H), 7.85 (t, 1H), 7.90 (m, 2H), 8.01 (m, 4H).

[0395] Synthesis of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-N-(3,5-bis(5-(methylsulfonyl)- 1,3,4- oxadiazol-2-yl)phenyl)propenamide (i.e., L-1288-p)

[0396]

[0185] Compound 1 (106 mg, 0.33 mmol; prepared according to Sarbisheh, et al.; Bioconjugate Chemistry 2020 31 (12), 2789-2806), EDC’HCl (88 mg, 0.46 mmol), and K- Oxyma (89 mg, 0.49 mmol) were combined as solids and dried under high vacuum for 30 m. The solids were slurried in DMF (10 mL) under N2 at ambient temperature. Compound 2 (73 mg, 0.30 mmol) and triethylamine (126 uL, 0.90 mmol) were added as a solution in DMF (1 mL). The reaction mixture was heated at 50°C for 2 days. The reaction mixture was concentrated under reduced pressure to an oily residue then redissolved in water / ACN 0.1% TFA (40 mL). The solution was purified by semipreparative reverse phase HPLC (Phenomenex Gemini C18 21.2 mm x 250 mm) using a gradient of water / acetonitrile containing 0.1% TFA. Product-containing fractions were combined and concentrated under reduced pressure. Yield of compound 3: 62 mg (38%). Calculated mw for compound 3: 550.61 g / mol, found m / z (ESI. positive mode): 551.74.

[0397]

[0186] Compound 3 (62 mg. 0. 11 mmol) was dissolved in DCM (2 mL) and cooled to 0°C. A 100 mg / mL solution of m-CPBA solution was prepared by dissolving 1.3 g m-CPBA (77 wt%) in 10 mL DCM and drying with sodium sulfate until clear. To the solution of compound 3 was added 2 mL m-CPBA (194 mg, 1.13 mmol) dropwise at 0°C. The reaction mixture was warmed to ambient temperature and allowed to proceed overnight. The reaction mixture was diluted with DCM (20 mL) and washed with 100 mM NaOH (10 mL). The organic phase was further washed with water (2 x 10 mL), dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in water: ACN 0.1% TFA (8 mL) and methanol (0.5 mL). The solution was purified by semipreparative reverse phase HPLC (Phenomenex Gemini Cl 8 21.2 mm x 250 mm) using a gradient of water / acetonitrile containing 0.1% TFA. Productcontaining fractions were combined and concentrated under reduced pressure. Yield of L- 1288-p: 31 mg (45%). Calculated mw for compound L-1288-p: 614.61 g / mol, found m / z (ESI, positive mode): 615.66. ‘H NMR (400 MHz,[D6]DMSO, 25°C): 5 = 2.65 (t, 2H), 3.36 (t, 2H) 3.55 (m, 10H), 3.74 (m, 8H), 8.34 (t, 1H), 8.68 (d, 2H), 10.68 (s, 1H).

[0398] Synthesis of N-methyl-4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)benzamide (i.e., CP-

[0399] 1113-p)

[0400]

[0187] To a slurry of L20 (5.0 g, 12.0 mmol) in THF (80 mL) was added a solution of methylamine (1.3 mL, 15.0 mmol) in THF (20 mL) dropwise at ambient temperature. After 30 m, SiCh (35 mL) was added and the reaction was concentrated under reduced pressure. The residue was purified by normal phase SiCh chromatography with a gradient of ethyl acetate in DCM (0-70%). Product-containing fractions were concentrated under reduced pressure. Yield of CP-1113-p: 1.76 g (52%). Calculated mw for compound CP-1113-p: 281.29 g / mol, found m / z (ESI, positive mode): 282.45. ’H NMR (400 MHz,[D6]DMSO, 25°C): 5 = 2.80 (d. 3H), 3.70 (s, 3H), 8.06 (m. 2H),. 8.18 (m, 2H), 8.68 (br q. 1H).

[0401] Synthesis of N-methyl-2-(methylsulfonyl)benzo[d]thiazole-6-carboxamide (i.e., CP-1154-

[0402] To a slurry of L-1064-p (300 mg, 0.74 mmol) in THF (6 mL) was added a solution of methylamine (40 wt% in water, 80 uL, 0.93 mmol) dropwise at ambient temperature. After 1 h, SiCh (3 mL) was added and the reaction was concentrated under reduced pressure. The too residue was purified by normal phase SiCh chromatography with a gradient of ethyl acetate in DCM (0-60%). Product-containing fractions were concentrated under reduced pressure. Yield of CP-1154-p: 166 mg (83%). Calculated mw for compound CP-1154-p: 270.32 g / mol, found m / z (ESI, positive mode): 271.25. 'H NMR (400 MHz,[D6]DMSO, 25°C): 5 = 2.83 (d, 3H), 3.61 (s, 3H), 8.11 (m, 1H), 8.32 (m, 1H), 8.72 (br q, 1H), 8.80 (d, 1H).

[0403] Example 3. Synthesis of RNAi Agents and TfRl-Speciflc Conjugates

[0404]

[0188] RNAi agent duplexes disclosed herein were synthesized in accordance with the following:

[0405] A. Synthesis.

[0406]

[0189] The sense and antisense strands of the RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 A or 600A. obtained from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the 2'-O-methyl phosphoramidites that were used included the following: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'-O-methyl-adenosine- 3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5 '-O-dimethoxy-trityl-N4-(acetyl)- 2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5'-O- dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N- diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2- cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidites carried the same protecting groups as the 2'-O-methyl RNA amidites. 5'-dimethoxytrityl-2'- O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia). The inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O- (2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA. USA). The following UNA phosphoramidites were used: 5'-(4.4’- Dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 5'-(4,4'-Dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'- benzoyl-3'-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite, 5'-(4,4'-Dimethoxytrityl)- N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, and 5'-(4,4'-Dimethoxy-trityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2- cyanoethyl)-(N,N- diiso-propyl)] -phosphorami dite. TFA aminolink phosphorami dites were also commercially purchased (ThermoFisher). The cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017 / 214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).

[0407] B. Cleavage and deprotection of support bound oligomer.

[0408]

[0190] After finalization of the solid phase synthesis, the dried solid support was treated with a 1 :1 volume solution of 40 wt. % methylamine in water and 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30°C. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0409] C. Conjugation ofRNAi agents to Fabs and Capping with CP-1113.

[0410]

[0191] RNAi agents described herein comprising a free amine were conjugated to L20-p using standard amide reaction chemistry following cleavage from the solid phase. To a solution of Fab in PBS (0.2 umol, 1.0-10.0 mg / mL in PBS) was added a freshly prepared solution of (tris(2-carboxyethyl)phosphine) hydrochloride (TCEP-HC1) in PBS (5-20 eq, 70 mM). The reaction was held overnight at room temperature and covered from light. The next day, TCEP was removed by loading the reaction mixture on a PD-10 desalting column equilibrated with PBS and eluted with PBS. The concentration of Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L20-modified sense strand in sodium phosphate buffer was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L20-modified sense strand (1-1.3 eq, 0.5-2.5 mM), and the reaction was mixed end-over-end. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab, DARI, and DAR2. After 1 hour, a solution of CP-1113-p: added to the reaction mixture (3 eq, 36 mM). After

[0411] 1 hour, a solution of L-cysteine in PBS was added to the reaction mixture (6-10 eq, 165 mM). Finally, the conjugate was annealed by addition of antisense strand (1.2-1.5 eq, 0.5-2.5 mM). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A). 20 mM tris 1500 mM NaCl (Buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was pump loaded onto the column and eluted with a gradient of 10-40% Buffer B. DARI and DAR2 fractions were differentiated by SEC Method 1, AEX Method 1, and Nanodrop 260 / 280 readings. DARI fractions were pooled and buffer exchanged to PBS using a PD-10 desalting column. The purified conjugate was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 13.2 minutes.

[0412] D. Conjugation ofRNAi agents to Fabs and Capping with N-ethyl maleimide.

[0413]

[0192] RNAi agents described herein comprising a free amine were conjugated to L20-p using standard amide reaction chemistry following cleavage from the solid phase. To a solution of Fab in PBS (10 mg, 0.2 umol, 1.0-10.0 mg / mL in PBS) was added a freshly prepared solution of TCEP-HC1 in PBS (5-20 eq, 70 mM). The reaction was held overnight at room temperature and covered from light. The next day, TCEP was removed by loading the reaction mixture on to a PD-10 desalting column equilibrated with 20 mM tris pH 8 and eluted with 20 mM tris pH 8. The concentration of Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L20-modified sense strand in sodium phosphate buffer was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L20-modified sense strand (1-1.3 eq, 0.5-2.5 mM), and the reaction was mixed end-over-end. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab, DARI, and DAR2. After 1 hour, a solution of N-ethyl maleimide (NEM) in 20 mM tris pH 8 was added to the reaction mixture (12 eq. 160 mM). After 1 h, the conjugate was annealed by addition of antisense strand (1.2-1.5 eq, 0.5-2.5 mM). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10-40% Buffer B. DARI and DAR2 fractions were differentiated by SEC Method 1, AEX Method 1, and UV-Vis 260 / 280 measurements. DARI fractions were pooled and buffer exchanged to PBS using a PD-10 column. The purified conjugate was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 13.2 minutes.

[0414] SEC Method 1

[0415] AIEX Method 1

[0416]

[0193] RNAi agents described herein comprising a free amine were first conjugated to

[0417] L1026-p. i.e.: using standard solid phase synthesis coupling techniques to generate the L-1026-modified sense strand:

[0418] (NH2-C6)s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb) (i.e., CS915332, SEQ ID NO: 159). Following cleavage from the solid phase, the RNAi agents described herein were synthesized according to the following procedure as exemplified by the synthesis of AC006448:

[0419]

[0194] To a solution of Fab0070 (28 mg, 0.59 pmol, 5.55 mg / mL in PBS) was added a freshly prepared solution of TCEP-HC1 in PBS (5 eq. 70 mM, 42 pL). The reduction was mixed end-over-end at ambient temperature for 15 minutes then held at 5°C overnight without agitation. The next day, TCEP was removed by loading the reaction mixture on two PD-10 desalting columns (Cytiva) equilibrated with 20 mM tris 50 mM NaCl pH 7.6 (alternatively, 20 mM tris pH 8 or PBS buffer may be used) and eluted with the same buffer. The concentration of the Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L-1026-modified sense strand (i.e., CS915332) in 10 mM sodium phosphate buffer pH 6.0-6.5 was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L- 1026-modified CS915332 (1.15 eq, 2.75 mM, 240 uL), and the reaction was mixed end-over- end at ambient temperature. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab0070, DARI product, and DAR2 product. After 30 m, a solution of L-cysteine in 20 mM tris 50 mM NaCl pH 7.6 (alternatively, some L-1026 conjugates have been prepared in 20 mM tris pH 8 or PBS buffer solutions) was added to the reaction mixture (10 eq, 165 mM, 36 uL). After 30 m, the conjugate was annealed by addition of antisense strand (CA003820) (L3 eq, L45 mM in water, 529 uL). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pEI 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10-40% Buffer B. DARI and DAR2 fractions were differentiated by SEC Method 1, AIEX Method 1, and UV-Vis 260 / 280 measurements. DARI fractions were pooled and buffer exchanged to PBS using two PD-10 columns. The purified conjugate. AC006448, was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 7.2 minutes.

[0420] SEC Method 1

[0421] AIEX Method 1 E. Conjugation ofRNAi agents to Fabs.

[0422] Synthesis of Fab0070-L-1288

[0423]

[0195] To a solution of Fab0070 in PBS (82 mg, 8.2 mg / mL) was added a freshly prepared solution of TCEP-HC1 in PBS (5 eq). The reaction mixture was held overnight at 5°C. The next day, the reaction mixture was buffer exchanged to 20 mM tris 50 mM NaCl pH 7.6 using four PD-10 columns equilibrated with the same buffer. The concentration was determined by Nanodrop, and the solution was diluted to 2 mg / mL with 20 mM tris 50 mM NaCl pH 7.6. The solution was diluted with DMSO (1.95 mL). A solution of L-1288 was prepared (2 mg / mL in DMSO) and charged (1.6 mL, 3.19 mg, 3 eq) over 15 m. After 10 m, the reaction was quenched with cysteine (10 eq). Insoluble material was removed by centrifugation. The supernatant removed, fdtered, diluted 1 : 1 with PBS, and concentrated / desalted using Pierce 10K MWCO spin columns to a volume of 8 mL. Finally, the solution was buffer exchanged by loading onto a HiPrep 26 / 10 desalting column and eluting with PBS. The final yield was approximately 80 mg.

[0424]

[0196] Bioconjugation of L-1289

[0425]

[0197] Lyophilized CS915332 (86 mg, 11.6 umol) was brought up in DMF (1.58 mL) and water (215 uL) and sonicated. To the solution was added triethylamine (9.7 uL, 6 eq) and a solution of L-1289 in DMF (50 mg / mL, 344 uL, 3 eq). The progress of the reaction was monitored by LCMS. After 1 h, added additional triethylamine (4 eq) and L-1289 solution (2 eq). After 20 m, LCMS showed complete conversion. The reaction mixture was acidified with phosphoric acid (200 mg / mL in water, 5 eq). The crude solution was added dropwise to a mixture of acetonitrile (43 mL) and PBS (1.7 mL). The precipitate was collected by centrifugation and the supernatant was discarded. The pellet was dissolved in water (1.5 mL) and added to acetonitrile (43 mL). The precipitate w as collected by centrifugation and dissolved in PBS (5 mL). The yield was 88 mg (98%).

[0426]

[0198] Conjugation of Fab0070-L-1288 with L-1307 modified sense strand

[0427]

[0199] To a solution of Fab0070-L-1288 in PBS (12.5 mg, 6.97 mg / mL, 1.79 rnL, 1 eq) was added a solution of L1307-modified sense strand: L-1307s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb), (i.e., CS009529, SEQ ID NO: 160) in PBS (6. 18 mg, 17.9 mg / mL. 345 uL, 3 eq). The combined solution was mixed end-over-end for 10 m then stored at 5 °C without agitation. The next day, the reaction was warmed to room temperature and annealed with antisense strand: cPrpusUfsgauuAfgcagGfuCfaAfaagsusg (i.e„ CA003820, SEQ ID NO: 161), 6.23 mg, 10.4 mg / mL, 599 uL, 3.3 eq. The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was diluted to 40 mL with MPA, loaded onto the column, and eluted with a gradient of 10-40% Buffer B. DARI -containing fractions were pooled and buffer exchanged to PBS using two PD-10 columns. The conjugate was analyzed by SEC Method 1 and found to be 99% pure with a retention time of 7.2 m. This procedure was also followed to generate the Fab-L-1288-L-1289-RNAi conjugates disclosed herein.

[0428] SEC Method 1

[0429] Example 4. Relative Expression of mRNA After Delivering TfRl Fab-Conjugated siRNA Antisense for the Target Androgen Receptor (AR) mRNA

[0430]

[0200] The siRNA duplexes are shown in sense and antisense in Tables 6 and 7, respectively. The antisense strand has complementary' to AR mRNA. The sense siRNA strand is conjugated to the Fabs shown in the Table 6 below.

[0431] Table 6. siRNA sense sequence for targeting AR mRNA.

[0432]

[0433]

[0434] Key: (a) represents 2'-O-methyl adenosine, (c) represents 2'-O-methyl cytidine, (g) represents 2'-O-methyl guanosine, and (u) represents 2'-O-methyl uridine; (Af) represents 2'-fluoro adenosine, (Cf) represents 2'-fluoro cytidine, (Gf) represents 2'-fluoro guanosine, and (Uf) represents 2'-fluoro uridine; (s) represents a phosphorothioate linkage; (invAb) and (invAb)s represents inverted abasic nucleotide (structure in table 13 below); cPrpus represents 5 '-cyclopropyl phosphonate-2'-O-methyluridine-3'- phosphorothioate (structure in table 13 below); (NH-C6)s structure in table 13 below; L20 structure in table 13 below; [CP- 1113] indicates that the Fab has been capped using CP-1113p according to the procedure of Example 6C; [NEM] indicates that the Fab has been capped using N-ethyl maleimide according to the procedure of Example 6D.

[0435] Table 7. siRNA Antisense Sequence for Targeting AR mRNA

[0436]

[0437] Key: (a) represents 2'-O-methyl adenosine, (c) represents 2'-O-methyl cytidine, (g) represents 2'-O-methyl guanosine, and (u) represents 2'-O-methyl uridine: (Af) represents 2'-fluoro adenosine, (Cf) represents 2'-fluoro cytidine, (Gf) represents 2'-fluoro guanosine, and (Uf) represents 2'-fluoro uridine; (s) represents a phosphorothioate linkage; cPrpus represents 5 '-cyclopropyl phosphonate-2'-O-methyluridine-3'- phosphorothioate (structure in table 13 below).

[0438] Table 8. Shows the structures for cPrpus, (invAb)s, (invAb), L20, L1026, L1100, L1176, L1064, L1063, and NH-C6 defined in the Key above for sense and antisense sequences

[0439] Example 5. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice

[0440]

[0201] On Study day 1, Tg B-hTfrl mice were injected intravenously with either a dosing volume of 250 pL / 25g PBS (group 1) or 1.5 mg / kg of a TfRl-Fab conjugated siRNA at a dosing volume of 250 uL / 25g (groups 2-7), and dosed again on day 2 with the same formulation, according to Table 9 below:

[0441] Table 9: Dosing groups for the mice of Example 5.

[0442]

[0202] On day 15, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including thoracic spinal cord, cortex, cerebellum and striatum. Samples were analyzed by qPCR for Androgen Receptor (AR) mRNA knockdown. FIG. 1 shows relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0443] Example 6. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice

[0444]

[0203] On Study day 1, Tg B-hTfrl mice were injected intravenously with either a dosing volume of 250 pL / 25g PBS (group 1) or 1.5 mg / kg of a TfRl-Fab conjugated siRNA at a dosing volume of 250 pL / 25g (groups 2-10), and dosed again on day 2 with the same formulation, according to Table 12 below:

[0445] Table 10: Dosing groups for the mice of Example 6.

[0446]

[0204] On day 15, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including thoracic spinal cord, cortex, cerebellum and striatum. Samples were analyzed by qPCR for Androgen Receptor (AR) mRNA knockdown. FIG. 2 shows relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0447] Example 7. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice

[0448]

[0205] On Study day 1, Tg B-hTfrl mice were injected intravenously with either a dosing volume of 250 pL / 25g PBS (group 1) or 1.5 mg / kg of a TfRl-Fab conjugated siRNA at a dosing volume of 250 pL / 25g (groups 2-6), and dosed again on day 2 with the same formulation, according to Table 11 below:

[0449] Table 11: Dosing groups for the mice of Example 7.

[0450]

[0206] On day 15. mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including thoracic spinal cord, cortex, cerebellum and striatum. Samples were analyzed by qPCR for Androgen Receptor (AR) mRNA knockdown. FIG. 3 shows relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0451] Example 8. In vivo AR mRNA Gene Expression in Different Antibody Conjugate Groups Relative to the AC003313 (Fab0002) Control Group in Select Brain Regions

[0452]

[0207] In vivo AR mRNA gene Knock Down of Fab-duplexes was measured in select brain regions. The AR mRNA gene expression in each group was normalized to the AC003313 (Fab0002-conjugated siRNA) group. FIG. 4 shows a graph of the relative expression values. The Fab0070-siRNA duplex group appears to have the lowest relative expression (highest gene knock down).

[0453] Example 9. In Vivo Knockdown of AR mRNA Gene Expression in Cynomolgus Monkeys

[0208] On Study day 0, cynomolgus monkeys were injected subcutaneously with either PBS or a compound formulation containing 3 mg / kg at a concentration of 1.5 mg / mL of an anti- TfRl Fab-siRNA conjugate, according to Table 12 below. On Study day 7, groups 2, 3 and 4 were dosed with the same formulation as on day 0. All animals were dosed at a volume of 2 mL / kg.

[0454] Table 12: Dosing groups for the non-human primates of Example 9.

[0455]

[0209] Three (n=3) monkeys were dosed in groups 1 (control), 3 and 4 (trigger treated), and two (n=2) monkeys were dosed in group 2. On study day 28, animals from all groups were euthanized and several tissues including Frontal Cortex, Temporal Cortex, Caudate, Cerebellum (cortex). Putamen, Motor Cortex, Medulla, Pons. Hippocampus, Thalamus, Hypothalamus, Midbrain, Substantia Nigra, Cervical dorsal root ganglion (DRG), Thoracic DRG, Lumbar DRG, Cervical Spinal Cord, Lumbar Spinal Cord, Thoracic Spinal Cord, and Visual Cortex were collected from each animal. Samples were analyzed by qPCR for AR mRNA knockdown. Average results for each group, relative to Group 1, are shown in Table 13 below: Table 13. Relative expression of AR mRNA in various tissues analyzed by qPCR for each of the dosing groups of Example 19.

[0456]

[0210] As show n in Table 13, above, durable reduction of AR mRNA expression was observed in multiple tissues for non-human primates treated with anti-TfRl antibody-siRNA conjugate. Groups 3 (Fab0061 -siRNA conjugate) and 4 (Fab0070-siRNA conjugate) outperformed Group 2 (Fab002-siRNA conjugate) in nearly every tissue analyzed, with the exception of Thoracic DRG and Lumbar DRG.

[0457] Example 10. In Vivo Knockdown of MAPI in in Cynomolgus Monkeys

[0458] [2H] As shown in Table 13. above, durable reduction of AR mRNA expression was observed in multiple tissues for non-human primates treated with anti-TfRl antibody-siRNA conjugate. Groups 3 (Fab0061 -siRNA conjugate) and 4 (Fab0070-siRNA conjugate) outperformed Group 2 (Fab002-siRNA conjugate) in nearly every tissue analyzed, with the exception of Thoracic DRG and Lumbar DRG.

[0459]

[0212] On Study day 0, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound formulation containing either 3 mg / kg at a concentration of 1.5 mg / mL and a volume of 2 mL / kg, 15 mg / kg at a concentration of 7.5 mg / mL and a volume of 2 mL / kg, or 30 mg / kg at a concentration of 7.5 mg / mL and a volume of 4 mL / kg of AC007414 in aCSF according to Table 14 below: Table 14: Dosing groups for the non-human primates of Example 10.

[0460]

[0213] Four (n=4) monkeys were dosed in each group. Monkeys were injected subcutaneously on days 0, 7. and 14. On study day 29, animals from each group were euthanized and brain and spinal cord tissue was collected from each animal. Samples were analyzed by qPCR for MAPT mRNA knockdown. Samples were analyzed by JESS for protein knockdown. Average mRNA knockdown for frontal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1. are shown in Table 15 below:

[0461] Table 15. Relative expression of MAPT mRNA in various tissues analyzed by qPCR for each of the dosing groups of Example 10

[0462]

[0214] Average protein knockdown for frontal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1, are shown in FIG. 5.

[0215] As can be seen in Table 15 and FIG. 5, MAPT RNAi agent bound to anti-Transferrin Fab070 achieved dose dependent and deep knockdown in CNS tissues when injected subcutaneously. The results demonstrate that treatment of MAPT -related diseases and disorder may be mitigated by RNAi agents administered subcutaneously.

[0463]

[0216] It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

[0464] Example 11. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice.

[0465]

[0217] On study day 1, Tg B-hTfrl mice were injected subcutaneously with either a dosing volume of 250 pL / 25g PBS (group 1) or 1.5 mg / kg of a TfRl-Fab conjugated siRNA at a dosing volume of 250 pL / 25g (groups 2-10), and dosed again on day 2 with the same formulation, according to Table 16 below:

[0466] Table 16. Dosing groups for the mice of Example 11.

[0467]

[0218] On day 15, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including thoracic spinal cord, cortex, cerebellum and striatum. Samples were analyzed by qPCR for Androgen Receptor (AR) mRNA knockdown. Table 17, below, shows the knockdown of AR in various tissues.

[0468] Table 17. Average mAR expression in CNS tissues of Example 11

[0219] As shown in Table 17, all dosing groups showed deep and stable knockdown using Fabs linked to RNAi agents through linkers of the present invention.

[0469] Example 12. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice.

[0470]

[0220] On study day 1, Tg B-hTfrl mice were injected subcutaneously with either a dosing volume of 250 pL / 25g PBS (group 1) or 1.5 mg / kg of a TfRl-Fab conjugated siRNA at a dosing volume of 250 iiL / 25g (groups 2-10), and dosed again on day 2 with the same formulation, according to Table 18 below:

[0471] Table 18. Dosing groups for the mice of Example 12.

[0472] 1221] On day 15. mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain, including thoracic spinal cord, cortex, cerebellum and striatum. Samples were analyzed by qPCR for Androgen Receptor (AR) mRNA knockdown. Table 19 shows relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0473] Table 19. mAR expression in CNS tissues of Example 12

[0474]

[0222] As shown in Table 19, both groups showed knockdown of AR using Fab-siRNA conjugates linked with linkers disclosed herein.

[0475] Example 13. In Vivo Administration ofHTT RNAi Agents in Cynomolgus Monkeys.

[0476]

[0223] HTT RNAi agents were evaluated in vivo in Cynomolgus monkeys. On Days 1. 8, and 15, four (n=4) male Cynomolgus monkeys for each test group were dosed with HTT RNAi agents formulated in PBS at 3.0 mg / kg (adjusted for individual animal body weight), 2.0 ml / kg dose volume, at 1.5 mg / ml dose concentration, or dosed with PBS. Each dose was administered via subcutaneous (SC) injection, and each dose was administered based on each respective animal’s most recent body weight. The dosing was in accordance with the following Table 20.

[0477] Table 20. Dosing for Cynomolgus monkeys of Example 13.

[0478]

[0224] The test animals were of non-human primate, Cynomolgus fascicularis monkeys. male, non-naive, aged 3-5 years. The test animals were acclimated to laboratory housing, per facility and acclimation standard operating procedures, for at least 14 days prior to the initiation of dosing. The RNAi agent test articles were administered via subcutaneous (SC) administration with a syringe and needle in the mid-scapular region.

[0479]

[0225] Dose sites were shaved prior to dosing. Day 1 dose was delivered to the animals’ left scapular region, Day 8 dose was delivered to the right scapular region, and Day 15 dose was delivered to the left scapular region. Each dose was given using syringe with a 23-25 gauge needle.

[0480]

[0226] The test animals’ individual body weights were recorded once pre-treatment Day -7, and then weekly through the duration of the study, and once prior to necropsy.

[0227] Cerebrospinal fluid (CSF), ~0.5 mL, was collected on Day -7 for all Groups. On Day 43 (day of necropsy), -2.0 mL CSF was collected for all Groups. For the CSF collection procedure, all test animals were anesthetized by intravenous (IV) injection of ketamine (10 mg / kg, IV) and dexmedetomidine (0.02 mg / kg, intramuscular IM) and positioned in lateral recumbency. The skin covering the insertion point was shaved and wiped several times with individual chlorhexidine scrubs. The head of the animal was gently flexed to where the chin nearly touches the chest, but the airway was not obstructed. The professional palpated the occipital protuberance and wings of the atlas (C l). The needle was inserted perpendicularly above the atlas through the skin to access the subarachnoid space. If the bone was encountered, the needle was redirected either anteriorly or posteriorly until the designated access point was found. The CSF would flow freely through the needle once the proper placement has been achieved and collected into a cryotube tube. The needle was removed, and direct pressure was applied to the puncture site for at least two minutes. Once the procedure was complete, the animals were administered Carprofen 2-4 mg / kg SC every' 12 hours for 1 day. Atipamezole (0.225 mg / kg, IM), was administered if necessary’.

[0481]

[0228] At Day 43. the Cynomolgus monkeys were euthanized. From the test animals, the following tissues were collected: left and right brain hemisphere, spinal cord, dorsal root ganglion (DRG). Tissues other than the aforementioned tissues may also be collected, and should such extra tissues be collected, their biological data were similarly presented below. The collected tissues were analyzed for biological parameters.

[0482]

[0229] From the collected tissues, cHTT mRNA transcript levels were quantified via qPCR, with cPPIB as endogenous control gene, normalized to Group 1 cynos dosed with PBS. The cHTT expression data is shown in the following Table 21.

[0483] Table 21. cHTT expression in cyno tissues of Example 13.

[0484]

[0230] In the temporal cortex, frontal cortex, motor cortex, caudate, hippocampus, Groups 2- 5 showed reduction in HTT transcripts out to at least Day 43 post dose. In the putamen, Groups 2, 4, and 5 showed reduction in HTT transcripts out to at least Day 43 post dose. Most notably, the most significant HTT transcript reduction was seen in the hippocampus, three doses of 3.0 mg / kg AC007867 showed -69% HTT transcript inhibition (0.306), and three doses of 3.0 mg / kg AC007865 showed -70% HTT transcript inhibition (0.302).

[0485]

[0231] HTT protein expression was analyzed via Jess protein assay in the cyno tissues, normalized to Group 1 cynos dosed with PBS. HTT protein was quantified using Anti- Huntingtin Protein Antibody (Sigma- Aldrich®, Cat. MAB2166). The HTT protein expression data is shown in the following Table 22. Table 22. HTT protein expression in cyno tissues of Example 13.

[0486]

[0232] In the temporal cortex, frontal cortex, motor cortex, caudate, putamen, hippocampus, and cerebellum, Groups 2-5 showed reduction in HTT protein out to at least Day 43 post dose. In the liver, Groups 2, 3, and 5 showed reduction in HTT protein out to at least Day 43 post dose. Most notably, the most significant HTT protein reduction is seen in the motor cortex, three doses of 3.0 mg / kg AC007867 showed -86% HTT protein inhibition (0.143). and three doses of 3.0 mg / kg AC007865 showed -85% HTT protein inhibition (0. 146).

[0487] Example 14. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice.

[0488]

[0233] AR RNAi agents were evaluated in vivo in mouse. On Day 1 and Day 2, five (n=5) female B-hTFRl mice were administered, via subcutaneous (SC) injection, either saline or AR RNAi agents (formulated in saline at 1.5 mg / kg animal body weight), at total dosing volume of 250 pL / 25g. Dosing was in accordance with Table 23 below.

[0489] Table 23. Dosing groups for the mice of Example 14.

[0490]

[0234] B-hTFRl mice, also known as C57BL / 6-Tfrctml(TFRC)Bcgen / Bcgen mice (Biocytogen), have the exons 4-19 of mouse Tfrl gene that encode the extracellular region replaced by human TFR1 exons 4-19.

[0491]

[0235] Each of the AR RNAi agents included modified nucleotides that were conjugated at the 5' terminal end of the sense strand to a targeting ligand that included an antigen binding moiety having the modified sequences as set forth in the duplex structures herein (see Tables 3, 4, 5, 6, 7, 8, 9 and 10 for specific modifications and structure infomiation related to the AR RNAi agents; including the antigen binding moieties).

[0492]

[0236] On Day 15, the mice were euthanized. From the mice, thoracic spinal cord and cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed via qPCR, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with saline. The mAR expression data is shown in the following Table 24. Table 24. Relative expression of mAR in mice thoracic spinal cord, cerebellum, striatum, and cortex, of Example 14

[0493]

[0237] In the thoracic spinal cord, cerebellum, cortex, and striatum, Groups 2-10 showed reduction in mAR out to at least Day 15. Most notably, two doses of 1.5 mg / kg AC006266 achieved -81 % inhibition (0.192) in the cerebellum. Example 15. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice.

[0494]

[0238] AR RNAi agents were evaluated in vivo in mouse. On Day 1 and Day 2, five (n=5) female B-hTFRl mice were administered, via subcutaneous (SC) injection, either saline or AR RNAi agents (formulated m saline at 1.5 mg / kg animal body weight), at total dosing volume of 250 pL / 25g. Dosing was in accordance with Table 25 below.

[0495]

[0239] Table 25. Dosing groups for the mice of Example 15.

[0496]

[0240] B-hTFRl mice, also known as C57BL / 6-Tfrctml(TTRC)Bcgon / Bcgen mice (Biocytogen), have the exons 4-19 of mouse Tfrl gene that encode the extracellular region replaced by human TFR1 exons 4-19.

[0497]

[0241] Each of the AR RNAi agents included modified nucleotides that were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included an antigen binding moiety having the modified sequences as set forth in the duplex structures herein (see Tables 3, 4, 5, 6, 7. 8, 9 and 10 for specific modifications and structure information related to the AR RNAi agents; including the antigen binding moieties).

[0498]

[0242] On Day 15. the mice were euthanized. From the mice, thoracic spinal cord and cerebellum, striatum, and cortex were harvested and collected for analysis. Additionally, muscle tissues were also collected for analysis. mAR mRNA transcript expression was analyzed via qPCR, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with saline. The mAR expression data is shown in the following Table 26.

[0499] Table 26. Relative expression of mAR in mice CNS and muscle tissues, of Example 15.

[0500]

[0243] In both the CNS and muscle tissues, Groups 2-7 showed reduction in mAR out to at least Day 15. Most notably, two doses of 1.5 mg / kg AC009278 achieved -80% inhibition (0.179) in the right cerebellum, and two doses of 1.5 mg / kg AC009279 achieved -74% inhibition (0.259) in the right gastrocnemius.

[0501] Example 16. In Vivo Knockdown of AR in Transgenic Tg B-hTfrl Mice.

[0502]

[0244] AR RNAi agents were evaluated in vivo in mouse. On Day 1 and Day 2, five (n=5) female B-hTFRl mice were administered, via subcutaneous (SC) injection, either saline or AR RNAi agents (formulated in saline at 1.5 mg / kg animal body weight), at total dosing volume of 250 pL / 25g. Dosing was in accordance with Table 27 below.

[0503] Table 27. Dosing groups for the mice of Example 16.

[0504]

[0245] B-hTFRl mice, also known as C57BL / 6-TfrctmllTFRC)Bcgen / Bcgen mice (Biocytogen), have the exons 4-19 of mouse Tfrl gene that encode the extracellular region replaced by human TFR1 exons 4-19.

[0505]

[0246] Each of the AR RNAi agents included modified nucleotides that were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included an anti gen binding moiety having the modified sequences as set forth in the duplex structures herein (see Tables 3, 4, 5, 6. 7, 8, 9 and 10 for specific modifications and structure information related to the AR RNAi agents; including the antigen binding moieties).

[0506]

[0247] On Day 15, the mice were euthanized. From the mice, thoracic spinal cord and cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed via qPCR, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with saline. The mAR expression data is shown in the following Table 28.

[0507]

[0248] Table 28. Relative expression of mAR in mice thoracic spinal cord, cerebellum, striatum, and cortex, of Example 16.

[0508]

[0249] In the thoracic spinal cord, cerebellum, cortex, and striatum, Groups 2-7 showed reduction in mAR out to at least Day 15. Most notably, two doses of 1.5 mg / kg AC006895 achieved ~76% inhibition (0.237) in the cerebellum.

[0509] OTHER EMBODIMENTS

[0510]

[0250] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CL IMSWhat is claimed is:

1. A compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein:Fab is an antibody or antibody fragment;R1is -ORla, -C(O)NHRla’ or -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyd, or substituted or unsubstituted aryl;Z is a bivalent or trivalent ar l group, or a bivalent or trivalent heteroaryl group;Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene: each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalky 1 chain;L1is absent or a linker comprising a PEG chain;L2is absent or a linker:R2comprises an oligonucleotide-based agent; m is 0 or 1 ; and n is 0 or 1 as valency permits.

2. The compound of claim 1 , wherein m is 0.

3. The compound of claim 1, wherein m is 1.

4. The compound of any one of claims 1-3, wherein n is 0.

5. The compound of any one of claims 1-3, wherein n is 1.

6. The compound of claim 1, wherein n is 0 and m is 1.

7. The compound of any one of claims 1, 3, 4, and 6 wherein the compound is of Formula (I-a):or a pharmaceutically acceptable salt thereof.

8. The compound of claim 1, wherein n is 1 and m is 0.

9. The compound of any one of claims 1. 2, 5, and 8 wherein the compound is of Formula (I-b):or a pharmaceutically acceptable salt thereof.

10. The compound of claim 9, wherein Y is substituted or unsubstituted heteroarylene.

11. The compound of claim 9 or 10, wherein Y is of the formula:

12. The compound of any one of claims 9-11, wherein Z is a bivalent aryl group.

13. The compound of any one of claims 9-12, wherein Z is a bivalent phenyl group.The compound of any one of claims 9-13, wherein Z is of the formula:

15. The compound of any one of claims 9-14, wherein R1is -C(O)NHR,a.

16. The compound of any one of claims 1-15, wherein Rlais substituted or unsubstituted C1-C6 alkyl.

17. The compound of any one of claims 1-16, wherein Rlais substituted or unsubstituted methyl.

18. The compound of any one of claims 1-17, wherein Rlais unsubstituted methyl.

19. A compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;L1is absent or a linker comprising a PEG chain;L2is absent or a linker;R2comprises an oligonucleotide-based agent; p is 0 or 1; andX is a leaving group.

20. A compound of Formula (III):or a pharmaceutically acceptable salt thereof, wherein:Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyd chain;L1is absent or a linker comprising a PEG chain;L2is absent or a linker;R3is a reactive moiety; p is 0 or 1; andX is a leaving group.

21. The compound of claim 19 or 20, wherein X is a sulfonate leaving group.

22. The compound of any one of claims 19-21, wherein X is -SChMe.

23. The compound of any one of claims 20-22, wherein R3is an activated ester.

24. The compound of any one of claims 20-23, wherein R3is of the formula:

25. The compound of any one of claims 20-22, wherein R3is an azide.

26. The compound of any one of claims 1-25, wherein Y1is a substituted or unsubstituted heteroarylene.

27. The compound of any one of claims 1-26, wherein Y1is a substituted or unsubstituted benzothiazole or a substituted or unsubstituted oxadiazole.

28. The compound of any one of claims 1-27. wherein Y1is of the formula:

29. The compound of any one of claims 1-28, wherein L1is a linker comprising a PEG chain.

30. The compound of any one of claims 1-29, wherein L1comprises 1-10 PEG units.

31. The compound of any one of claims 1-30, wherein L1is of the formula:

32. The compound of any one of claims 1-28, wherein L1is absent.

33. The compound of any one of claims 1-32, wherein L2is of the formula:

34. The compound of any one of claims 1-32, wherein L2is absent.

35. The compound of any one of claims 1-34, wherein Z1is a bivalent aryl group, or a bivalent aryl group.

36. The compound of any one of claims 1-35, wherein Z1is a bivalent phenyl group, or a trivalent phenyl group.

37. The compound of any one of claims 1-36, wherein Z1is of the formula:ne of claims 1-34, wherein Z1comprises a branched alkyl or heteroalkyl chain.

39. The compound of any one of claims 1-34, or 38 wherein Z1comprises a branched heteroalkyl chain.

40. The compound of any one of claims 1-34, or 38-39 wherein Z1is of the formula:

41. The compound of any one of claims 1-34, wherein Z1is absent.

42. A compound comprising an antibody or antibody fragment; and a structure selectedL2is absent or a linker,R2comprises an oligonucleotide-based agent, and represents an attachment point to the antibody or antibody fragment.or a pharmaceutically acceptable salt thereof, wherein: L2is absent or a linker,R2comprises an oligonucleotide-based agent, andX is a leaving group.

44. The compound of claim 43, wherein X is a sulfonate leaving group.

45. The compound of claim 43 or 44, wherein X is -SChMe.

46. The compound of any one of claims 42-45, wherein L2is of the formula:

47. The compound of any one of claims 42-45, wherein L2is absent.

48. The compound of any one of claims 1-19, or 26-47, wherein R2comprises an RNAi agent.

49. The compound of any one of claims 1-19, or 26-48, wherein R2comprises a sense strand containing one or two inverted abasic residues.

50. The compound of any one of claims 1-19, or 26-49, wherein R2comprises a sense strand containing two inverted abasic residues.

51. The compound of any one of claims 1-19, or 26-50, wherein R2comprises a sense strand containing one or two inverted abasic residues.

52. The compound of claim 50 or 51, wherein the Fab is conjugated to the 5’ terminal end of the sense strand.A represents a point of connection of the structure to an antibody or antibody fragment, and represents a point of connection of the structure to a moiety comprising an RNAi agent.or a salt thereof.

55. A method of synthesizing a compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein:Fab is an antibody or antibody fragment;R1is -0Rla, -C(O)NHRla’ or -NHC(O)Rla, wherein Rlais substituted or unsubstituted Ci-Ce alkyl, or substituted or unsubstituted aryl;Z is a bivalent or trivalent aryl group, or a bivalent or trivalent heteroaryl group:Y is substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; each instance of Y1is independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;Z1is absent, a bivalent or tnvalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;L1is absent or a linker comprising a PEG chain;L2is absent or a linker;R2comprises an oligonucleotide-based agent; m is 0 or 1; and n is 0 or 1 as valency permits; comprising, reacting a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Z1is absent, a bivalent or trivalent aryl group, a bivalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain;L1is absent or a linker comprising a PEG chain;L2is absent or a linker;R2comprises an oligonucleotide-based agent; p is 0 or 1; andX is a leaving group; with an antibody comprising a cysteine residue.

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