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

Abstract

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

Description

[0001] Cross-references to related applications This application claims priority to the following U.S. Provisional Patent Application Serial No. 63 / 624,417, filed January 24, 2024; U.S. Provisional Patent Application Serial No. 63 / 573,151, filed April 2, 2024; U.S. Provisional Patent Application Serial No. 63 / 662,243, filed June 20, 2024; U.S. Provisional Patent Application Serial No. 63 / 662,272, filed June 20, 2024; and U.S. Provisional Patent Application Serial No. 63 / 718,062, filed November 8, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to improved linkers for conjugating oligonucleotide-based reagents (e.g., RNAi reagents) to antibodies or antibody fragments (e.g., anti-transferrin receptor (TfR1) antibodies or fragments).

[0003] sequence list This application contains a sequence list (compliant with standard ST26), which has been submitted in XML format and is incorporated herein by reference in its entirety. The XML sequence list file is named 30745-WO_SeqListing.xml, was created on January 22, 2025, and has a size of 990 kb. Background Technology

[0004] In the medical field, delivering therapeutic payloads to specific tissues and cells of interest to a subject in vivo remains a significant challenge. This is particularly true for oligonucleotide-based therapeutics, such as antisense oligonucleotides (ASOs) and RNA interference (RNAi) agents (typically containing small (or short) interfering RNAs made of chemically modified nucleotides), which have shown great promise and potential to revolutionize medicine and provide effective therapeutic options for diseases that were previously untreatable; provided, of course, that the therapeutic oligonucleotides can reach the desired cells and tissues in vivo. Indeed, achieving suitable delivery of oligonucleotide-based therapeutics remains one of the most pressing challenges in discovering and identifying viable therapeutics.

[0005] Several other methods for delivering interfering RNA are being tested / developed for in vivo use. For example, siRNA can be delivered: “naked” in saline solution; conjugated with polycations, cationic lipids / lipid transfection reagents, or cationic peptides; as a component of defined molecular conjugates (e.g., cholesterol-modified siRNA, TAT-DRBD / siRNA complexes); as a component of liposomes; and as a component of nanoparticles. These methods have shown varying degrees of success. Another approach is to conjugate siRNA with antibodies or antibody fragments. However, new and improved methods and connectors are still needed for conjugating siRNA and other oligonucleotide-based reagents with antibodies and antibody fragments to provide targeted in vivo delivery of RNAi molecules, which could enhance the therapeutic potential of RNAi. Summary of the Invention

[0006] This article provides novel adapters for conjugating oligonucleotide-based reagents (e.g., RNAi reagents) to antibodies or antibody fragments (e.g., anti-transferrin receptor (TfR1) antibodies or fragments).

[0007] In some respects, compounds of formula (I) are provided herein: (I), Or its pharmaceutically acceptable salt, wherein: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and If the chemical valence allows, n can be 0 or 1.

[0008] On the other hand, this document provides compounds comprising antibodies or antibody fragments; and structures selected from the following: ,and or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0009] In some embodiments, the compounds provided herein have the following formula: L 2 For non-existent or connector, R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0010] In some embodiments, the compounds provided herein have the following formula: , where R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0011] In some embodiments, the compounds provided herein have the following formula: L 2 For non-existent or connector, R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0012] In some embodiments, the compounds provided herein have the following formula: , where R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0013] In some respects, this paper provides compounds of formula (II): (II), Or its pharmaceutically acceptable salt, wherein Z 1It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 For non-existent or connector; R 2 It contains oligonucleotide-based reagents; p is 0 or 1; and X is a leaving group.

[0014] On the other hand, this paper provides compounds of formula (III): (III), Or its pharmaceutically acceptable salt, wherein Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 For non-existent or connector; R 3 is the reactive part; p is 0 or 1; and X is the leaving group.

[0015] In some embodiments, compounds of the following formula are provided herein: , or its pharmaceutically acceptable salt, wherein L 2 For non-existent or connector, R 2 It contains oligonucleotide-based reagents, and X is a leaving group.

[0016] In some embodiments, compounds of the following formula are provided herein: (L-1026-p), or (L20-p), or its salt.

[0017] In some respects, this article also provides methods for synthesizing compounds of formula (I) or pharmaceutically acceptable salts thereof: (I), Where Fab represents an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a Z is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted arylene group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 For non-existent or connector; R 2 The reagent comprises an oligonucleotide; m is 0 or 1; and n is 0 or 1 where chemical valence permits; wherein the method comprises reacting a compound of formula (II) or a pharmaceutically acceptable salt thereof with an antibody containing a cysteine ​​residue: (II), Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 For non-existent or connector; R 2 It contains oligonucleotide-based reagents; p is 0 or 1; and X is a leaving group.

[0018] definition For convenience, certain terms used herein, in the specification, embodiments, and claims are collected herein. The following definitions and descriptions are intended and contemplated to be controlling in any future interpretation, unless explicitly and unambiguously modified in the embodiments described below, or when the application of their meaning renders any interpretation meaningless or substantially meaningless. Where the interpretation of a term renders it meaningless or substantially meaningless, the definition should be taken from Webster's Dictionary, Third Edition, or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (edited by Anthony Smith, Oxford University Press, Oxford, 2004).

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

[0020] As used herein and unless otherwise stated, the terms “an” and “a kind” are to be regarded as meaning “one / kind,” “at least one / kind,” or “one or more / a combination of.” Unless the context requires otherwise, singular terms used herein shall include the plural, and plural terms shall include the singular.

[0021] As used herein, the term "receptor" is intended to encompass the entire receptor or its ligand-binding portion. These portions of the receptor specifically include those regions sufficient for ligand-specific binding to occur, including those regions capable of being recognized by antibodies or antibody fragments.

[0022] Antibodies (used interchangeably in plural form) are immunoglobulin molecules that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, peptides, etc., by means of at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term “antibody,” such as an anti-TfR1 antibody, encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, biantibodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., VH-only antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site of desired specificity, including glycosylated variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies.

[0023] Antibodies, such as anti-TfR1 antibodies, include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to belong to any particular class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the antibody's constant domain of its heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0024] As used herein, the term “Fab” refers to the antigen-binding fragment (Fab) region that binds to the TfR1 antigen and includes six complementarity-determining regions (CDRs), three of which are on the variable light chain (VL) and three of which are on the variable heavy chain (VH).

[0025] The term "ligand" is defined as any molecule or atom that binds to a receptor molecule, such as a protein, nucleotide, or fragment thereof, binding domain, or complement.

[0026] As used herein, the term “protein” includes peptides, polypeptides, common molecules, fusion proteins, purified naturally occurring proteins, artificially synthesized proteins, recombinant proteins, antibodies, antibody fragments and their analogues, derivatives or combinations thereof.

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

[0028] As used herein, the term "antibody" may refer to an antigen-binding fragment region (Fab) or an antibody that includes the Fc portion of the antibody, depending on the context. Examples of TfR1 antibodies are described herein.

[0029] The term "conjugate" is defined as consisting of two or more molecules; or two or more entities coupled together. Preferably, the two molecules or entities are conjugated through nonspecific or specific protein-protein interactions, covalent bonding, non-covalent bonding, or coordination chemistry. In the context of this invention, the first molecule may be an interfering RNA molecule (i.e., an RNAi reagent), and the second molecule may be a ligand of a receptor on a target cell as defined herein. In some embodiments, the term conjugate includes a TfR1 Fab coupled to an oligonucleotide-based reagent (e.g., siRNA), which may also include a linker and / or peptide or protein for releasing the oligonucleotide-based reagent from endosomes as described herein.

[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.

[0031] Unless otherwise stated, the term "siRNA" as used herein refers to double-stranded interfering RNA.

[0032] As used herein, the term "therapeuticly effective amount" refers to the amount of interfering RNA or a pharmaceutical composition containing interfering RNA determined to produce a therapeutic response in mammals. Such therapeutically effective amounts can be readily determined by those skilled in the art and using the methods described herein.

[0033] As used in this article, the phrase “attenuated expression” when referring to a gene or mRNA means the administration or expression of a certain amount of interfering RNA (e.g., siRNA) to reduce the translation of the target mRNA into a protein by means of mRNA cleavage or by direct inhibition of translation.

[0034] As used herein, the terms “suppress,” “silence,” and “attenuate” refer to a measurable reduction in the expression of the target mRNA or corresponding protein compared to the expression of the target mRNA or corresponding protein in the absence of the interfering RNA of the present invention.

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

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

[0037] As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size properties of the protein in which the substitution occurs. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those found in references compiling such methods, for example, *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., eds., 2nd ed., ColdSpring Harbor Laboratory Press, ColdSpring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conservative substitution of amino acids includes substitutions in amino acids within the following group: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0038] The "percentage of identity" between two amino acid sequences was determined using the algorithm described by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, which was modified as described by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This type of algorithm was incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of interest. When there are gaps between two sequences, methods such as those described by Altschul et al. can be used. Nucleic Acids Res As described in 25(17):3389-3402, 1997, the use of Gapped BLAST is permitted. When using the BLAST and Gapped BLAST procedures, the default parameters of their respective procedures (e.g., XBLAST and NBLAST) can be used.

[0039] As used herein, the terms “oligonucleotide” and “polynucleotide” refer to polymers of linked nucleosides, each of which may be independently modified or unmodified.

[0040] As used herein, an “RNAi reagent” (also referred to as an “RNAi trigger”) means a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading or inhibiting (e.g., under appropriate conditions, degrading or inhibiting) the translation of the mRNA transcript of a target messenger RNA (mRNA). As used herein, an RNAi reagent may act via an RNA interference mechanism (i.e., by interacting with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC) or via one or more alternative mechanisms or pathways. Although it is believed that RNAi reagents (as used herein) act primarily through an RNA interference mechanism, the disclosed RNAi reagents are not bound to or limited to any particular pathway or mechanism of action. The RNAi reagents disclosed herein contain sense and antisense strands and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi reagents described herein is at least partially complementary to the targeted mRNA. RNAi reagents may include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0041] As used herein, the terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” when referring to the expression of a given gene mean that, when a cell, cell group, tissue, organ, or subject in which the gene is transcribed is treated with the RNAi reagent described herein, the expression of the gene is reduced, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from mRNA, compared to a second cell, cell group, tissue, organ, or subject that has not been treated in this way.

[0042] As used herein, the terms “sequence” and “nucleotide sequence” refer to a series or sequence of nucleobases or nucleotides, described by a series of letters in standard nomenclature.

[0043] As used herein, “base,” “nucleotide base,” or “nucleobase” refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide and includes the major purine bases adenine and guanine, and the major pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, hybrid bases, size-enlarged bases, and fluorinated bases. (See, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidides containing modified nucleobases) is known in the art.

[0044] As used herein, the term "nucleotide" has the same meaning as commonly understood in the art, and therefore refers to a glycoside comprising a sugar moiety, a base moiety, and a covalently linked group (linking group) such as a phosphate ester or thiophosphate internucleotide linking group, and encompasses naturally occurring nucleotides such as DNA or RNA, as well as non-naturally occurring nucleotides comprising modified sugar and / or base moieties, also referred to herein as nucleotide analogs or modified nucleotides. In this document, a single nucleotide may be referred to as a monomer or unit.

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

[0046] Those skilled in the art will be able to select the most suitable set of conditions for hybridization testing. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include native or modified nucleotides or nucleotide mimics, at least to the extent that the hybridization requirements described above are met. Sequence identity or complementarity is independent of modification. For example, to determine identity or complementarity, as defined herein, a and Af are complementary to U (or T) and identical to A.

[0047] Unless otherwise stated, the symbols used herein shall be as follows. This means that any one or more groups can be attached to it, which is consistent with the scope of the invention described herein.

[0048] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or sequence of their atomic bonding or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," and stereoisomers that are non-overlapping mirror images are called "enantiomers," or sometimes optical isomers. A carbon atom bonded to four different substituents is called a "chiral center."

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

[0050] As used in the claims herein, the phrase “consisting of…” excludes any element, step, or ingredient not specified in the claims. When used in the claims herein, the phrase “consisting substantially of…” limits the scope of the claims to the specified materials or steps and those materials or steps that do not substantially affect one or more essential and novel features of the claimed invention.

[0051] It will be readily understood and appreciated by those skilled in the art 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 on the environment in which the compound or composition exists. Therefore, as used herein, the structures disclosed contemplate that certain functional groups (e.g., OH, SH, or NH) can be protonated or deprotonated. As will be readily understood by those skilled in the art, the disclosure herein is intended to cover the disclosed compounds and compositions regardless of their protonated state based on the environment (e.g., pH). Accordingly, compounds described herein having unstable protons or basic atoms should also be understood to represent the salt form of the respective compounds.

[0052] The compounds described herein may be in the form of free acids, free bases, or salts. Pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of this invention.

[0053] As used herein, the terms “link” or “combination” refer to the connection between two compounds or molecules via a covalent bond. Unless otherwise stated, the terms “link” and “combination” as used herein may refer to a link between a first compound and a second compound, with or without any inserted atoms or groups.

[0054] As used herein, the term "including" is used to mean the phrase "including but not limited to" and is used interchangeably with it. Unless the context clearly indicates otherwise, the term "or" is used to mean the term "and / or" and is used interchangeably with it.

[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. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0056] When numerical values ​​are explicitly stated, it should be understood that values ​​of approximately the same quantity or amount as the stated values ​​are also within the scope of this disclosure. When a combination is disclosed, each sub-combination of the elements in that combination is also specifically disclosed and within the scope of this disclosure. Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. When any element of the disclosure is disclosed to have multiple alternatives, instances of the disclosure in which each alternative is excluded individually or in combination with other alternatives are also disclosed; more than one element in the disclosure may have such exclusions, and all combinations of elements having such exclusions are also disclosed.

[0057] Other objects, features, aspects and advantages of the invention will become apparent from the following detailed description, the accompanying drawings and the claims. Attached Figure Description

[0058] These and other features, aspects, and advantages of this disclosure can be better understood when the following detailed description is read in conjunction with the accompanying drawings.

[0059] Figure 1 The study showed that, in different brain regions, relative androgen receptor (AR) mRNA expression was knocked down after delivery of selected TfR1-targeted Fab conjugated to siRNA (with an antisense strand complementary to androgen receptor mRNA) compared to control siRNA.

[0060] Figure 2 The study showed that, in different brain regions, relative androgen receptor mRNA expression was knocked down after delivery of selected TfR1-targeting Fab conjugated to siRNA (with an antisense strand complementary to androgen receptor mRNA) compared to control siRNA.

[0061] Figure 3 The study showed that, in different brain regions, relative androgen receptor mRNA expression was knocked down after delivery of selected TfR1-targeting Fab conjugated to siRNA (with an antisense strand complementary to androgen receptor mRNA) compared to control siRNA.

[0062] Figure 4 Showing the standardization for Fab0002, in K D The binding affinity of the selected TfR1-targeting Fab was calculated.

[0063] Figure 5 The knockdown of MAPT protein in various tissues is shown in accordance with the description in Example 10. Detailed Implementation

[0064] This disclosure relates to improved linkers for conjugating oligonucleotide-based reagents (e.g., RNAi reagents) to antibodies or antibody fragments (e.g., anti-transferrin receptor (TfR1) antibodies or fragments).

[0065] In some respects, this paper provides compounds of formula (I): (I), Or its pharmaceutically acceptable salt, wherein: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and If the chemical valence allows, n can be 0 or 1.

[0066] In some implementations, m is 0. In some implementations, m is 1.

[0067] In some implementations, n is 0. In some implementations, n is 1.

[0068] In some implementations, n is 0 and m is 1. In some implementations, n is 1 and m is 0.

[0069] In some embodiments, the compound of formula (I) has formula (Ia): (Ia), Or its pharmaceutically acceptable salt, wherein: Fab is an antibody or antibody fragment; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 For non-existent or joint; and R 2 Reagents containing oligonucleotides.

[0070] In some embodiments, the compound of formula (I) has formula (Ib): (Ib), Or its pharmaceutically acceptable salt, wherein: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 For substituted or unsubstituted aryl groups, or substituted or unsubstituted heteroaryl groups; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 For non-existent or joint; and R 2 Reagents containing oligonucleotides.

[0071] In some embodiments, the compounds of formula (I) disclosed herein contain a substituent R. 1 In some implementations, R 1 For –OR 1a , where R 1aIt is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group. In some embodiments, R 1 –C(O)NHR 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group. In some embodiments, R 1 –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group. In some embodiments, R 1 –C(O)NHR 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 1a It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 1a The methyl group may be substituted or unsubstituted. In some embodiments, R 1a The methyl group is unsubstituted.

[0072] In some embodiments, the compounds of formula (I) disclosed herein contain a substituent Y. In some embodiments, Y is a substituted or unsubstituted aryl group. In some embodiments, Y is a substituted or unsubstituted heteroaryl group. In some embodiments, Y has the following formula: ,or .

[0073] In some embodiments, the compounds of formula (I) disclosed herein contain a substituent Z. In some embodiments, Z is a divalent aryl group. In some embodiments, Z is a divalent phenyl group. In some embodiments, Z has the following formula: In some embodiments, Z is a trivalent aryl group. In some embodiments, Z is a divalent heteroaryl group. In some embodiments, Z is a trivalent heteroaryl group.

[0074] This article further discloses compounds of formula (II): (II), Or its pharmaceutically acceptable salt, wherein: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; p is 0 or 1; and X is a leaving group.

[0075] This article also discloses compounds of formula (III): (III), Or its pharmaceutically acceptable salt, wherein: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 3 The reactive part; p is 0 or 1; and X is a leaving group.

[0076] In some embodiments, the compounds of formula (III) disclosed herein contain a substituent X. In some embodiments, X is a sulfonate leaving group. In some embodiments, X is –SO2Me.

[0077] In some embodiments, the compounds of formula (III) disclosed herein contain a substituent R. 3 In some implementations, R 3 To activate the ester. In some embodiments, R 3 It has the following formula: In some implementations, R 3 It is an azide.

[0078] In some embodiments, the compounds of formulas (I), (II), and (III) disclosed herein contain the substituent Y. 1 In some implementations, Y 1 At least one of them is a substituted or unsubstituted aryl group. In some embodiments, Y 1 At least one of them is a substituted or unsubstituted heteroaryl group. In some embodiments, Y 1 Both cases involve substituted or unsubstituted aryl groups. In some embodiments, Y... 1 Both cases involve substituted or unsubstituted heteroaryl groups. In some embodiments, Y 1 The two scenarios are the same. In some implementations, Y 1 The two scenarios are different. In some implementations, Y 1 One or both of these are substituted or unsubstituted benzothiazoles or substituted or unsubstituted oxadiazoles. In some embodiments, Y1 One or both of these cases have the following formula: In some implementations, Y 1 One or both of the following conditions have the following formula: .

[0079] In some embodiments, the compounds of formula (I), (II) and (III) disclosed herein contain the substituent L. 1 In some implementations, L 1 It does not exist. In some implementations, L 1 For connectors containing PEG chains. In some embodiments, L 1 It contains 1-10 PEG units. In some embodiments, L 1 It contains 3-6 PEG units. In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: In some implementations, L 1 It has the following formula: .

[0080] In some embodiments, the compounds of formula (I), (II) and (III) disclosed herein contain the substituent L. 2 In some implementations, L 2 It does not exist. In some implementations, L 2 For connectors. In some implementations, L 2 It has the following formula: In some implementations, L2 It has the following formula: .

[0081] In some embodiments, the compounds of formulas (I), (II), and (III) disclosed herein contain the substituent Z. 1 In some implementations, Z 1 It does not exist. In some implementations, Z 1 It is a divalent aryl group. In some embodiments, Z 1 It is a divalent phenyl group. In some embodiments, Z 1 It has the following formula: In some implementations, Z 1 It is a trivalent aryl group. In some embodiments, Z 1 It is a trivalent phenyl group. In some embodiments, Z 1 It has the following formula: In some implementations, Z 1 It is a divalent heteroaryl group. In some embodiments, Z 1 It is a trivalent heteroaryl group.

[0082] In some implementations, Z 1 It is a branched alkyl chain. In some embodiments, Z 1 It is a branched heteroalkyl chain. In some embodiments, Z 1 It has the following formula: In some implementations, Z 1 It has the following formula: In some implementations, Z 1 It has the following formula: .

[0083] In some embodiments, the compounds of formula (I) and formula (II) disclosed herein contain a substituent R. 2 In some implementations, R 2 It contains RNAi reagents. In some implementations, R 2 It comprises a sense chain containing a reverse debase residue. In some embodiments, R 2 It comprises a sense chain containing two reverse debasing residues. In some embodiments, R 2 The sense chain is attached to the Fab at the 5' end of the sense chain.

[0084] In some embodiments, this document provides compounds comprising an antibody or antibody fragment; and structures selected from the following: Or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0085] On the other hand, this article provides compounds of the following formula: Or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and X is a leaving group.

[0086] In some respects, this article provides compounds of the following formula: Or its salt.

[0087] This article further provides methods for synthesizing compounds of formula (I) or pharmaceutically acceptable salts thereof: (I), in: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and n can be 0 or 1, provided that the chemical valence allows it; The method comprises reacting a compound of formula (II) or a pharmaceutically acceptable salt thereof with an antibody containing a cysteine ​​residue: (II), in: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; p is 0 or 1; and X is a leaving group.

[0088] Modified nucleotides When used in various oligonucleotide constructs, the modified nucleotides can maintain the activity of the compound in cells, while improving the serum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans when the oligonucleotide constructs are administered.

[0089] In some embodiments, the RNAi reagent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2’-hydroxynucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides may include, but are not limited to, deoxyribonucleotides, nucleotide mimics, debased nucleotides, 2’-modified nucleotides, reverse nucleotides, nucleotides containing modified nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2’,3’-open-ring nucleotide mimics (unlocked nucleotide analogs), locked nucleotides, 3’-methoxy (2’-nucleotide-linked) nucleotides, 2’-F-arabinonucleotides, 5’-methyl,2’-fluoronucleotides, morpholinonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. 2’-modified nucleotides ( Right now Nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring include, but are not limited to, 2'-O-methyl nucleotides (also known as 2'-methoxynucleotides), 2'-fluoronucleotides (also known herein as 2'-deoxy-2'-fluoronucleotides), 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-(2-methoxyethyl)) nucleotides (also known as 2'-MOE), 2'-aminonucleotides, and 2'-alkylnucleotides. All positions in a given compound need not be uniformly modified. Instead, more than one modification may be incorporated into a single RNAi reagent or even into a single nucleotide. A modification at one nucleotide is independent of a modification at another nucleotide. The nucleobases of various modifications are well known and described in the art.

[0090] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine (hypoxanthine), xanthine, 2-aminoadenine, adenine, and guanine 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives, adenine and guanine 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives, 2-thiouracil, 2-Thiothymine, 2-Thiocytosine, 5-Haloururamine, Cytosine, 5-Protyynyluramine, 5-Protyynylcytosine, 6-Azouramine, 6-Azocytosine, 6-Azothymine, 5-uramine (pseudouramine), 4-Thiouramine, 8-Halogen, 8-Amino, 8-Mercapto, 8-Thioalkyl, 8-Hydroxy and other 8-substituted adenine and guanine, 5-Halogen (e.g., 5-bromo), 5-Trifluoromethyl and other 5-substituted uramine and cytosine, 7-Methylguanine and 7-Methyladenine, 8-azaguanine and 8-azaadenine, 7-Deadenine, 7-Deadenine, 3-Deadenine and 3-Deadenine.

[0091] In some embodiments, the 5' and / or 3' ends of the antisense strand may include a debase residue (Ab), which may also be referred to as a "debase site" or "debase nucleotide". A debase residue (Ab) is a nucleotide or nucleoside lacking a nucleobase at the 1' position of the sugar moiety. In some embodiments, the debase residue may be located inside the nucleotide sequence. In some embodiments, Ab or AbAb may be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand may include one or more additional debase residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab is added to the 3' end of the sense strand. In some embodiments, the debase (deoxyribose) residue may be replaced with a ribitol (debase ribose) residue.

[0092] In some embodiments, all or substantially all of the nucleotides in the RNAi reagent are modified nucleotides. An RNAi reagent, as used herein, in which substantially all of the present nucleotides are modified nucleotides, is an RNAi reagent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are ribonucleotides (i.e., unmodified) in both the sense and antisense strands. A sense strand, as used herein, in which substantially all of the present nucleotides are modified nucleotides, is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the sense strand. An antisense strand, as used herein, in which substantially all of the present nucleotides are modified nucleotides, is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the sense strand. In some embodiments, one or more nucleotides in the RNAi reagent are unmodified ribonucleotides. The chemical structures of some modified nucleotides are described in Table A herein.

[0093] Modified nucleoside interbonds In some embodiments, one or more nucleotides of the RNAi reagent are linked by a non-standard bond or backbone (i.e., a modified internucleotide bond or modified backbone). Modified internucleotide bonds or backbones include, but are not limited to, thiophosphate groups (represented herein as lowercase "s"), chiral thiophosphates, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, thiodiphosphates, alkylphosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, hypophosphonates, phosphoramidites (e.g., 3'-aminophosphatidyl, aminoalkylphosphatidyl, or thiophosphatidyl), thioalkyl-phosphonates, thioalkyl phosphate triesters, morpholino bonds, borophosphates having a normal 3'-5' bond, 2'-5' linked analogs of borophosphates, or borophosphates having reverse polarity, wherein adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified nucleoside interbonds or backbones lack a phosphorus atom. Modified nucleoside interbonds lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl sugar interbonds, mixed heteroatom and alkyl or cycloalkyl sugar interbonds, or one or more short-chain heteroatom or heterocyclic sugar interbonds. In some embodiments, the modified nucleoside interskeletons include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formyl and thioformyl skeletons, methyleneformyl and thioformyl skeletons, alkene-containing skeletons, aminosulfonate skeletons, methyleneimino and methylenehydrazine skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0094] In some implementations, the sense strand of the RNAi reagent may contain 1, 2, 3, 4, 5 or 6 phosphate thioester bonds, and the antisense strand of the RNAi reagent may contain 1, 2, 3, 4, 5 or 6 phosphate thioester bonds, or both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5 or 6 phosphate thioester bonds.

[0095] In some embodiments, the sense strand of the RNAi reagent contains at least two phosphate-thioester nucleoside bonds. In some embodiments, the phosphate-thioester nucleoside bonds are located between 1-3 nucleotides from the 3' end of the sense strand. In some embodiments, one phosphate-thioester nucleoside bond is located at the 5' end of the sense strand nucleotide sequence, while the other phosphate-thioester bond is located at the 3' end of the sense strand nucleotide sequence. In some embodiments, two phosphate-thioester nucleoside bonds are located at the 5' end of the sense strand, while the other phosphate-thioester bond is located at the 3' end of the sense strand. In some embodiments, the sense strand does not contain any phosphate-thioester nucleoside bonds between nucleotides, but contains one, two, or three phosphate-thioester bonds between the terminal nucleotides at both the 5' and 3' ends and the optionally present reverse debasement residue terminal cap.

[0096] Capped residues or parts In some embodiments, the sense chain may include one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capped residue.” As used herein, a “capped residue” is a nonnucleotide compound or other portion that may be incorporated into one or more ends of a nucleotide sequence of an RNAi reagent disclosed herein. In some cases, capping residues may provide certain beneficial properties to the RNAi reagent, such as protection against exonuclease degradation. In some embodiments, an inverse debasement residue (invAb) (also referred to in the art as an “inverse debasement site”) is added as a capping residue (see Table A). (See, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16; U.S. Patent No. 5,998,203). Capping residues are generally known in the art and include, for example, inverse debasement residues and carbon chains, such as terminal C3H7 (propyl), C6H… 13 (Hexyl) or C 12 H 25 (Dodecyl) group. In some embodiments, the capping residue is present at the 5' end, 3' end, or both of the 5' and 3' ends of the sense chain. In some embodiments, the 5' end and / or 3' end of the sense chain may include more than one reverse debased deoxyribose moiety as a capping residue.

[0097] In some embodiments, one or more inverse debase residues (invAbs) are added to the 3' end of the sense strand. In some embodiments, one or more inverse debase residues (invAbs) are added to the 5' end of the sense strand. In some embodiments, one or more inverse debase residues or inverse debase sites are inserted between the adapter and the nucleotide sequence of the sense strand of the RNAi reagent. In some embodiments, including one or more inverse debase residues or inverse debase sites at or near one or more ends of the sense strand of the RNAi reagent allows for enhanced RNAi reagent activity or other desired properties.

[0098] In some embodiments, one or more inverse debase residues (invAbs) are added to the 5' end of the sense strand. The inverse debase residues may be linked via phosphate esters, thiophosphate esters (e.g., shown herein as (invAbs)s), or other nucleoside bonds. In some embodiments, including one or more inverse debase residues at or near one or more ends of the sense strand of the RNAi reagent may allow for enhanced RNAi reagent activity or other desired properties. In some embodiments, the inverse debase (deoxyribose) residues may be replaced with inverse ribitol (debase ribose) residues. In some embodiments, the 3' end of the antisense strand core sequence or the 3' end of the antisense strand sequence may include inverse debase residues. The chemical structures of the inverse debase deoxyribose residues are shown in Table A below.

[0099] The following symbols are used in this article to indicate modified nucleotides, linkers, and linking groups: A = adenosine-3′-phosphate C = cytidine-3′-phosphate G = guanosine-3′-phosphate U = uridine-3′-phosphate I = Inosine-3′-phosphate a = 2′-O-methyladenosine-3′-phosphate as = 2′-O-methyladenosine-3′-thiophosphate c = 2′-O-methylcytidine-3′-phosphate cs = 2′-O-methylcytidine-3′-thiophosphate g = 2′-O-methylguanosine-3′-phosphate gs = 2′-O-methylguanosine-3′-thiophosphate i = 2′-O-methylinosine-3′-phosphate is = 2′-O-methylinosine-3′-thiophosphate t = 2′-O-methyl-5-methyluridine-3′-phosphate ts = 2′-O-methyl-5-methyluridine-3′-thiophosphate u = 2′-O-methyluridine-3′-phosphate us = 2′-O-methyluridine-3′-thiophosphate Af = 2′-fluoroadenosine-3′-phosphate Afs = 2′-fluoroadenosine-3′-thiophosphate Cf = 2′-fluorocytidine-3′-phosphate Cfs = 2′-Fluorocytidine-3′-Thiophosphate Gf = 2′-Fluoroguanosine-3′-phosphate Gfs = 2′-Fluoroguanosine-3′-Thiophosphate Tf = 2′-fluoro-5′-methyluridine-3′-phosphate Tfs = 2′-fluoro-5′-methyluridine-3′-thiophosphate Uf = 2′-fluorouridine-3′-phosphate Ufs = 2′-fluorouridine-3′-thiophosphate a_2N = See Table A a_2Ns = See Table A (invAb) = reverse debasing deoxyribonucleotide-5′-phosphate, see Table A (invAb)s = reverse debased deoxyribonucleotide-5′-thiophosphate, see Table A s = thiophosphate bond p = terminal phosphate ester (e.g., synthetic) (Alk-SS-C6) = See Table A (C6-SS-C6) = See Table A (C6-SS-Alk-Me) = See Table A (NH2-C6) = See Table A L20 = See Table A L-1026 = See Table A L-1274 = See Table A L-1288 = See Table A L-1166 = See Table A L-1275 = See Table A L-1100 = See Table A L-1045 = See Table A L-1176 = See Table A L-1064 = See Table A L-1063 = See Table A As will be readily understood by those skilled in the art, unless otherwise indicated by the sequence (e.g., by a thiophosphate bond "s"), when present in oligonucleotides, nucleotide monomers are linked to each other by 5'-3'-phosphodiester bonds. As will be clearly understood by those skilled in the art, thiophosphate bonds, as shown in the modified nucleotide sequences disclosed herein, replace the phosphodiester bonds typically present in oligonucleotides. Furthermore, it will be readily understood by those skilled in the art that the terminal nucleotides at the 3' end of a given oligonucleotide sequence typically have a hydroxyl group (-OH) at the respective 3' position of the given monomer, rather than an isolated phosphate moiety. Moreover, as will be readily understood and recognized by those skilled in the art, although the thiophosphate chemical structures shown herein generally indicate an anion on the sulfur atom, the invention disclosed herein covers all thiophosphate tautomers (e.g., where the sulfur atom has a double bond and the anion is on the oxygen atom). Unless otherwise expressly indicated herein, this understanding by those skilled in the art is adopted in the description of the RNAi reagents described herein.

[0100] Examples of certain linker groups used with the RNAi reagents disclosed herein are included in the chemical structures provided in Table A below. Each sense strand and / or antisense strand may have any of the linker groups listed herein, as well as other linker groups, conjugated to the 5′ and / or 3′ ends of the sequence.

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

[0102] Anti-TfR1 Fab and anti-TfR1 antibody This disclosure provides antibodies that bind to TfR1, such as human TfR1. In some embodiments, the anti-TfR1 antibodies disclosed herein are capable of binding to TfR1 expressed on cell surfaces, such as brain endothelial cells. Therefore, the antibodies disclosed herein can be used to facilitate the delivery of antibody-conjugated therapeutic or diagnostic agents to cells and tissues outside the liver. In some cases, the anti-TfR1 antibodies provided herein can also be used to detect the presence of TfR1 receptors in vitro or in vivo. As used herein, the term "anti-TfR1 antibody" refers to any antibody capable of binding to a TfR1 peptide (e.g., a TfR1 peptide expressed on a cell surface, such as brain endothelial cells), which may have a suitable source, such as human or non-human mammals (e.g., mice, rats, rabbits, primates such as monkeys, etc.).

[0103] Typical antibody molecules contain a heavy chain variable region (V). H ) and light chain variable region (V L This typically involves antigen binding. H and V L The region can be further subdivided into high-variability regions, also known as "complementary determinant regions" ("CDRs"), interspersed with more conservative regions called "frame regions" ("FRs"). Each V H and V L Typically, it consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methods known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, for example, Kabat, E.A., et al. (1991). Sequences of Proteins of Immunological Interest, Fifth Edition US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Allazikani et al. (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0104] The anti-TfR1 antibody described herein may be a full-length antibody containing two heavy chains and two light chains, each comprising a variable domain and a constant domain. Alternatively, the anti-TfR1 antibody may be an antigen-binding fragment of a full-length antibody. Examples of binding fragments covered within the term "antigen-binding fragment" of a full-length antibody include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of an antibody arm; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) a functionally separate complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using a synthetic linker via recombination, enabling the preparation of a single protein chain in which the VL and VH regions pair to form a monovalent molecule called a single-stranded Fv (scFv). See, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0105] In some embodiments, the anti-TfR1 antibody provided herein is a TfR1 antigen-binding fragment region (Fab) that specifically binds to TfR1. In some instances, the TfR1-specific Fab binds to TfR1 on brain endothelial cells. In some embodiments, the TfR1-specific Fab is capable of recognizing and specifically binding to one or more epitopes on TfR1.

[0106] The anti-TfR1 antibodies disclosed in this paper, such as TfR1-specific Fab, can competitively bind to TfR1 with transferrin (Tf), a physiological TfR1 ligand. After Fab binds to TfR1 on the cell surface, Fab and the attached interfering RNA are transferred into the cell via constitutive clathrin-mediated endocytosis.

[0107] The antibodies described herein may have a suitable origin, such as mice, rats, or humans. Such antibodies are not naturally occurring, meaning they are not produced in animals without human intervention (e.g., immunizing such animals with a desired antigen or a fragment thereof, or isolating them from an antibody library). Any antibody described herein, such as an anti-TfR1 antibody, may be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogeneous population of antibodies, and "polyclonal antibody" refers to a heterogeneous population of antibodies. Neither term limits the source of the antibody or the manner in which it is prepared.

[0108] In some implementations, the anti-TfR1 antibody is a human antibody, which can be isolated from a human antibody library or generated in transgenic mice. For example, a fully human antibody can be obtained using commercially available mice (which have been engineered to express a specific human immunoglobulin). Transgenic animals designed to produce a more desired (e.g., fully human antibody) or more robust immune response can also be used to generate humanized antibodies or human antibodies. Examples of such technologies are Xenomouse™ from Amgen, Inc. (Fremont, Calif.), and HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, the antibody can be recombinantly prepared using phage display or yeast technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12: 433-455. Alternatively, antibody library display techniques known in the art, such as phage, yeast, mammalian cell, or mRNA display techniques, can be used to produce human antibodies and antibody fragments in vitro from a gene bank of immunoglobulin variable (V) domains derived from non-immunized donors.

[0109] In other embodiments, the anti-TfR1 antibody may be a humanized antibody. A humanized antibody refers to the form of a non-human (e.g., mouse) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the receptor's CDR are replaced with residues of a CDR from a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and capability. In some cases, one or more Fv frame region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not found in the receptor antibody or in the input CDR or frame sequence, but are included to further improve and optimize antibody performance. In some cases, humanized antibodies may contain at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the CDR regions correspond to those regions of non-human immunoglobulins, and all or substantially all of the FR regions are those regions of the common sequence of human immunoglobulins. Humanized antibodies preferably also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically a constant region or domain of a human immunoglobulin. The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, said CDRs also referred to as one or more CDRs “derived” from one or more CDRs of the original antibody. Humanized antibodies may also involve affinity maturation. Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989).

[0110] In some embodiments, the anti-TfR1 antibody described herein binds specifically to the corresponding target antigen (here, TfR1) or its epitope. The term "specifically binds" to an antigen or epitope is a well-known term in the art. It is said to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with a higher affinity than the molecule reacts with alternative targets. It binds "specifically" to a target antigen or epitope if it binds with higher affinity, stronger affinity, easier binding, and / or for a longer duration than the antibody binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (TfR1) or an epitope therein is an antibody that binds to that target antigen with higher affinity, stronger affinity, easier binding, and / or for a longer duration than it binds to other antigens or other epitopes of the same antigen. It should also be understood under this definition that, for example, an antibody that specifically binds to a first target antigen may bind specifically or preferentially to a second target antigen, or may not bind specifically or preferentially. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some instances, antibodies that "specifically bind" to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen (i.e., baseline binding activity can only be detected in conventional methods).

[0111] In some implementations, the anti-TfR1 antibody, as described herein, has a suitable binding affinity for the target antigen (e.g., TfR1) or its antigenic epitope. As used herein, "binding affinity" refers to the apparent binding constant or K0. A K A It is the dissociation constant (K) D The inverse of ) . The anti-TfR1 antibodies described herein may have a binding affinity (K) for TfR1 of at least 2 nM, 1 nM, 0.5 nM, 0.1 nM or lower. D Increased binding affinity corresponds to decreased K. D The higher affinity of an antibody for the first antigen compared to the second antigen can be achieved by binding to the K+ receptor of the second antigen. A (or numerical value K) D Compared to higher K binding to the first antigen A (or a smaller value for K) DThis is indicated by [the specific antigen]. In such cases, the antibody is specific to the first antigen (e.g., the first protein in the first conformation or its mimicry) relative to the second antigen (e.g., the same first protein in the second conformation or its mimicry; or the second protein). The difference in binding affinity (e.g., specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 90, 100, 500, 1000, 10,000, or 10 [units of measurement missing]. 5 In some implementations, any anti-TfR1 antibody can be further subjected to affinity maturation to increase the antibody's binding affinity to the target antigen or its epitope.

[0112] Binding affinity (or binding specificity) can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopic methods (e.g., using fluorescence assays). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of the bound protein relative to the target protein concentration. The concentration of the bound protein ([bound]) is generally correlated with the concentration of the free target protein ([free]) by the following equation: [bound] = [free] / (Kd + [free]).

[0113] However, it is not always necessary to make K. A The precise determination is necessary because sometimes a quantitative measurement of affinity is obtained (e.g., determination is made using methods such as ELISA or FACS analysis, with K...). A It is proportional and therefore can be used for comparison, such as determining whether the affinity is higher (e.g., 2 times higher), obtaining a qualitative measurement of affinity, or obtaining an inferred value of affinity (e.g., by functional assays such as activity in vitro or in vivo assays) is sufficient.

[0114] Example anti-TfR1 antibody In some implementations, the anti-TfR1 antibodies provided herein are humanized antibodies derived from mouse monoclonal antibody clones. Briefly, the heavy and light chain complementarity-determining domains of a mouse parent are transplanted into the shared human VH and VL frames to generate a parental humanized antibody. A humanized library is generated using Kunkel mutagenesis to introduce variants at certain frame region locations, and CDR-FR linker sites are constructed and clones exhibiting high binding affinity for human TfR1 are screened. The selected mab1G clone showed the highest binding affinity and was therefore selected for further affinity maturation. See the examples below. Exemplary anti-TfR1 antibodies derived from affinity maturation, containing variants in certain CDRs relative to the mab1G parent, exhibit further enhanced binding affinity.

[0115] Also within the scope of this disclosure are functional variants of any exemplary anti-TfR1 antibodies as disclosed herein. Such functional variants are substantially similar to the exemplary antibodies in both structure and function. The functional variants contain a V that is substantially the same as that of the exemplary antibodies. H and V L CDR. For example, it can be a variation of up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residues contained only in the total CDR region of the antibody, and with substantially similar affinity (e.g., having K at the same order of magnitude). D The value) binds to the same epitope of TfR1. In some cases, the functional variant may have the same heavy chain CDR3 as the exemplary antibody, and optionally the same light chain CDR3 as the exemplary antibody. Alternatively or additionally, the functional variant may have the same heavy chain CDR2 as the exemplary antibody. (The last sentence appears to be incomplete and possibly refers to a different context.) H In contrast, such anti-TfR1 antibodies can contain V only with CDR amino acid residue variations in the heavy chain CDR1. H Fragment. In some instances, anti-TfR1 antibodies may further comprise V having the same V as the exemplary antibody. L CDR3, and optionally the same V L V of CDR1 or VL CDR2 L Excerpt.

[0116] Alternatively or additionally, amino acid residue variations can be conserved amino acid residue substitutions.

[0117] In some implementations, anti-TfR1 antibodies may contain such heavy chain CDRs as the V of the exemplary antibodies described herein. HCompared to the CDR, the heavy chain CDR individually or collectively has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. Alternatively or additionally, anti-TfR1 antibodies may comprise such light chain CDRs as the V of the exemplary antibodies described herein. L Compared to the CDR, the light chain CDR individually or collectively has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. As used herein, “individually” means that, relative to the corresponding CDR of the exemplary antibody, one CDR of the antibody shares the indicated sequence identity. “Collectively” means that, with respect to the three V of the combined antibody… H or V L CDR and the corresponding three V of the exemplary antibody in combination H or V L CDRs share the sequence identity indicated.

[0118] In some embodiments, the heavy chain of any anti-TfR1 antibody as described herein may further include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). The heavy chain constant region may have any suitable origin, such as human, mouse, rat, or rabbit. Alternatively or additionally, the light chain of the anti-TfR1 antibody may further include a light chain constant region (CL), which may be any CL known in the art. In some instances, the CL is a κ light chain. In other instances, the CL is a λ light chain. Antibody heavy chains and light chain constant regions are well known in the art, such as those available in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0119] In some cases, anti-TfR1 antibodies are in Fab form, comprising a heavy chain containing any VH region and CH1 fragment disclosed herein, and a light chain containing any VL region and light chain constant region (LC) disclosed herein. The CH1 fragment can belong to any immunoglobulin (Ig) heavy chain constant region (e.g., human Ig heavy chain constant region). In some cases, CH1 can belong to an IgG (e.g., IgG1, IgG2, or IgG4) heavy chain. In some cases, the LC can be of human origin. It can be a κ chain. Alternatively, it can be a λ chain.

[0120] In some embodiments, the anti-TfR1 antibody disclosed herein may be a single-chain antibody (scFv). The scFv antibody may comprise a VH fragment and a VL fragment, which may be linked via a flexible peptide linker. In some cases, the scFv antibody may be VH→VL oriented (from the N-terminus to the C-terminus). In other cases, the scFv antibody may be VL→VH oriented (from the N-terminus to the C-terminus).

[0121] Fab libraries with mutant CDRs generated by Kunkel mutagenesis were sorted for TfR1 affinity as described in Example 2.

[0122] Preparation of anti-TfR1 antibody As described herein, antibodies capable of binding to TfR1 can be prepared by any method known in the art, such as via recombinant techniques. See, for example, Harlow and Lane (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0123] When needed, antibodies of interest (monoclonal or polyclonal) (e.g., produced by hybridoma cell lines or isolated from antibody libraries) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or amplification. The sequence encoding the antibody of interest can be maintained in a vector within host cells, and the host cells can then be amplified and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to, for example, humanize the antibody or improve affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is of a non-human origin and intended for use in clinical trials and treatments in humans, the constant region can be modified to more closely resemble the human constant region to avoid an immune response. Alternatively or additionally, it may be desirable to genetically manipulate the antibody sequence to obtain higher affinity and / or specificity for the target antigen, as well as greater efficacy in enhancing TfR1 activity. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to the antibody while still maintaining its binding specificity to the target antigen.

[0124] Alternatively, antibodies capable of binding to the target antigen (TfR1 molecule) as described herein can be isolated from a suitable antibody library via routine practice. The antibody library can be used via a routine screening process to identify proteins that bind to the target antigen (e.g., human TfR1, such as cell surface TfR1). During selection, peptide components are probed with the target antigen or fragments thereof, and if a peptide component binds to the target, it is typically identified by retention on a support. Retained display library members are recovered from the support and analyzed. Analysis may include amplification and subsequent selection under similar or different conditions. For example, positive and negative selection may alternate. Analysis may also include determining the amino acid sequence of the peptide component and purifying the peptide component for detailed characterization.

[0125] There are a variety of conventional methods known in the art for identifying and isolating antibodies that can bind to the target antigens described herein, including phage display, yeast display, ribosome display, or mammalian display techniques.

[0126] Genetically modified antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced via, for example, conventional recombinant technologies. In one instance, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a monoclonal antibody). Once isolated, the DNA can be placed in one or more expression vectors, which are then transfected into host cells to obtain monoclonal antibody synthesis in recombinant host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or otherwise non-immunoglobulin-producing myeloma cells. See, for example, PCT Publication WO 87 / 04462. DNA can then be modified, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking immunoglobulin coding sequences to all or part of the coding sequences of non-immunoglobulin polypeptides. In this way, genetically modified antibodies with binding specificity to target antigens, such as “chimeric” or “hybrid” antibodies, can be prepared.

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

[0128] The CDR region within the selected human receptor gene can be replaced by a CDR region from a parental nonhuman antibody or a functional variant thereof. If necessary, residues predicted to be important in interacting with the CDR region within the framework region of the parental strand (see description above) can be used to replace the corresponding residues in the human receptor gene.

[0129] Single-chain antibodies can be prepared via recombinant technology by linking nucleotide sequences encoding the heavy chain variable region with nucleotide sequences encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the techniques described above for single-chain antibody production (US Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce scFv libraries that display phages, yeast, mammalian cells, or mRNA, and TfR1-specific scFv clones can be identified from the libraries following standard procedures. Positive clones can undergo further screening to identify those that enhance CD19 activity.

[0130] Antibodies obtained following the methods known in the art and described herein can be characterized using methods well-known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” Many methods known in the art exist for mapping and characterizing epitope localization on proteins, including resolving the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In another instance, epitope mapping can be used to determine the sequence to which the antibody binds. Epitopes can be linear epitopes, i.e., contained in a single amino acid segment, or conformational epitopes formed by the three-dimensional interactions of amino acids, which are not necessarily contained in a single segment (a linear sequence of primary structure). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for antibody binding assays. In another example, epitopes to which antibodies bind can be identified in systematic screening by using overlapping peptides derived from the target antigen sequence and determining antibody binding. Based on gene fragment expression assays, open reading frames encoding the target antigen are randomly or through specific genetic constructs, and the reactivity of the expressed antigen fragment with the antibody to be tested is determined. For example, gene fragments can be produced by PCR, then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Some epitopes can also be identified using large libraries (phage libraries) of random peptide sequences displayed on the surface of phage particles.

[0131] Alternatively, a defined library of overlapping peptide fragments can be used to test antibody binding in a simple binding assay. In another instance, mutagenesis of the antigen-binding domain, domain exchange assays, and alanine scan mutagenesis can be performed to identify sufficient and / or necessary residues required for epitope binding. For example, domain exchange assays can be performed using mutants of the target antigen, where various fragments of TfR1 have been replaced (exchanged) with sequences from closely related but antigenically different proteins (e.g., another member of the tumor necrosis factor receptor family). By evaluating antibody binding to mutant TfR1, the importance of specific antigen fragments for antibody binding can be assessed.

[0132] Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[0133] In some instances, anti-TfR1 antibodies (e.g., in Fab form) are prepared using recombinant techniques illustrated below.

[0134] Nucleic acids encoding the heavy and light chains of the anti-TfR1 antibody as described herein can be cloned into an expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one example, the nucleotide sequences encoding the heavy and light chains are operatively linked to different promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operatively linked to a single promoter, such that both the heavy and light chains are expressed by the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

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

[0136] Generally, nucleic acid sequences encoding one or all strands of an antibody can be cloned into a suitable expression vector and operatively ligated to a suitable promoter using methods known in the art. For example, under suitable conditions, the nucleotide sequence and the vector can be contacted with a restriction endonuclease to generate complementary ends on each molecule, which can then pair with each other and be ligated together with a ligase. Alternatively, synthetic nucleic acid adapters can be ligated to the ends of genes. These synthetic adapters contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used for antibody production.

[0137] Various promoters can be used to express the antibodies described herein, including but not limited to the cytomegalovirus (CMV) immediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter, and herpes simplex virus TK promoter.

[0138] Adjustable promoters can also be used. Such adjustable promoters include those that use lac repressors from E. coli as transcription regulators to regulate transcription from mammalian cell promoters carrying lac operons [Brown, M. et al., Cell, 49: 603-612 (1987)], and those that use tetracycline repressors (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)]. Other systems include the FK506 dimer using estradiol, RU486, dihydroxymilosterone, or rapamycin, VP16, or p65. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0139] A tunable promoter comprising a repressor and an operon can be used. In one embodiment, a lac repressor from *E. coli* can act as a transcription regulator to regulate transcription from mammalian cell promoters carrying the lac operon [M. Brown et al., Cell, 49: 603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992)]. A tetracycline repressor (tetR) is combined with a transcription activator (VP 16) to produce a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), which is then combined with a minimal promoter carrying tetO derived from the major immediate early promoter of human cytomegalovirus (hCMV) to produce a tetR-tet operon system controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor alone (tetR), rather than a tetR-mammalian cell transcription factor fusion derivative, can act as a potent trans-regulator to control gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16): 1392-1399 (2003)). A particular advantage of this tetracycline-induced switch is that it does not require the use of a tetracycline repressor-mammalian cell trans-activator or a repressor fusion protein, which can be toxic to cells in some cases (Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)) to achieve its regulated effect.

[0140] Additionally, the vector may contain some or all of the following: selectable marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyomavirus origin of replication and ColE1 for appropriate episome replication; internal ribosome binding sites (IRESes), multifunctional multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing transgenic vectors are well known and available in the art.

[0141] Examples of polyadenylation signals that can be used to practice the methods described herein include, but are not limited to, human type I collagen polyadenylation signals, human type II collagen polyadenylation signals, and SV40 polyadenylation signals.

[0142] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any antibody can be introduced into suitable host cells for antibody production. The host cells can be cultured under suitable conditions for expressing the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered from the cultured cells (e.g., cells or culture supernatant) via conventional methods (e.g., affinity purification). If necessary, the antibody polypeptide chain can be incubated under suitable conditions for a suitable period of time to allow antibody production.

[0143] In some embodiments, the methods described herein for preparing antibodies involve a recombinant expression vector encoding both the heavy and light chains of the anti-TfR1 antibody as described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains that form the antibody, the polypeptide chains of which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow antibody formation.

[0144] In one example, two recombinant expression vectors are provided: one encoding a heavy chain of an anti-TfR1 antibody, and the other encoding a light chain of an anti-TfR1 antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each expression vector can be introduced into a suitable host cell individually. Positive transformants can be selected and cultured under suitable conditions that allow for antibody polypeptide chain expression. When both expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow for antibody formation. When the two expression vectors are introduced into different host cells, each expression vector can be recovered from the respective host cell or culture medium. The two polypeptide chains can then be incubated under suitable conditions for antibody formation.

[0145] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. For example, some antibodies can be separated by affinity chromatography using a protein A or protein G-conjugated matrix.

[0146] Any nucleic acid encoding the heavy chain, light chain, or both of the anti-TfR1 antibody as described herein, a vector containing such nucleic acid (e.g., an expression vector), and a host cell containing the vector are all within the scope of this disclosure. Methods for producing anti-TfR1 antibodies via expressing such nucleic acids in host cells and harvesting the resulting antibodies from the host cells and / or culture supernatant are also within the scope of this disclosure.

[0147] Linking groups and delivery media In some embodiments, RNAi reagents such as those disclosed herein contain or are conjugated to one or more nonnucleotide groups (including, but not limited to, Fab, linker groups, delivery polymers, or delivery media). Fab can enhance the targeting, delivery, or attachment of the RNAi reagent. The nonnucleotide group can be covalently linked to the 3′ and / or 5′ ends of the sense and / or antisense strands. In some embodiments, the RNAi reagent contains a nonnucleotide group linked to the 3′ and / or 5′ end of the sense strand. In some embodiments, the nonnucleotide group is linked to the 5′ end of the sense strand of the RNAi reagent. The nonnucleotide group can be directly or indirectly linked to the RNAi reagent via a linker / connector group. In some embodiments, the nonnucleotide group is linked to the RNAi reagent via an unstable, cleavable, or reversible bond or linker.

[0148] In some embodiments, non-nucleotide groups (e.g., Fab) enhance the pharmacokinetic or biodistribution properties of the RNAi reagent or conjugate to which it is attached, thereby improving the cell-specific or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, non-nucleotide groups enhance the endocytosis of the RNAi reagent.

[0149] RNAi reagents described herein can be synthesized with reactive groups (e.g., amino groups (also referred to herein as amines) at the 5′ and / or 3′ ends.

[0150] For example, in some embodiments, the RNAi reagent disclosed herein is synthesized having an NH2-C6 group (represented as (NH2-C6) in the modified sequence herein) at the 5′ end of the sense strand of the RNAi reagent. The terminal amino group may then react to form a conjugate with, for example, a group containing Fab. In some embodiments, the RNAi reagent disclosed herein is synthesized having one or more alkynyl groups at the 5′ end of the sense strand of the RNAi reagent. The terminal alkynyl group may then react to form a conjugate with, for example, a group containing Fab.

[0151] In some embodiments, the linker group is conjugated to the RNAi reagent. The linker group facilitates the covalent linking of the oligonucleotide-based reagent to a Fab, pharmacokinetic modifier, delivery polymer, or delivery medium. The linker group may be attached to the 3′ and / or 5′ end of the sense or antisense strand of the RNAi reagent. In some embodiments, the linker group is attached to the sense strand of the RNAi reagent. In some embodiments, the linker group is conjugated to the 5′ or 3′ end of the sense strand of the RNAi reagent. In some embodiments, the linker group is conjugated to the 5′ end of the sense strand of the RNAi reagent. Examples of linker 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, debased residues / nucleotides, amino acids, triyne functionalized groups, ribitols, and / or PEG groups.

[0152] A linker or connecting group is a connection between two atoms that links a chemical group (e.g., an RNAi reagent) or fragment of interest to another chemical group (e.g., Fab) or fragment of interest via one or more covalent bonds. Unstable links contain unstable bonds. Links may optionally include spacer groups that increase the distance between the two linked atoms. Spacer groups may further increase the flexibility and / or length of the link. Spacer groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, areneyl, and arynyl; each of these may contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the foregoing list is not intended to limit the scope of this specification.

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

[0154] Table A shows the structures of the various modified nucleotides, linkers, and linking groups used throughout this disclosure. Alternatively, other linking groups known in the art can be used. In many cases, the linking group is commercially available or incorporated into a commercially available nucleotide phosphorylamide.

[0155] In some embodiments, the delivery medium can be used to deliver RNAi reagents to cells or tissues. The delivery medium is a compound that improves the delivery of RNAi reagents to cells or tissues. The delivery medium may include, but is not limited to, substances composed of, but not limited to, polymers such as amphiphilic polymers, membrane-active polymers, peptides, melittin peptides, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines.

[0156] In some embodiments, the RNAi reagents described herein can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art for nucleic acid delivery. RNAi reagents can also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol groups and cholesterol derivatives), encapsulated in nanoparticles, liposomes, micelles, conjugated to polymers, or DPCs (see, for example, WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169 and WO2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each incorporated herein by reference), either by iontophoresis or by incorporation into other delivery media or systems available in the art, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or protein carriers.

[0157] The following provides alternative implementations of this disclosure: Implementation Scheme 1. A compound of formula (I): (I), Or its pharmaceutically acceptable salt, wherein: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and If the chemical valence allows, n can be 0 or 1.

[0158] Implementation Scheme 2. The compound of Implementation Scheme 1, wherein m is 0.

[0159] Implementation Scheme 3. The compound of Implementation Scheme 1, wherein m is 1.

[0160] Implementation Scheme 4. A compound from any one of Implementation Schemes 1-3, where n is 0.

[0161] Implementation Scheme 5. A compound from any one of Implementation Schemes 1-3, where n is 1.

[0162] Implementation Scheme 6. The compound of Implementation Scheme 1, wherein n is 0 and m is 1.

[0163] Implementation Scheme 7. A compound of any one of Implementation Schemes 1, 3, 4 and 6, wherein said compound has formula (Ia): (Ia), Or its pharmaceutically acceptable salt.

[0164] Implementation Scheme 8. The compound of Implementation Scheme 1, wherein n is 1 and m is 0.

[0165] Implementation Scheme 9. A compound of any one of Implementation Schemes 1, 2, 5 and 8, wherein said compound has the formula (Ib): (Ib), Or its pharmaceutically acceptable salt.

[0166] Implementation Scheme 10. The compound of Implementation Scheme 9, wherein Y is a substituted or unsubstituted heteroaryl group.

[0167] Implementation Scheme 11. The compound of Implementation Scheme 9 or 10, wherein Y has the following formula: ,or .

[0168] Implementation Scheme 12. A compound of any one of Implementation Schemes 9-11, wherein Z is a divalent aryl group.

[0169] Implementation Scheme 13. A compound of any one of Implementation Schemes 9-12, wherein Z is a divalent phenyl group.

[0170] Implementation Scheme 14. A compound of any one of Implementation Schemes 9-13, wherein Z has the following formula: .

[0171] Implementation Scheme 15. A compound of any one of Implementation Schemes 9-14, wherein R 1 –C(O)NHR 1a .

[0172] Implementation Scheme 16. A compound of any one of Implementation Schemes 1-15, wherein R 1a It is a substituted or unsubstituted C1-C6 alkyl group.

[0173] Implementation Scheme 17. A compound of any one of Implementation Schemes 1-16, wherein R 1a The methyl group can be substituted or unsubstituted.

[0174] Implementation Scheme 18. A compound of any one of Implementation Schemes 1-17, wherein R 1a The methyl group is unsubstituted.

[0175] Implementation Scheme 19. A compound of formula (II): (II), Or its pharmaceutically acceptable salt, wherein: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; p is 0 or 1; and X is a leaving group.

[0176] Implementation Scheme 20. A compound of formula (III): (III), Or its pharmaceutically acceptable salt, wherein: Z 1It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 3 The reactive part; p is 0 or 1; and X is a leaving group.

[0177] Implementation Scheme 21. The compound of Implementation Scheme 19 or 20, wherein X is a sulfonate leaving group.

[0178] Implementation Scheme 22. A compound of any one of Implementation Schemes 19-21, wherein X is –SO2Me.

[0179] Implementation Scheme 23. A compound of any one of Implementation Schemes 20-22, wherein R 3 It is an activated ester.

[0180] Implementation Scheme 24. A compound of any one of Implementation Schemes 20-23, wherein R 3 It has the following formula: .

[0181] Implementation Scheme 25. A compound of any one of Implementation Schemes 20-22, wherein R 3 It is an azide.

[0182] Implementation Scheme 26. A compound of any one of Implementation Schemes 1-25, wherein Y 1 For substituted or unsubstituted heteroaryl groups.

[0183] Implementation Scheme 27. A compound of any one of Implementation Schemes 1-26, wherein Y 1 It is either a substituted or unsubstituted benzothiazole or a substituted or unsubstituted oxadiazole.

[0184] Implementation Scheme 28. A compound of any one of Implementation Schemes 1-27, wherein Y 1 It has the following formula: ,or .

[0185] Implementation Scheme 29. A compound of any one of Implementation Schemes 1-28, wherein L 1 For connectors containing PEG chains.

[0186] Implementation Scheme 30. A compound of any one of Implementation Schemes 1-29, wherein L 1 It contains 1-10 PEG units.

[0187] Implementation Scheme 31. A compound of any one of Implementation Schemes 1-30, wherein L 1 It has the following formula: .

[0188] Implementation Scheme 32. A compound of any one of Implementation Schemes 1-28, wherein L 1 It does not exist.

[0189] Implementation Scheme 33. A compound of any one of Implementation Schemes 1-32, wherein L 2 It has the following formula: ,or .

[0190] Implementation Scheme 34. A compound of any one of Implementation Schemes 1-32, wherein L 2 It does not exist.

[0191] Implementation Scheme 35. A compound of any one of Implementation Schemes 1-34, wherein Z 1 It is a divalent aryl group or a trivalent aryl group.

[0192] Implementation Scheme 36. A compound of any one of Implementation Schemes 1-35, wherein Z 1 It is a divalent phenyl group or a trivalent phenyl group.

[0193] Implementation Scheme 37. A compound of any one of Implementation Schemes 1-36, wherein Z 1 It has the following formula: ,or .

[0194] Implementation Scheme 38. A compound of any one of Implementation Schemes 1-34, wherein Z 1 An alkyl or heteroalkyl chain containing branches.

[0195] Implementation Scheme 39. A compound of any one of Implementation Schemes 1-34 or 38, wherein Z 1 A heteroalkyl chain containing branches.

[0196] Implementation Scheme 40. A compound of any one of Implementation Schemes 1-34 or 38-39, wherein Z 1 It has the following formula: .

[0197] Implementation Scheme 41. A compound of any one of Implementation Schemes 1-34, wherein Z 1 It does not exist.

[0198] Implementation Scheme 42. A compound comprising an antibody or an antibody fragment; and a structure selected from the following: Or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

[0199] Implementation Scheme 43. A compound of the following formula: Or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and X is a leaving group.

[0200] Implementation Scheme 44. The compound of Implementation Scheme 43, wherein X is a sulfonate leaving group.

[0201] Implementation scheme 45. The compound of implementation scheme 43 or 44, wherein X is –SO2Me.

[0202] Implementation Scheme 46. A compound of any one of Implementation Schemes 42-45, wherein L 2 It has the following formula: ,or .

[0203] Implementation Scheme 47. A compound of any one of Implementation Schemes 42-45, wherein L 2 It does not exist.

[0204] Implementation Scheme 48. A compound of any one of Implementation Schemes 1-19 or 26-47, wherein R 2 Includes RNAi reagents.

[0205] Implementation Scheme 49. A compound of any one of Implementation Schemes 1-19 or 26-48, wherein R 2 It contains a sense chain with one or two reverse debasing residues.

[0206] Implementation Scheme 50. A compound of any one of Implementation Schemes 1-19 or 26-49, wherein R 2 It contains a sense chain with two reverse debasing residues.

[0207] Implementation Scheme 51. A compound of any one of Implementation Schemes 1-19 or 26-50, wherein R 2 It contains a sense chain with one or two reverse debasing residues.

[0208] Implementation scheme 52. The compound of implementation scheme 50 or 51, wherein Fab is connate with the 5' end of the sense chain.

[0209] Implementation Scheme 53. A compound comprising the structure of the following formula: Or its pharmaceutically acceptable salt, wherein: A This indicates the connection point between the structure and the antibody or antibody fragment, and R This indicates the connection point between the structure and the portion containing the RNAi reagent.

[0210] Implementation Scheme 54. A compound of the following formula: Or its salt.

[0211] Implementation Scheme 55. A method for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof: (I), in: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and n can be 0 or 1, provided that the chemical valence allows it; The method comprises reacting a compound of formula (II) or a pharmaceutically acceptable salt thereof with an antibody containing a cysteine ​​residue: (II), in: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; p is 0 or 1; and X is a leaving group.

[0212] The above-provided implementation schemes and projects will now be illustrated by the following non-limiting examples. Example

[0213] Example 1. Anti-transferrin antibody fragment (Fab) Antibodies and antibody fragments used in combination with the linkers described herein can be produced by methods known in the art. Some examples of antitransferrin Fab are described below.

[0214] CDR3 is the same sequence in 15 Fab clones VL (QHFWGTPLT; SEQ ID NO: 13) and VH (GTRAYHY; SEQ ID NO: 24).

[0215] Table 1. Complementarity Determining Regions (CDRs) of Light Chains Table 2. Complementarity Determining Regions (CDRs) of Heavy Chains Table 3 shows exemplary light chain framework regions used in the exemplary anti-TfR1 antibody provided herein. Exemplary CL sequences for use in the Fab light chain are also provided in Table 3.

[0216] Table 3. Exemplary Light Chain Framework Regions and Constant Regions Table 4 shows exemplary heavy chain framework regions used in the exemplary anti-TfR1 antibodies provided herein. Table 4 also provides exemplary CH1 sequences for use in the Fab heavy chain.

[0217] Table 4. Exemplary Heavy Chain Frame Region and Constant Region Fragments Table 5 below lists the VH and VL sequences of the exemplary anti-TfR1 antibodies provided herein, as well as their heavy and light chain sequences in Fab form.

[0218] Table 5. Sequences of exemplary anti-TfR1 Fab antibodies Example 2. Synthesis of antibody-siRNA adapter The following provides the synthesis of various Fab connectors used throughout this application.

[0219] Synthesis of 16-((3,5-bis(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)amino)-16-oxo-4,7,10,13-tetraoxahexadecanoic acid 2,3,5,6-tetrafluorophenyl ester (i.e., L-1026-p) Compound 6 (2.35 g, 7.31 mmol; according to Sarbisheh et al.) Bioconjugate Chemistry Prepared in 202031 (12), 2789-2806) 、 EDC-HCl (2.38 g, 12.43 mmol) and K-Oxyma (2.50 g, 13.9 mmol) were used as a solid mixture and slurried in DMF (190 mL) at ambient temperature under N2. Compound 7 (1.192 g, 5.48 mmol) was added as a solution (10 mL) in DMF. After 5 minutes, triethylamine (4.5 mL, 32.2 mmol) was added dropwise at ambient temperature. The reaction mixture was heated at 50°C for 2 days. The reaction mixture was concentrated under reduced pressure to a red oil, which was slurried in DCM (250 mL) and washed with a saturated aqueous sodium bicarbonate solution (200 mL). The layers were separated, and the aqueous layer was further extracted with DCM (100 mL). The combined organic phases were washed with water (200 mL) and brine (200 mL). The organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by normal-phase SiO2 chromatography using a gradient of ethyl acetate in DCM (0-100%). Yield of compound 8: 1.77 g (49%), partially contaminated with compound 6. Calculated mw of compound 8: 653.77 g / mol, measured m / z (ESI, positive ion mode): 654.83.

[0220] Compound 8 (1.77 g, 2.71 mmol) was dissolved in TFA:DCM [1:1] (18 mL) and stirred at ambient temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and then co-evaporated with toluene (3 x 30 mL). The residue was purified by normal-phase SiO2 chromatography using a gradient (0-7%) of DCM containing 0.1% formic acid and methanol. Yield of compound 9: 1.30 g (80%). Calculated mw of compound 9: 597.66 g / mol, measured m / z (ESI, positive ion mode): 598.79.

[0221] Compound 9 (1.30 g, 2.18 mmol) was dissolved in DCM (50 mL) and cooled to 0°C. By dissolving 10.38 g... m -CPBA (77 wt%) was dissolved in 80 mL of DCM and dried over sodium sulfate until clear to prepare 100 mg / mL. m -CPBA solution. At 0°C, 58 mL of the solution of compound 9 was added dropwise. m -CPBA (5.85 g, 26.1 mmol) 。 The reaction mixture was heated to ambient temperature and allowed to stand overnight. The reaction mixture was concentrated, slurried in 0.1% formic acid (50 mL) in DCM, and filtered. The filtrate was purified by normal-phase SiO2 chromatography using a gradient (0-10%) of DCM containing 0.1% formic acid and methanol. Yield of compound 10: 1.03 g (72%). Calculated mw of compound 10: 661.65 g / mol, measured m / z (ESI, positive ion mode): 662.65.

[0222] At 0°C, EDC (0.448 g, 2.34 mmol) was added to a solution of compound 10 (1.03 g, 1.56 mmol) in DCM:ACN [4:1] (15 mL), followed by a solution of TFP (0.310 g, 1.87 mmol) in DCM:ACN [4:1] (5 mL). After 5 minutes, the reaction mixture was heated to ambient temperature. After 1.5 hours, the reaction mixture was concentrated to dryness. The crude product was purified by preparative reversed-phase HPLC (Phenomenex Gemini C18 50 mm x 250 mm, 10 μm) using a water / acetonitrile gradient containing 0.1% TFA. The fraction containing the product was concentrated under reduced pressure. Yield of L-1026-p: 1.10 g (87%). The calculated mw of compound L-1026-p is 809.71 g / mol, and the measured m / z (ESI, positive ion mode) is 810.62. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 2.64 (t, 2H), 3.00 (t, 2H), 3.49(m, 12H), 3.74 (m, 10H), 7.92 (m, 1H), 8.34 (t, 1H), 8.68 (d, 2H), 10.67 (s,1H).

[0223] Synthesis of 1-(4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)-14-(1-(4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-yl)-1-oxo-5,8,11,17,20,23-hexaoxa-2,14-diazahexacosane-26-acid 2,3,5,6-tetrafluorophenyl ester (i.e., L-1045-p) At 0°C, DIEA (190 μL, 1.09 mmol) was added to a solution of compound 5 (250 mg, 0.44 mmol) in DCM (5 mL), followed by compound L20-p (400 mg, 0.96 mmol) as a slurry in DCM (5 mL). After 5 minutes, the reaction was heated to ambient temperature. After 2.5 hours, the mixture was concentrated under reduced pressure. The residue was co-evaporated with ACN (5 mL x 2) and used for the next step without purification, assuming 100% yield. Calculated mw for compound 6: 1072.17 g / mol, measured m / z (ESI, positive ion mode): 1073.05.

[0224] At 0°C, EDC (100 mg, 0.52 mmol) was added to a solution of compound 6 (467 mg, 0.44 mmol) in DCM (4 mL). After 5 minutes, TFP (87 mg, 0.52 mmol) was added as a solution in DCM (0.5 mL). After 5 minutes, the reaction mixture was heated to ambient temperature. After 4 hours, an additional portion of EDC (25 mg, 0.13 mmol) was added. After an additional 30 minutes, the reaction mixture was concentrated. The residue was co-evaporated with ACN (2 x 5 mL).

[0225] The crude reaction mixture was dissolved in water / ACN 0.1% TFA (25 mL) and MeOH (5 mL). The mixture contained solids, which were removed by centrifugation and the solution was filtered. The solution was purified by semi-preparative reversed-phase HPLC (Phenomenex Gemini C18 21.2 mm x 250 mm) using a water / acetonitrile gradient containing 0.1% TFA. The fraction containing the product (60 mL) was diluted 1:1 with DCM. The pH of the mixture was adjusted to pH 7 with saturated sodium bicarbonate aqueous solution (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 co-evaporated with ACN (50 mL). Yield of L-1045-p: 76 mg (14% in 2 steps). The calculated mw of compound L-1045-p is 1220.22 g / mol, and the measured m / z (ESI, positive ion mode) is 1221.02. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 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).

[0226] Synthesis of 2,3,5,6-tetrafluorophenyl ester of 2-(methanesulfonyl)benzo[d]thiazolyl-6-carboxylic acid (i.e., L-1064-p) Sodium methanesulfinate (85% wt%, 162 mg, 1.16 mmol, technical grade) was added to a solution of compound 1 (200 mg, 0.77 mmol) in DMF (3 mL). The reaction mixture was heated at 70°C for 30 min and 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 phases were washed with water (3 x 10 mL) and then with brine (10 mL). The organic phases were dried over sodium sulfate, filtered, and concentrated. Calculated mw for compound 2: 257.28 g / mol, measured m / z (ESI, positive ion mode): 258.29.

[0227] At ambient temperature, EDC (203 mg, 1.06 mmol) was added to a slurry of compound 2 (182 mg, 0.71 mmol) in DCM (2 mL), followed by the addition of a solution of TFP (129 mg, 0.78 mmol) in DCM (1 mL). After 2 hours, the reaction solution became clear. The reaction solution was concentrated to dryness. The residue was adsorbed onto SiO2 (3 mL) and purified by normal-phase SiO2 chromatography with a gradient of DCM in hexane (0–50% DCM). Yield of L-1064: 191 mg (67%). Calculated mw of compound L-1064-p: 405.34 g / mol, measured m / z (ESI, positive ion mode): 406.23. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 3.66 (s, 3H), 8.06 (m, 1H), 8.4-8.5 (m, 2H), 9.35 (d, 1H).

[0228] Synthesis of 1-(2-(methanesulfonyl)benzo[d]thiazolyl)-14-(1-(2-(methanesulfonyl)benzo[d]thiazolyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-yl)-1-oxo-5,8,11,17,20,23-hexaoxa-2,14-diazahexacosane-26-acid 2,3,5,6-tetrafluorophenyl ester (i.e., L-1100-p) At 0°C, DIEA (190 μL, 1.09 mmol) was added to a solution of compound 1 (250 mg, 0.44 mmol) in DCM (5 mL), followed by dropwise addition of a solution of L-1064 (400 mg, 0.96 mmol) in DCM (5 mL). The reaction mixture was heated to ambient temperature. After 1 hour, EDC (168 mg, 0.87 mmol) was added to the reaction mixture. After an additional 1.5 hours, the reaction mixture was concentrated into an oil. The residue was purified by normal-phase SiO2 chromatography using a methanol gradient (0–10%) in DCM. Yield of L-1100-p: 299 mg (57%). Calculated mw of compound L-1100-p: 1198.29 g / mol, measured m / z (ESI, positive ion mode): 1198.70. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 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).

[0229] Synthesis of 1-(4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-acid 2,3,5,6-tetrafluorophenyl ester (i.e., L-1063-p) A solution of compound 1 (500 mg, 2.40 mmol) in THF (6 mL) was bubbled under nitrogen for 10 min. Potassium carbonate (664 mg, 4.80 mmol) was added to the solution. The mixture was stirred at ambient temperature for 30 min. A solution of compound 2 (2.78 g, 7.2 mmol) in THF (2 mL) was added dropwise under rt. The reaction was allowed to proceed overnight. After 24 hours, LCMS showed a mixture of product and starting material; 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 saturated sodium bicarbonate aqueous solution (15 mL). The organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by normal-phase SiO2 chromatography using a gradient of ethyl acetate in DCM (0–50%). Yield of compound 3: 980 mg (80%). The calculated mw of compound 3 is 512.62 g / mol, and the measured m / z (ESI, positive ion mode) is 513.69.

[0230] Compound 3 (980 mg, 1.91 mmol) was dissolved in TFA:DCM [1:1] (6 mL) at rt. After 30 min, toluene (30 mL) was added and the mixture was concentrated under reduced pressure. The residue was co-evaporated with toluene (3 x 20 mL) and then with 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, measured m / z (ESI, positive ion mode): 457.05.

[0231] Compound 4 (1.30 g, 2.18 mmol) was dissolved in DCM (2 mL) and cooled to 0°C. By dissolving 1.3 g... m -CPBA (77 wt%) was dissolved in 10 mL of DCM and dried over sodium sulfate until clear, while preparing m A 100 mg / mL solution of CPBA was added dropwise to a solution of compound 4 at 0°C. m -CPBA (654 mg, 2.92 mmol). The reaction mixture was heated to ambient temperature and allowed to stand overnight. The reaction mixture was quenched by adding an aqueous sodium sulfite solution (10% w / w, 4 mL) under vigorous stirring. The mixture was diluted with DCM (20 mL) and water (10 mL). The organic phase was collected and washed 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 showed 60% contamination with 3-chlorobenzoic acid. Corrected yield of compound 5: 213 mg (97%). Calculated mw of compound 5: 488.51 g / mol, measured m / z (ESI, positive ion mode): 489.57.

[0232] At 0°C, EDC (293 mg, 1.53 mmol) was added to a solution of compound 5 (213 mg, 0.44 mmol) in DCM (2.2 mL), followed by the addition of TFP (217 mg, 1.31 mmol) in DCM (1 mL). The reaction mixture was allowed to warm to ambient temperature. After 2 hours, the reaction mixture was concentrated under reduced pressure. The residue was purified by normal-phase SiO2 chromatography with a gradient of ethyl acetate in DCM (0–50%). Yield of L-1063-p: 121 mg (44%). Calculated mw of compound L-1063: 636.57 g / mol, measured m / z (ESI, positive ion mode): 637.66. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 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).

[0233] Synthesis of (S)-1-(4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)-18-(1-(4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-acylamino)-15,19-dioxo-3,6,9,12,23,26-hexaoxa-16,20-diazanonadecan-29-acid 2,3,5,6-tetrafluorophenyl ester (i.e., L-1176-p) Under rt, diethylamine (2.70 mL, 26.15 mmol) was added to a solution of 1,1-dimethyl ethyl ester of 3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-L-alanine (CAS 2084868-72-2) (1.00 g, 2.61 mmol) in ACN (6.3 mL). After 30 min, the reaction mixture was concentrated under reduced pressure. The residue was co-evaporated 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, measured m / z (ESI, positive ion mode): 161.36.

[0234] DIEA (408 μL, 2.34 mmol) was added to a slurry of compound 2 (125 mg, 0.78 mmol) in DMF (2.5 mL), followed by a solution of compound 1 (784 mg, 1.72 mmol; prepared as described above) in DCM (5 mL). TBTU (626 mg, 1.95 mmol) was added to the turbid mixture at ambient temperature. After 30 minutes, the reaction mixture was concentrated under reduced pressure (to remove DCM) and EtOAc (30 mL) was added. The mixture was washed with saturated aqueous sodium bicarbonate solution (2 x 15 mL), saturated ammonium chloride solution (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 SiO2 (7 mL) and purified by normal-phase SiO2 chromatography using a gradient of methanol in DCM (0–7%). Yield of compound 3: 492 mg (61%). The calculated mw of compound 3 is 1037.21 g / mol, and the measured m / z (ESI, positive ion mode) is 1037.89.

[0235] 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 and then co-evaporated 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, measured m / z (ESI, positive ion mode): 981.84.

[0236] At ambient temperature, DIEA (77 uL, 0.44 mmol) was added to a solution of compound 4 (291 mg, 0.30 mmol) and compound 5 (58 mg, 0.33 mmol) in DMF (4.4 mL), followed by HATU (169 mg, 0.44 mmol). The reaction mixture was allowed to stand overnight. The reaction mixture was diluted with 0.1% TFA (5 mL) and purified by semi-preparative RP HPLC (Phenomenex Gemini C18 21.2 x 250 mm) using a water / acetonitrile gradient containing 0.1% TFA. The fraction containing the product was concentrated under reduced pressure. Yield of compound 6: 229 mg (68%). Calculated mw of compound 6: 1140.28 g / mol, measured m / z (ESI, positive ion mode): 1140.97.

[0237] At 0°C, EDC (77 mg, 0.40 mmol) was added to a solution of compound 6 (229 mg, 0.20 mmol) in DCM (3.4 mL), followed by TFP (67 mg, 0.40 mmol). After 1 hour, additional TFP (267 mg, 1.61 mmol) and EDC (77 mg, 0.40 mmol) were added. After an additional 2 hours, additional EDC (96 mg, 0.50 mmol) was added. Finally, after 30 minutes, the reaction mixture was concentrated to 2 mL and purified by normal-phase SiO2 chromatography with a methanol gradient (0–7%) in DCM. Yield of compound 7: 69 mg (27%). Calculated mw of compound 7: 1288.34 g / mol, measured m / z (ESI, positive ion mode): 1288.83.

[0238] A solution of compound 7 (69 mg, 0.054 mmol) in DCM (2 mL) was prepared and cooled to 0°C. This was achieved by adding 1.3 g of the solution. m -CPBA (77 wt%) was dissolved in 10 mL of DCM and dried over sodium sulfate until clear, while preparing m A 100 mg / mL solution of CPBA was added dropwise to a solution of compound 7 at 0°C. m-CPBA (140 mg, 0.80 mmol). The reaction mixture was heated to ambient temperature and allowed to stand overnight. The reaction mixture was concentrated and then suspended in water / ACN (10 mL) containing 0.1% TFA and purified by a semi-preparative RP HPLC (Phenomenex Gemini C18 21.2 x 250 mm) using a water / acetonitrile gradient containing 0.1% TFA. The fraction containing the product was concentrated under reduced pressure. Yield of L-1176-p: 60 mg (83%). Calculated mw of compound L-1176-p: 1352.34 g / mol, measured m / z (ESI, positive ion mode): 1352.79. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 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).

[0239] Synthesis of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-N-(3,5-bis(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide (i.e., L-1288-p) Compound 1 (106 mg, 0.33 mmol; according to Sarbisheh et al.); Bioconjugate ChemistryCompound 2 (73 mg, 0.30 mmol) and triethylamine (126 μL, 0.90 mmol) were combined as solids and dried under high vacuum for 30 min. The solids were then slurried in DMF (10 mL) at ambient temperature under N2. Compound 2 (73 mg, 0.30 mmol) and triethylamine (126 μL, 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 and then dissolved in water / ACN 0.1% TFA (40 mL). The solution was purified by semi-preparative reversed-phase HPLC (Phenomenex Gemini C18 21.2 mm x 250 mm) using a water / acetonitrile gradient containing 0.1% TFA. The fractions containing the product were combined and concentrated under reduced pressure. Yield of compound 3: 62 mg (38%). Calculated mw of compound 3: 550.61 g / mol, measured m / z (ESI, positive ion mode): 551.74.

[0240] Compound 3 (62 mg, 0.11 mmol) was dissolved in DCM (2 mL) and cooled to 0°C. By using 1.3 g... m -CPBA (77 wt%) was dissolved in 10 mL of DCM and dried over sodium sulfate until clear, and the preparation was carried out. m A 100 mg / mL solution of CPBA was added dropwise to the solution of compound 3 at 0°C. m -CPBA (194 mg, 1.13 mmol). The reaction mixture was heated to ambient temperature and allowed to stand 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: 0.1% ACN TFA (8 mL) and methanol (0.5 mL). The solution was purified by semi-preparative reversed-phase HPLC (Phenomenex Gemini C18 21.2 mm x 250 mm) using a water / acetonitrile gradient containing 0.1% TFA. Fractions containing the product were combined and concentrated under reduced pressure. Yield of L-1288-p: 31 mg (45%). Calculated mw of compound L-1288-p: 614.61 g / mol, measured m / z (ESI, positive ion mode): 615.66. 1H NMR (400 MHz, [D6]DMSO, 25°C): δ = 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).

[0241] Synthesis of N-methyl-4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)benzamide (i.e., CP-1113-p) At ambient temperature, a solution of methylamine (1.3 mL, 15.0 mmol) in THF (20 mL) was added dropwise to a slurry of L20 (5.0 g, 12.0 mmol) in THF (80 mL). After 30 minutes, SiO2 (35 mL) was added and the reaction mixture was concentrated under reduced pressure. The residue was purified by normal-phase SiO2 chromatography using a gradient of ethyl acetate in DCM (0–70%). The fraction containing the product was concentrated under reduced pressure. Yield of CP-1113-p: 1.76 g (52%). Calculated mw of compound CP-1113-p: 281.29 g / mol, measured m / z (ESI, positive ion mode): 282.45. 1 H NMR (400 MHz, [D6]DMSO, 25°C): δ = 2.80 (d, 3H), 3.70 (s, 3H), 8.06 (m, 2H),, 8.18 (m, 2H), 8.68 (br q, 1H).

[0242] Synthesis of N-methyl-2-(methanesulfonyl)benzo[d]thiazole-6-carboxamide (i.e., CP-1154-p) At ambient temperature, a methylamine solution (40 wt%, in water, 80 μL, 0.93 mmol) was added dropwise to a slurry of L-1064-p (300 mg, 0.74 mmol) in THF (6 mL). After 1 hour, SiO2 (3 mL) was added and the reaction mixture was concentrated under reduced pressure. The residue was purified by normal-phase SiO2 chromatography with a gradient of ethyl acetate in DCM (0–60%). The fraction containing the product was concentrated under reduced pressure. Yield of CP-1154-p: 166 mg (83%). Calculated mw of compound CP-1154-p: 270.32 g / mol, measured m / z (ESI, positive ion mode): 271.25. 1H NMR (400 MHz, [D6]DMSO, 25°C): δ = 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).

[0243] Example 3. Synthesis of RNAi reagents and TfR1-specific conjugates The double strands of the RNAi reagent disclosed herein were synthesized according to the following procedure: A. Synthesis .

[0244] The sense and antisense strands of the RNAi reagent were synthesized using a solid-phase phosphoramidite technique employed in oligonucleotide synthesis. Depending on scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was performed on a solid support made of controlled-porosity glass (CPG, 500 Å or 600 Å, from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the 2'-O-methylphosphoramidite used comprised the following: (5'-O-dimethoxytriphenylmethyl-N... 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-O-dimethoxy-triphenylmethyl-N 4 -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphamide, (5'-O-dimethoxytriphenylmethyl-N 2-(isobutyryl)-2'-O-methylguanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide and 5'-O-dimethoxytriphenylmethyl-2'-O-methyluridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2'-Deoxy-2'-fluorophosphamide carries the same protecting group as 2'-O-methylRNAphosphamide (amidite). 5'-Dimethoxytriphenylmethyl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from Glen Research (Virginia). Reverse debasing (3'-O-dimethoxytriphenylmethyl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide was purchased from ChemGenes. (Wilmington, MA, USA). The following UNA phosphoramids were used: 5'-(4,4'-dimethoxytriphenylmethyl)-N6-(benzoyl)-2',3'-open-ring-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-2',3'-open-ring-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-2',3'-open-ring-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-(4,4'-dimethoxytriphenylmethyl)-N-isobutyryl-2 ',3'-open-ring-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide and 5'-(4,4'-dimethoxytriphenylmethyl)-2',3'-open-ring-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide. The TFA-linked phosphoramide was also commercially available (ThermoFisher). Cyclopropylphosphonate phosphoramide was synthesized according to International Patent Application Publication No. WO2017 / 214112 (see also Altenhofer et al., Chem. Communications (Royal Soc. Chem.), 57(55): 6808-6811 (July 2021)).

[0245] B. Cleavage and deprotection of oligomers bound to support agents.

[0246] After solid-phase synthesis was complete, the dried solid support was treated with a 1:1 volume solution of 40% by weight aqueous methylamine and 28% to 31% ammonium hydroxide solution (Aldrich) at 30°C for 1.5 hours. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0247] C. Conjugation of RNAi reagents with Fab and capping with CP-1113.

[0248] The RNAi reagent containing free amines described in this article, after cleavage from the solid phase, is reacted with L20-p using standard amide reaction chemistry. Conjugation. Freshly prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) in PBS (5-20 equivalents, 70 mM) was added to a solution of Fab in PBS (0.2 μmol, 1.0-10.0 mg / mL in PBS). The reaction was allowed to proceed overnight at room temperature in the dark. The next day, TCEP was removed by loading the reaction mixture onto a PD-10 desalting column equilibrated with PBS and eluted with PBS. The concentration of Fab in the eluent was determined using the theoretical absorbance at 280 nm. A solution of the L20-modified sense chain in sodium phosphate buffer was prepared, and the concentration was determined using the theoretical absorbance at 260 nm. The L20-modified sense chain (1-1.3 equivalents, 0.5-2.5 mM) was added to the desalted Fab solution, and the reaction was carried out by inverted mixing. Analysis by SEC Method 1 and AIEX Method 1 showed the mixture of starting Fab, DAR1, and DAR2. After 1 hour, CP-1113-p: The solution in DMSO was added to the reaction mixture (3 equivalents, 36 mM). After 1 hour, the solution of L-cysteine ​​in PBS was added to the reaction mixture (6–10 equivalents, 165 mM). Finally, the conjugate was annealed by adding antisense (1.2–1.5 equivalents, 0.5–2.5 mM). The conjugate was purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was pumped onto the column and eluted with a gradient of 10–40% buffer B. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AEX Method 1, and Nanodrop 260 / 280 readings. The DAR1 fraction was combined, and PBS was replaced with PD-10 desalting column buffer. The purified conjugate was analyzed by SEC method 1 and eluted as a monomer peak with a retention time of 13.2 minutes.

[0249] D. Conjugation of RNAi reagents with Fab and capping with N-ethylmaleimide.

[0250] The RNAi reagent containing free amines described in this article, after cleavage from the solid phase, is reacted with L20-p using standard amide reaction chemistry. Conjugation. A freshly prepared solution of TCEP-HCl in PBS (5–20 equivalents, 70 mM) was added to a solution of Fab in PBS (10 mg, 0.2 μmol, 1.0–10.0 mg / mL in PBS). The reaction was allowed to proceed overnight at room temperature in the dark. The next day, TCEP was removed by loading the reaction mixture onto a PD-10 desalting column equilibrated with 20 mM tris pH 8 and eluted with 20 mM tris pH 8. The concentration of Fab in the eluent was determined using the theoretical absorbance at 280 nm. A solution of the L20-modified sense chain in sodium phosphate buffer was prepared, and its concentration was determined using the theoretical absorbance at 260 nm. The L20-modified sense chain (1–1.3 equivalents, 0.5–2.5 mM) was added to the desalted Fab solution, and the reaction was carried out by inverted mixing. Analysis by SEC Method 1 and AIEX Method 1 showed the mixture of starting Fab, DAR1, and DAR2. One hour later, a solution of N-ethylmaleimide (NEM) in 20 mM tris pH 8 was added to the reaction mixture (12 equivalents, 160 mM). One hour later, the conjugate was annealed by adding antisense (1.2–1.5 equivalents, 0.5–2.5 mM). The conjugate was purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10–40% buffer B. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AEX Method 1, and UV-Vis 260 / 280 measurements. The DAR1 fraction was combined, and PBS was replaced with PD-10 column buffer. The purified conjugate was analyzed by SEC method 1 and eluted as a monomer peak with a retention time of 13.2 minutes.

[0251] SEC Method 1 AIEX Method 1 The RNAi reagent containing free amines described in this article is first coupled with L1026-p using standard solid-phase synthesis techniques: Concatenation is performed to generate a semantic chain modified by L-1026: (NH2-C6)s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb) (i.e., CS915332, SEQ ID NO: 159). After cleavage from the solid phase, the RNAi reagent described herein is synthesized according to the following procedure, as exemplified by the synthesis of AC006448: A freshly prepared solution of TCEP-HCl in PBS (5 equivalents, 70 mM, 42 µL) was added to a solution of Fab0070 (28 mg, 0.59 µmol, 5.55 mg / mL in PBS). The reaction was inverted and mixed at ambient temperature for 15 minutes, then kept overnight at 5°C without stirring. The next day, TCEP was removed by loading the reaction mixture onto two PD-10 desalting columns (Cytiva), which were equilibrated with 20 mM tris 50 mM NaCl pH 7.6 (alternatively, 20 mM tris pH 8 or PBS buffer) and eluted with the same buffer. The concentration of Fab in the eluent was determined using the theoretical absorbance at 280 nm. A solution of the L-1026-modified sense chain (i.e., CS915332) was prepared in 10 mM sodium phosphate buffer pH 6.0–6.5, and its concentration was determined using the theoretical absorbance at 260 nm. L-1026-modified CS915332 (1.15 equivalents, 2.75 mM, 240 μL) was added to the desalted Fab solution, and the reaction was carried out by inverted mixing at ambient temperature. Analysis by SEC Method 1 and AIEX Method 1 showed a mixture of starting Fab0070, DAR1 product, and DAR2 product. After 30 minutes, an L-cysteine ​​solution in 20 mM tris 50 mM NaCl pH 7.6 (alternatively, some L-1026 conjugates have been prepared in 20 mM tris pH 8 or PBS buffer) was added to the reaction mixture (10 equivalents, 165 mM, 36 μL). After 30 minutes, the conjugate was annealed by adding antisense chain (CA003820) (1.3 equivalents, 1.45 mM in water, 529 μL). The conjugates were purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ5PW (20 μm). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10–40% buffer B. The DAR1 and DAR2 fractions were distinguished by SEC Method 1, AIEX Method 1, and UV-Vis 260 / 280 measurements. The DAR1 fraction was combined, and two PD-10 column buffers were replaced with PBS. The purified conjugate AC006448 was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 7.2 min.

[0252] SEC Method 1 AIEX Method 1 E. Conjugation of RNAi reagents with Fab.

[0253] Synthesis of Fab0070-L-1288 Add freshly prepared TCEP-HCl solution (5 equivalents) to a Fab0070 solution in PBS (82 mg, 8.2 mg / mL). Incubate the reaction mixture overnight at 5°C. The next day, using four PD-10 columns equilibrated with the same buffer, buffer-exchange the reaction mixture to 20 mM Tris 50 mM NaCl pH 7.6. Determine the concentration using Nanodrop and dilute the solution to 2 mg / mL with 20 mM Tris 50 mM NaCl pH 7.6. Dilute the solution with DMSO (1.95 mL). Prepare an L-1288 solution (2 mg / mL in DMSO) and load it over 15 minutes (1.6 mL, 3.19 mg, 3 equivalents). Quench the reaction with cysteine ​​(10 equivalents) after 10 minutes. Remove insoluble material by centrifugation. The supernatant was collected, filtered, diluted 1:1 with PBS, and concentrated / desalted to a volume of 8 mL using a Pierce 10K MWCO centrifuge column. Finally, the solution was buffer-exchanged by loading it onto a HiPrep 26 / 10 desalting column and eluting with PBS. The final yield was approximately 80 mg.

[0254] L-1289 bioconjugation Lyophilized CS915332 (86 mg, 11.6 μmol) was dissolved in DMF (1.58 mL) and water (215 μL) and sonicated. Triethylamine (9.7 μL, 6 equivalents) and a solution of L-1289 in DMF (50 mg / mL, 344 μL, 3 equivalents) were added to the solution. The reaction was monitored by LCMS. After 1 hour, additional triethylamine (4 equivalents) and L-1289 solution (2 equivalents) were added. Complete conversion was observed by LCMS after 20 minutes. The reaction mixture was acidified with phosphoric acid (200 mg / mL in water, 5 equivalents). The crude solution was added dropwise to a mixture of acetonitrile (43 mL) and PBS (1.7 mL). The precipitate was collected by centrifugation, and the supernatant was discarded. The precipitate was dissolved in water (1.5 mL) and added to acetonitrile (43 mL). The precipitate was collected by centrifugation and dissolved in PBS (5 mL). The yield was 88 mg (98%).

[0255] The concatenation of semantic chains modified by Fab0070-L-1288 and L-1307 Add the L1307-modified sense chain solution to the Fab0070-L-1288 solution in PBS (12.5 mg, 6.97 mg / mL, 1.79 mL, 1 equivalent): L-1307s(invAb)scacuuuugAfCfCfugcuaaucaas(invAb) (i.e., CS009529, SEQ ID NO: 160) in PBS (6.18 mg, 17.9 mg / mL, 345 uL, 3 equivalents). The combined solutions were inverted and mixed for 10 minutes, then stored at 5°C without stirring. The next day, the reaction was heated to room temperature and annealed with the antisense chain: cPrpusUfsgauuAfgcagGfuCfaAfaagsusg (i.e., CA003820, SEQ ID NO: 161), 6.23 mg, 10.4 mg / mL, 599 uL, 3.3 equivalents. The conjugates were purified using an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (buffer A), 20 mM tris 1500 mM NaCl (buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 μm). The crude reaction mixture was diluted to 40 mL with MPA, loaded onto the column, and eluted with a gradient of 10–40% buffer B. Fractions containing DAR1 were combined and exchanged for PBS using two PD-10 column buffers. The conjugates were analyzed by SEC Method 1 and found to be 99% pure with a retention time of 7.2 min. This procedure was also followed to generate the Fab-L-1288-L-1289-RNAi conjugates disclosed herein.

[0256] SEC Method 1 Example 4. After delivery of a TfR1 Fab-conjugated siRNA antisense targeting androgen receptor (AR) mRNA, Relative expression of mRNA The siRNA duplexes are shown as sense and antisense in Tables 6 and 7, respectively. The antisense strand is complementary to the AR mRNA. The sense siRNA strand is conjugated to the Fab shown in Table 6 below.

[0257] Table 6. siRNA sense sequences used to target AR mRNA.

[0258] Symbol explanation: (a) represents 2′-O-methyladenosine, (c) represents 2′-O-methylcytidine, (g) represents 2′-O-methylguanosine, and (u) represents 2′-O-methyluridine; (Af) represents 2′-fluoroadenosine, (Cf) represents 2′-fluorocytidine, (Gf) represents 2′-fluoroguanosine, and (Uf) represents 2′-fluorouridine; (s) represents a phosphate thioester bond; (invAb) and (invAb)s represent reverse debasing nucleotides (bonds). The structures are shown in Table 13 below; cPrpus represents 5′-cyclopropylphosphonate-2′-O-methyluridine-3′-thiophosphate (structures are shown in Table 13 below); (NH-C6)s structure is shown in Table 13 below; L20 structure is shown in Table 13 below; [CP-1113] indicates that Fab has been capped using CP-1113p according to the procedure of Example 6C; [NEM] indicates that Fab has been capped using N-ethylmaleimide according to the procedure of Example 6D.

[0259] Table 7. siRNA antisense sequences for targeting AR mRNA Symbol explanation: (a) represents 2′-O-methyladenosine, (c) represents 2′-O-methylcytidine, (g) represents 2′-O-methylguanosine, and (u) represents 2′-O-methyluridine; (Af) represents 2′-fluoroadenosine, (Cf) represents 2′-fluorocytidine, (Gf) represents 2′-fluoroguanosine, and (Uf) represents 2′-fluorouridine; (s) represents a thiophosphate bond; cPrpus represents 5′-cyclopropylphosphonate-2′-O-methyluridine-3′-thiophosphate (structures are shown in Table 13 below).

[0260] Table 8 shows the structures of cPrpus, (invAb)s, (invAb), L20, L1026, L1100, L1176, L1064, L1063, and NH-C6 as defined in the symbol descriptions of the meaningful and antisense sequences in Tables 6 and 7 above. Example 5. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice According to Table 9 below, on day 1 of the study, Tg B-hTfr1 mice were administered intravenously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-7) at a dose of 250 μL / 25g, and were administered the same formulation again on day 2. Table 9: The drug administration groups of mice in Example 5.

[0261] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 1 The relative AR mRNA knockdown was shown for each group in the four analyzed tissues.

[0262] Example 6. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice According to Table 12 below, on day 1 of the study, Tg B-hTfr1 mice were administered intravenously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-10) at a dose of 250 μL / 25g, and were administered the same formulation again on day 2. Table 10: Drug administration groups in mice of Example 6.

[0263] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 2 The relative AR mRNA knockdown was shown for each group in the four analyzed tissues.

[0264] Example 7. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice According to Table 11 below, on day 1 of the study, Tg B-hTfr1 mice were administered intravenously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-6) at a dose of 250 μL / 25g, and were administered the same formulation again on day 2. Table 11: Drug administration groups in mice of Example 7.

[0265] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed by qPCR for androgen receptor (AR) mRNA knockdown. Figure 3 The relative AR mRNA knockdown was shown for each group in the four analyzed tissues.

[0266] Example 8. The effect of different antibody conjugate groups on the selected brain regions compared to the AC003313 (Fab0002) control group AR mRNA gene expression in vivo In vivo AR mRNA gene knockdown of the Fab-doublet was measured in selected brain regions. AR mRNA gene expression in each group was normalized for the AC003313 (Fab0002 conjugated siRNA) group. Figure 4 A graph showing relative expression values ​​is displayed. The Fab0070-siRNA duplex group appears to have the lowest relative expression (highest gene knockdown).

[0267] Example 9. In vivo knockdown of AR mRNA gene expression in cynomolgus monkeys According to Table 12 below, on day 0 of the study, cynomolgus monkeys were administered subcutaneously with PBS or a compound formulation containing 3 mg / kg of anti-TfR1 Fab-siRNA conjugate at a concentration of 1.5 mg / mL. On day 7 of the study, groups 2, 3, and 4 were administered the same formulation as on day 0. All animals were administered at a volume of 2 mL / kg.

[0268] Table 12: Drug administration groups in non-human primates in Example 9.

[0269] Three monkeys (n=3) were administered the drug in groups 1 (control), 3, and 4 (trigger treatment), and two monkeys (n=2) were administered the drug in group 2. On day 28 of the study, animals from all groups were euthanized, and tissues from each animal were collected including: frontal cortex, temporal cortex, caudate nucleus, cerebellum (cortex), putamen, motor cortex, medulla oblongata, pons, hippocampus, thalamus, hypothalamus, midbrain, substantia nigra, dorsal cervical root ganglion (DRG), thoracic DRG, lumbar DRG, cervical spinal cord, lumbar spinal cord, thoracic spinal cord, and visual cortex. Samples were analyzed by qPCR for AR mRNA knockdown. The mean results for each group relative to group 1 are shown in Table 13 below: Table 13. Relative expression of AR mRNA in various tissues by qPCR analysis for each dosing group in Example 19.

[0270] As shown in Table 13 above, a persistent decrease in AR mRNA expression was observed in multiple tissues in non-human primates treated with anti-TfR1 antibody-siRNA conjugates. Except for the thoracic and lumbar DRGs, groups 3 (Fab0061-siRNA conjugate) and 4 (Fab0070-siRNA conjugate) outperformed group 2 (Fab002-siRNA conjugate) in almost every tissue analyzed.

[0271] Example 10. In vivo knockdown of MAPT in cynomolgus monkeys As shown in Table 13 above, a persistent decrease in AR mRNA expression was observed in multiple tissues in non-human primates treated with anti-TfR1 antibody-siRNA conjugates. Except for the thoracic and lumbar DRGs, groups 3 (Fab0061-siRNA conjugate) and 4 (Fab0070-siRNA conjugate) outperformed group 2 (Fab002-siRNA conjugate) in almost every tissue analyzed.

[0272] According to Table 14 below, on day 0 of the study, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound preparation containing AC007414 at a concentration of 1.5 mg / mL and 2 mL / kg, 15 mg / kg at a concentration of 7.5 mg / mL and 2 mL / kg, or 30 mg / kg at a concentration of 7.5 mg / mL and 4 mL / kg. Table 14: Dosing groups in non-human primates in Example 10 Group ID Animals given the drug Group 1 (aCSF) n=4 Group 2 (3 x 3 mg / kg AC007414) n=4 Group 3 (3 x 15 mg / kg AC007414) n=4 Group 4 (3 x 30 mg / kg AC007414) n=4 Four monkeys (n=4) in each group were administered the drug. Subcutaneous injections were given on days 0, 7, and 14. On day 29 of the study, animals from each group were euthanized, and brain and spinal cord tissues were collected from each animal. Samples were analyzed for MAPT mRNA knockdown by qPCR. Samples were analyzed for protein knockdown by JESS. The mean mRNA knockdown in the frontal cortex, hippocampus, and thoracic spinal cord for each group relative to group 1 is shown in Table 15 below: Table 15. Relative expression of MAPT mRNA in various tissues by qPCR analysis for each dosing group in Example 10. Compared to Group 1, the mean protein knockdown in the frontal cortex, hippocampus, and thoracic spinal cord of each group was shown in Figure 5 middle.

[0273] As shown in Table 15 and Figure 5 As can be seen, when injected subcutaneously, the MAPT RNAi reagent, which binds to anti-transferrin Fab070, achieved dose-dependent and deep knockdown in CNS tissues. The results confirm that treatment of MAPT-related diseases and symptoms can be achieved through subcutaneous administration of the RNAi reagent.

[0274] It should be understood that the foregoing disclosure highlights certain specific embodiments of the invention, and all equivalent modifications or alternatives are within the spirit and scope of the invention as set forth in the appended claims.

[0275] Example 11. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0276] According to Table 16 below, on day 1 of the study, Tg B-hTfr1 mice were administered subcutaneously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-10) with either 250 μL / 25g. The same formulation was administered again on day 2. Table 16. Drug administration groups in mice of Example 11.

[0277] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed for androgen receptor (AR) mRNA knockdown by qPCR. Table 17 below shows the AR knockdown in various tissues.

[0278] Table 17. Mean mAR expression in CNS tissues of Example 11 As shown in Table 17, all dosing groups showed deep and stable knockdown of Fab using the RNAi reagent linked to the adapter of the present invention.

[0279] Example 12. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0280] According to Table 18 below, on day 1 of the study, Tg B-hTfr1 mice were administered subcutaneously with either 250 μL / 25g PBS (Group 1) or 1.5 mg / kg TfR1-Fab conjugated siRNA (Groups 2-10) with either 250 μL / 25g. The same formulation was administered again on day 2. Table 18. Drug administration groups in mice of Example 12.

[0281] On day 15, mice were euthanized, and the left hemisphere of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were obtained from the right hemisphere of the brain, including the thoracic spinal cord, cortex, cerebellum, and striatum. Samples were analyzed for androgen receptor (AR) mRNA knockdown by qPCR. Table 19 shows the relative AR mRNA knockdown in each of the four analyzed tissues for each group.

[0282] Table 19. mAR expression in CNS tissues of Example 12 As shown in Table 19, both groups showed AR knockdown using Fab-siRNA conjugates linked by the adapters disclosed herein.

[0283] Example 13. In vivo administration of HTT RNAi reagent in cynomolgus monkeys.

[0284] In vivo evaluation of HTT RNAi reagent in cynomolgus monkeys 。 On days 1, 8, and 15, four (n=4) male cynomolgus monkeys in each test group were administered HTT RNAi reagent at doses of 1.5 mg / ml, 2.0 ml / kg, or 3.0 mg / kg (adjusted for individual animal weight) in PBS, or via PBS. Each dose was administered subcutaneously (SC) and based on the most recent body weight of each animal. Dosing was performed according to Table 20 below.

[0285] Table 20. Administration of drugs to cynomolgus monkeys in Example 13.

[0286] The test animals were non-human primates, male cynomolgus fascicularis, not first-time test subjects, aged 3–5 years. Prior to administration, the test animals were acclimatized to laboratory conditions for at least 14 days according to facility and acclimatization standard operating procedures. RNAi reagent test samples were administered subcutaneously (SC) via syringe and needle into the mid-scapular region.

[0287] The animal was shaved before administration. The day 1 dose was delivered to the left scapular region, the day 8 dose to the right scapular region, and the day 15 dose to the left scapular region. Each dose was administered using a syringe with a 23-25 ​​gauge needle.

[0288] Individual weights of the test animals were recorded once 7 days before treatment, then weekly throughout the study, and once before necropsy.

[0289] On day -7, ~0.5 mL of cerebrospinal fluid (CSF) was collected from all groups. On day 43 (the day of autopsy), ~2.0 mL of CSF was collected from all groups. For the CSF collection procedure, all test animals were anesthetized by intravenous (IV) administration of ketamine (10 mg / kg, IV) and dexmedetomidine (0.02 mg / kg, IM, IM) and placed in a lateral decubitus position. The skin covering the insertion site was shaved off and wiped several times with individual chlorhexidine swabs. The animal's head was gently bent until the chin almost touched the chest, but the airway was not obstructed. A professional palpated the occipital protuberance and the atlantoaxial (C1) wing. The needle was inserted vertically above the atlantoaxial vertebra, through the skin, into the subarachnoid space. If bone was encountered, the needle was reoriented forward or backward until the designated entry point was found. Once proper placement was achieved, CSF would flow freely through the needle and be collected in a cryotube. The needle was removed, and direct pressure was applied to the puncture site for at least two minutes. Once the procedure is complete, administer carbofen 2–4 mg / kg to the animals every 12 hours via SC for 1 day. If necessary, administer atemexazole (0.225 mg / kg, IM).

[0290] On day 43, the cynomolgus monkeys were euthanized. The following tissues were collected from the test animals: left and right cerebral hemispheres, spinal cord, and dorsal root ganglia (DRG). Other tissues besides those listed above may also be collected, and if such additional tissues are collected, their biological data are similarly presented below. The collected tissues were analyzed for biological parameters.

[0291] The cHTT mRNA transcription level was quantified from collected tissues by qPCR, with cPPIB used as an endogenous control gene, and normalized for group 1 cynomolgus monkeys administered PBS. cHTT expression data are shown in Table 21 below.

[0292] Table 21. cHTT expression in cynomolgus monkey tissues from Example 13.

[0293] In the temporal cortex, frontal cortex, motor cortex, caudate nucleus, and hippocampus, groups 2–5 showed a reduction in HTT transcripts up to at least day 43 post-dose. In the putamen, groups 2, 4, and 5 showed a reduction in HTT transcripts up to at least day 43 post-dose. Most notably, the most significant reduction in HTT transcripts was observed in the hippocampus, with three doses of 3.0 mg / kg AC007867 showing ~69% HTT transcript inhibition (0.306), and three doses of 3.0 mg / kg AC007865 showing ~70% HTT transcript inhibition (0.302).

[0294] HTT protein expression was analyzed in cynomolgus monkey tissues using the Jess protein assay, normalized for group 1 cynomolgus monkeys administered with PBS. HTT protein was quantified using an anti-huntingtin protein antibody (Sigma-Aldrich®, catalog MAB2166). HTT protein expression data are shown in Table 22 below.

[0295] Table 22. HTT protein expression in cynomolgus monkey tissues from Example 13.

[0296] In the temporal cortex, frontal cortex, motor cortex, caudate nucleus, putamen, hippocampus, and cerebellum, groups 2–5 showed a decrease in HTT protein up to at least day 43 post-dose. In the liver, groups 2, 3, and 5 showed a decrease in HTT protein up to at least day 43 post-dose. Most notably, the most significant decrease in HTT protein was observed in the motor cortex, with three doses of 3.0 mg / kg AC007867 showing ~86% HTT protein inhibition (0.143), and three doses of 3.0 mg / kg AC007865 showing ~85% HTT protein inhibition (0.146).

[0297] Example 14. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0298] The AR RNAi reagent was evaluated in vivo in mice. On days 1 and 2, five (n=5) female B-hTFR1 mice were administered saline or the AR RNAi reagent (prepared in saline at 1.5 mg / kg body weight) via subcutaneous (SC) injection at a total dose volume of 250 μL / 25 g. Dosage was performed according to Table 23 below.

[0299] Table 23. Drug administration groups in mice of Example 14.

[0300] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0301] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand, the targeting ligand comprising an antigen-binding moiety having a modified sequence as shown in the double-stranded structure herein (for specific modification and structural information (including the antigen-binding moiety) associated with AR RNAi reagents, see Tables 3, 4, 5, 6, 7, 8, 9 and 10).

[0302] Mice were euthanized on day 15. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 24 below.

[0303] Table 24. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 14.

[0304] In the thoracic spinal cord, cerebellum, cortex, and striatum, groups 2–10 showed a reduction in mAR up to at least day 15. Most notably, two doses of 1.5 mg / kg AC006266 achieved ~81% inhibition (0.192) in the cerebellum.

[0305] Example 15. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0306] The AR RNAi reagent was evaluated in vivo in mice. On days 1 and 2, five (n=5) female B-hTFR1 mice were administered saline or the AR RNAi reagent (prepared in saline at 1.5 mg / kg body weight) via subcutaneous (SC) injection at a total dose volume of 250 μL / 25 g. Dosage was performed according to Table 25 below.

[0307] Table 25. Drug administration groups in mice of Example 15.

[0308] B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0309] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand, the targeting ligand comprising an antigen-binding moiety having a modified sequence as shown in the double-stranded structure herein (for specific modification and structural information (including the antigen-binding moiety) associated with AR RNAi reagents, see Tables 3, 4, 5, 6, 7, 8, 9 and 10).

[0310] Mice were euthanized on day 15. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested and collected for analysis. Muscle tissue was also collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 26 below.

[0311] Table 26. Relative expression of mAR in mouse CNS and muscle tissue in Example 15.

[0312] In both the CNS and muscle tissue, groups 2–7 showed a reduction in mAR up 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 muscle.

[0313] Example 16. In vivo knockdown of AR in transgenic Tg B-hTfr1 mice.

[0314] The AR RNAi reagent was evaluated in vivo in mice. On days 1 and 2, five (n=5) female B-hTFR1 mice were administered saline or the AR RNAi reagent (prepared in saline at 1.5 mg / kg body weight) via subcutaneous (SC) injection at a total dose volume of 250 μL / 25 g. Dosage was performed according to Table 27 below.

[0315] Table 27. Drug administration groups in mice of Example 16 B-hTFR1 mice, also known as C57BL / 6-Tfrc tm1(TFRC)Bcgen / Bcgen mice (Biocytogen) have exon 4-19 of the mouse Tfr1 gene, which encodes the extracellular region, replaced by exon 4-19 of the human TFR1 gene.

[0316] Each AR RNAi reagent comprises a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand, the targeting ligand comprising an antigen-binding moiety having a modified sequence as shown in the double-stranded structure herein (for specific modification and structural information (including the antigen-binding moiety) associated with AR RNAi reagents, see Tables 3, 4, 5, 6, 7, 8, 9 and 10).

[0317] Mice were euthanized on day 15. Thoracic spinal cord, cerebellum, striatum, and cortex were harvested and collected for analysis. mAR mRNA transcript expression was analyzed by qPCR, with mPPIA as an endogenous gene, and normalized for group 1 mice treated with saline. mAR expression data are shown in Table 28 below.

[0318] Table 28. Relative expression of mAR in the thoracic spinal cord, cerebellum, striatum and cortex of mice in Example 16.

[0319] In the thoracic spinal cord, cerebellum, cortex, and striatum, groups 2–7 showed a reduction in mAR up to at least day 15. Most notably, two doses of 1.5 mg / kg AC006895 achieved ~76% inhibition (0.237) in the cerebellum.

[0320] Other implementation plans It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A compound of formula (I): (I)， Or its pharmaceutically acceptable salt, wherein: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a Or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and If the chemical valence allows, n can be 0 or 1.

2. The compound according to claim 1, wherein m is 0.

3. The compound according to claim 1, wherein m is 1.

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

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

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

7. The compound according to any one of claims 1, 3, 4 and 6, wherein the compound has formula (Ia): (I-a), Or its pharmaceutically acceptable salt.

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

9. The compound according to any one of claims 1, 2, 5 and 8, wherein the compound has the formula (Ib): (I-b), Or its pharmaceutically acceptable salt.

10. The compound according to claim 9, wherein Y is a substituted or unsubstituted heteroaryl group.

11. The compound according to claim 9 or 10, wherein Y has the following formula: ,or .

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

13. The compound according to any one of claims 9-12, wherein Z is a divalent phenyl group.

14. The compound according to any one of claims 9-13, wherein Z has the following formula: .

15. The compound according to any one of claims 9-14, wherein R 1 –C(O)NHR 1a .

16. The compound according to any one of claims 1-15, wherein R 1a It is a substituted or unsubstituted C1-C6 alkyl group.

17. The compound according to any one of claims 1-16, wherein R 1a The methyl group can be substituted or unsubstituted.

18. The compound according to any one of claims 1-17, wherein R 1a The methyl group is unsubstituted.

19. A compound of formula (II): (II), Or its pharmaceutically acceptable salt, wherein: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; p is 0 or 1; and X is a leaving group.

20. A compound of formula (III): (III), Or its pharmaceutically acceptable salt, wherein: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 3 The reactive part; p is 0 or 1; and X is a leaving group.

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

22. The compound according to any one of claims 19-21, wherein X is –SO2Me.

23. The compound according to any one of claims 20-22, wherein R 3 It is an activated ester.

24. The compound according to any one of claims 20-23, wherein R 3 It has the following formula: .

25. The compound according to any one of claims 20-22, wherein R 3 It is an azide.

26. The compound according to any one of claims 1-25, wherein Y 1 For substituted or unsubstituted heteroaryl groups.

27. The compound according to any one of claims 1-26, wherein Y 1 It is either a substituted or unsubstituted benzothiazole or a substituted or unsubstituted oxadiazole.

28. The compound according to any one of claims 1-27, wherein Y 1 It has the following formula: ,or .

29. The compound according to any one of claims 1-28, wherein L 1 For connectors containing PEG chains.

30. The compound according to any one of claims 1-29, wherein L 1 It contains 1-10 PEG units.

31. The compound according to any one of claims 1-30, wherein L 1 It has the following formula: 。 32. The compound according to any one of claims 1-28, wherein L 1 It does not exist.

33. The compound according to any one of claims 1-32, wherein L 2 It has the following formula: ,or .

34. The compound according to any one of claims 1-32, wherein L 2 It does not exist.

35. The compound according to any one of claims 1-34, wherein Z 1 It is a divalent aryl group or a trivalent aryl group.

36. The compound according to any one of claims 1-35, wherein Z 1 It is a divalent phenyl group or a trivalent phenyl group.

37. The compound according to any one of claims 1-36, wherein Z 1 It has the following formula: 。 38. The compound according to any one of claims 1-34, wherein Z 1 An alkyl or heteroalkyl chain containing branches.

39. The compound according to any one of claims 1-34 or 38, wherein Z 1 A heteroalkyl chain containing branches.

40. The compound according to any one of claims 1-34 or 38-39, wherein Z 1 It has the following formula: 。 41. The compound according to any one of claims 1-34, wherein Z 1 It does not exist.

42. A compound comprising an antibody or an antibody fragment; and a structure selected from the following: Or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and This indicates the attachment point to the antibody or antibody fragment.

43. A compound with the following formula: Or its pharmaceutically acceptable salt, wherein: L 2 If it is not present or is a connector, R 2 Includes oligonucleotide-based reagents, and X is a leaving group.

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

45. The compound according to claim 43 or 44, wherein X is –SO2Me.

46. ​​The compound according to any one of claims 42-45, wherein L 2 It has the following formula: ,or .

47. The compound according to any one of claims 42-45, wherein L 2 It does not exist.

48. The compound according to any one of claims 1-19 or 26-47, wherein R 2 Includes RNAi reagents.

49. The compound according to any one of claims 1-19 or 26-48, wherein R 2 It contains a sense chain with one or two reverse debasing residues.

50. The compound according to any one of claims 1-19 or 26-49, wherein R 2 It contains a sense chain with two reverse debasing residues.

51. The compound according to any one of claims 1-19 or 26-50, wherein R 2 It contains a sense chain with one or two reverse debasing residues.

52. The compound according to claim 50 or 51, wherein the Fab is connate with the 5' end of the sense chain.

53. A compound having the following structure: Or its pharmaceutically acceptable salt, wherein: A This indicates the connection point between the structure and the antibody or antibody fragment, and R This indicates the connection point between the structure and the portion containing the RNAi reagent.

54. A compound with the following formula: Or its salt.

55. A method for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof: (I)， in: Fab is an antibody or antibody fragment; R 1 For –OR 1a –C(O)NHR 1a 、or –NHC(O)R 1a , where R 1a It is a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group; Z is a divalent or trivalent aryl group, or a divalent or trivalent heteroaryl group; Y is a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Y 1 Each case is independently a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; m is 0 or 1; and n can be 0 or 1, provided that the chemical valence allows it; The method comprises reacting a compound of formula (II) or a pharmaceutically acceptable salt thereof with an antibody containing a cysteine ​​residue: (II), in: Z 1 It is absent, a divalent or trivalent aryl group, a divalent or trivalent heteroaryl group, a branched alkyl chain, or a branched heteroalkyl chain; L 1 For connectors that do not exist or contain PEG chains; L 2 If it is not present or is a connector; R 2 Reagents containing oligonucleotides; p is 0 or 1; and X is a leaving group.

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