Lysosome-targeted bifunctional molecules for the degradation of autoantibodies

Abstract

The present disclosure provides bifunctional molecules comprising a moiety that targets a lysosome-targeting molecule, a linker, and a polypeptide that specifically binds to an extracellular target molecule, such as an antibody, that is targeted for degradation. The present disclosure provides a class of lysosome-targeting bifunctional molecules comprising a ligand moiety that specifically binds to a lysosome-targeting receptor (also called a cellular internalization receptor) on the surface of a cell, a linker, and a polypeptide that specifically binds to the extracellular target molecule. The inventors have demonstrated that bifunctional molecules (also called conjugates) of the present disclosure, having a specific arrangement of ligand-binding moieties (X) with linkers of desired valency and / or length, can simultaneously specifically bind to both an internalization receptor and an extracellular target molecule, such as an autoantibody, with high affinity and exhibit high uptake activity of the target molecule. The conjugates of the present disclosure enable the sequestration and degradation of the target molecule in the lysosomes of the cell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 480,509, filed January 18, 2023, and U.S. Provisional Application No. 63 / 518,537, filed August 9, 2023, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Many therapeutic agents act by binding to functionally important sites on target proteins, thereby modulating the activity of those proteins, or by recruiting immune effectors to act on target proteins, as with many monoclonal antibody drugs. However, there exists an untapped reservoir of medically important human proteins that are considered "undruggable" because they are not readily adaptable to currently available therapeutic targeting approaches.

[0003] Autoimmune diseases occur when the body's immune response mistakenly targets an individual's tissues, ultimately leading to organ destruction or dysfunction. Autoantibodies recognize self-antigens and are often associated with autoimmune diseases. Conventional treatments for autoimmune diseases can control excessive immune responses and reduce inflammation or pain, but often cause unwanted side effects.

[0004] Many therapeutic agents act by binding to functionally important sites on target proteins, thereby modulating the activity of those proteins, or by recruiting immune effectors to act on target proteins, as with many monoclonal antibody drugs. However, there exists an untapped reservoir of medically important human proteins that are considered "undruggable" because they are not easily adaptable to currently available therapeutic targeting approaches. Therefore, there is a need for therapeutics that can target a broader range of proteins.

[0005] Lysosomal targeting chimeras (LYTACs) are bifunctional molecules that enable selective protein degradation through binding to cell surface receptors. The first LYTACs targeted extracellular proteins for degradation via binding to the cation-independent mannose-6-phosphate receptor (CI-M6PR) or the asialoglycoprotein receptor (ASGPR). ASGPR is a transmembrane glycoprotein receptor found primarily in hepatocytes and plays a key role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues. ASGPR cycles between endosomes and the cell surface. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a class of lysosome-targeted bifunctional molecules that include a ligand portion that specifically binds to a lysosome-targeting receptor (also called a cellular internalization receptor) on the surface of a cell, a linker, and a polypeptide that specifically binds to an extracellular target molecule.

[0007] The present inventors have demonstrated that bifunctional molecules (also called conjugates) of the present disclosure, which have a specific arrangement of ligand-binding moieties (X) with linkers of desired valency and / or length, can simultaneously bind specifically to both internalizing receptors and extracellular target molecules such as autoantibodies with high affinity and exhibit high uptake activity of the target molecules. The conjugates of the present disclosure enable the sequestration and degradation of target molecules in cellular lysosomes.

[0008] Lysosomal-targeting bifunctional molecules bind to an extracellular target molecule via a target-binding polypeptide and to a cellular internalization receptor via a ligand moiety. Binding of the ligand moiety to the cellular internalization receptor can trigger internalization of the bifunctional molecule and the bound target molecule. The bifunctional molecules described herein can promote intracellular transport of the target autoantibody and can promote sequestration and / or degradation of the target autoantibody in the lysosomes of the cell. In some embodiments, the extracellular target molecule is an autoantibody, and the polypeptide of the bifunctional molecule is a protein antigen of the autoantibody.

[0009] Also provided herein are compositions comprising such bifunctional molecules, as well as methods of using the bifunctional molecules to target extracellular target molecules of interest for sequestration and / or lysosomal degradation. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows an exemplary mechanism of action of an exemplary bifunctional molecule (e.g., a target-binding conjugate) that binds to a lysosomal targeting receptor (also called a cellular internalization receptor) on the surface of a cell, facilitating transport into the cell and facilitating sequestration and / or degradation of a target molecule (e.g., an autoantibody) in the lysosomes of the cell. [Figure 2] 2A, 2B, 2C, and 2D are schematic diagrams of various representations of immunogenic domains that can bind to antibodies. These domains can be designed and expressed according to various embodiments of the present disclosure as described herein, as gene fusions containing N / C-terminal tags including human Fc (FIGS. 2A and 2B), human serum albumin (FIG. 2C), and human serum albumin containing 6x-poly-histidine (FIG. 2D). [Figure 3] 1A and 1B show the in vitro uptake of patient-derived autoantibodies in human hepatocellular carcinoma (HepG2) cells using the target-binding conjugates described herein. [Figure 4]1A, B, C, and D show selective autoantibody uptake by exemplary target-binding conjugates. [Figure 5] A, B, and C show the ability of MuSK conjugates to mediate lysosomal delivery of patient-derived pathogenic anti-MuSK antibodies. [Figure 6] A and B show the ability of the conjugate to clear patient-derived antibody 13-3B5 from mouse serum. [Figure 7] A and B show the in vitro uptake of patient-derived autoantibodies in human hepatocellular carcinoma (HepG2) cells using a thermostable TSHR mutant-based target-binding conjugate with an Fc carrier polypeptide described herein. [Figure 8] A and B show the uptake of patient-derived autoantibodies in human HepG2 cells using target-binding conjugates based on thermostable TSHR mutants fused to HSA as described herein. [Figure 9] A and B show AChR conjugate-mediated cellular uptake of patient-derived antibody MAB637 and pathogenicity tool antibody MAB35. [Figure 10] FIG. 1 shows the ability of AChR conjugates to induce degradation of pathogenic antibodies in Hep G2 cells. [Figure 11] A and B show BP180 conjugate-mediated cellular uptake of patient-derived antibody 3-30G. [Figure 12] FIG. 1 shows the ability of BP180 conjugates to induce degradation of the pathogenic antibody 3-30G in Hep G2 cells. [Figure 13] A and B show PR3 conjugate-mediated cellular uptake of pathogenic antibody 4A5. [Figure 14] FIG. 1 shows the ability of PR3 conjugates to induce degradation of the pathogenic antibody 4A5 in Hep G2 cells. DETAILED DESCRIPTION OF THE INVENTION

[0011] Lysosome-targeting bifunctional molecules As summarized above, the present disclosure provides lysosomal-targeting bifunctional molecules (also referred to as LYTACs) that target disease-causing extracellular target molecules, such as autoantibodies, for degradation. The lysosomal-targeting bifunctional molecules comprise a ligand moiety that specifically binds to a lysosomal targeting receptor (i.e., an internalization receptor), which is linked to a bait polypeptide that specifically binds to a target molecule (e.g., an autoantibody). In certain embodiments, the lysosomal targeting receptor is a lysosomal cell surface receptor (e.g., an asialoglycoprotein receptor [ASGPR] or a cation-independent mannose-6-phosphate receptor [CI-M6PR]).

[0012] In some embodiments, LYTACs bind to circulating pathogenic autoantibodies and form a ternary complex with a cellular internalizing receptor (e.g., liver-specific internalizing receptor, ASGPR). After clathrin-mediated endocytosis, the protein complex proceeds through the endocytic pathway, whereby the cellular internalizing receptor (e.g., ASGPR) regulates pH and Ca. 2+ Upon decreasing levels, they dissociate and are recycled to the cell surface. LYTACs and pathogenic antibodies subsequently progress to lysosomes where they are degraded by lysosomal proteases.

[0013] The present inventors have demonstrated that bifunctional molecules (e.g., conjugates) of the present disclosure having a specific arrangement of ligand-binding moieties (X) with linkers of desired valency and / or length can simultaneously specifically bind to both an internalizing receptor and a target autoantibody with high affinity and exhibit high uptake activity of the target autoantibody. The conjugates of the present disclosure can enable sequestration and degradation of the target autoantibody in cellular lysosomes.

[0014] Accordingly, the present disclosure includes an autoantibody-degrading bifunctional molecule of formula (I), or a prodrug thereof, or a pharmaceutically acceptable salt thereof: [ka] (I) During the ceremony, X is a moiety that binds to a lysosomal targeting molecule; n is 1 to 20 (e.g., 1 to 10, 1 to 6, or 1 to 4 for M6PR ligands, or 1 to 3 for GalNAc ligands, for example); L is a linker, m is 1 to 10 (e.g., 1 to 6 or 1 to 4, where m can be a discrete loading or an average loading (i.e., ligand-to-polypeptide (LPR) ratio)); Y is any carrier polypeptide; B is a polypeptide that specifically binds to an extracellular target molecule (eg, an autoantibody).

[0015] In some embodiments of Formula (I), the bifunctional molecule is of Formula (Ia): [ka] (Ia) During the ceremony, Z is a residue moiety resulting from the covalent bond between the chemoselective ligation group of the linker and the compatibility group of YB; The linker L may be any linking moiety L 1 , L 2 and L 3 which together form a linear or branched linker between X and YB, wherein L 1 and L 3 are independently linear linking moieties, and L 2 is a branched linking moiety, a, b, and c are independently 0 or 1; n is 1, 2, or 3; When n is 1, b is 0 and at least one of a and c is 1; When n is 2 or 3, a, b, and c are each 1; m is bonded to YB (X n-L) moieties, where m is in the range of about 1 to about 80 (e.g., m is 1 to 20, 1 to 10, 1 to 8, 2 to 8, 3 to 6, or 4 to 5, or m is 1 to 3, e.g., 1, 2, or 3).

[0016] In certain embodiments of Formula (Ia), Z is a residue moiety resulting from the covalent bond between a thiol-reactive chemoselective ligation group and one or more cysteine ​​residues of YB, or Z is a residue moiety resulting from the covalent bond between an amine-reactive chemoselective ligation group and one or more lysine residues of YB.

[0017] In some embodiments, the target-binding conjugate is of formula (II'), or a prodrug thereof, or a salt thereof: [ka] (II') During the ceremony, n is 1 to 3, m is 1 to 3; X, Y, and B are each independently as defined herein; L 1 ~L 6 are each independently a linking moiety, and together they form Z 1 and Y, forming a linear or branched linker therebetween; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5.

[0018] In certain embodiments of Formulas (I)-(Ia), Y is absent and the linker is directly linked to polypeptide B. In certain embodiments of Formulas (I)-(Ia), the carrier polypeptide Y is present and the linker is directly linked to Y and / or polypeptide B.

[0019] In certain embodiments of formula (Ia), YB is a chimeric fusion protein comprising a carrier polypeptide and a polypeptide that specifically binds to a target autoantibody.

[0020] Each of the components of the lysosome-targeting bifunctional molecules, the preferred configuration of such molecules, and methods of their use are now described in more detail.

[0021] Lysosomal targeting moiety Lysosomal targeting receptors are cell surface receptors that allow for internalization of the conjugated compounds of the present disclosure.

[0022] In some embodiments, the lysosomal targeting receptor is selected from asialoglycoprotein receptor (ASGPR), cation-independent mannose-6-phosphate receptor (CI-M6PR, also referred to herein as M6PR), folate receptor, CD63, sortilin, IFITM3, molecules in the endosomal / lysosomal pathway, LIMP-1, and LIMP-2. In some embodiments, the lysosomal targeting receptor is ASGPR. In some embodiments, the lysosomal targeting receptor is CI-M6PR. In some embodiments, the lysosomal targeting receptor is a folate receptor.

[0023] A variety of ligand moieties that bind to lysosomal targeting molecules can be utilized in the conjugate compounds of the present disclosure.

[0024] ASGPR-binding compounds and conjugates are described in International Publication WO2023 / 288033, filed July 14, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0025] CI-M6PR binding compounds and conjugates are described in International Publication WO2023 / 288015, filed July 14, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0026] ASGPR-binding compounds and conjugates, or CI-M6PR-binding compounds and conjugates, are described in International Publication WO2022 / 142377, filed January 8, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0027] Folate receptor binding compounds and conjugates are described in International Publication WO2022 / 150721, filed January 10, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0028] ASGPR ligand An asialoglycoprotein receptor (ASGPR) ligand moiety is a moiety that binds to ASGPR (i.e., also referred to as an ASGPR-binding moiety) and promotes internalization of the bifunctional molecule of which it is a part, and any bound target molecule (e.g., an autoantibody), via ASGPR. The ASGPR ligand moiety of the present disclosure can be linked to a polypeptide construct via a linker without affecting the specific binding to or function of cell-surface ASGPR. The present inventors have demonstrated, for example, in the methods of the present disclosure, that lysosomal-targeted bifunctional molecules of the present disclosure comprising one or more linked ASGPR ligand moieties can exploit the function of cell-surface ASGPR in a biological system, for example, for internalization and / or sequestration of autoantibodies in cellular lysosomes and subsequent lysosomal degradation.

[0029] ASGPR, also known as the Ashwell-Morell receptor, is a transmembrane glycoprotein receptor found primarily in hepatocytes that mediates the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues. ASGPR cycles between intracellular endosomes and the cell surface. In some embodiments, the ASGPR is Homo sapiens asialoglycoprotein receptor 1 (ASGR1) (see, e.g., NCBI Reference Sequence: NM_001197216).

[0030] In some embodiments, the ASGPR-binding moiety (X) comprises an amino sugar ring analog of galactose (e.g., N-acetylgalactosamine, or an analog thereof), which is linked to a linker scaffold via an optional linking moiety at the 1-, 2-, or 6-position of the sugar ring analog. In some embodiments, the linking moiety comprises an oxygen, sulfur, nitrogen, or carbon atom linked to the 1-position of the ring. In some embodiments, the linking moiety comprises an oxygen, sulfur, nitrogen, or carbon atom linked to the 2-position of the ring. In some embodiments, the linking moiety comprises an oxygen, sulfur, nitrogen, or carbon atom linked to the 1-position of the ring. In certain embodiments, the linking moiety linked to the 1-, 2-, or 6-position of the ring comprises an optionally substituted aryl or heteroaryl group. In certain embodiments, the amino sugar ring analog of galactose has a bicyclic structure.

[0031] ASGPR ligand moieties of interest that may be adapted for use in the conjugates of the present disclosure are described in International Application No. WO2023 / 288033, filed July 14, 2022, the disclosure of which is incorporated herein by reference in its entirety. Preferred ASGPR ligand moieties of interest are described below.

[0032] In some embodiments, the ASGPR ligand portion of the bifunctional molecule (e.g., (XL) of Formula (I) n ) has an affinity (K ) of 300 nM or less, e.g., 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, or 1 nM or less. d ) specifically binds to ASGPR. In this context, the terms "bind," "bind," "specifically bind," or "specifically bind to" are used interchangeably.

[0033] In some embodiments of the conjugates described herein, X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II): [ka] (II) During the ceremony, R 1 -Z 1 selected from -*, -H, -OH, optionally substituted (C1-C6)alkyl, -OCH3, -OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R 2 -Z 1 -*, -NHCOCH3, -NHCOCF3, -NHCOCH2CF3, -OH, -NHR, and optionally substituted triazole; R 6 -Z 1 -*, -OH, -OR, optionally substituted (C1-C6) alkyl, -OC(O)R, -C(O)NHR, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl, -NHCOR, and -NRCOR; each R is independently an optionally substituted (C1-C6) alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C1-C6) alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl; R 1 , R 2 , and R 6 One of them is -Z 1 -*, and "*" is Z 1 represents the point of attachment to the linker (L), R 3 and R 4 are each independently H or a promoiety, or R 3 and R 4 are linked in a ring to form a promoiety, R 11 is H or a bridging moiety connecting the 5-carbon to the 1-carbon of the ring, Z1 -Z 11 -, -Z 11 -A 1 -, -A 2 -, -NR 21 CO-, -CONR 21 -, -NR 21 SO2-, -SO2NR 21 -, -NR 21 C(=O)NR 21 - and -NR 21 C(=S)NR 21 - a linking moiety selected from -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 )2, -A 1 -and-A 2 - is an optionally substituted heterocyclylene, an optionally substituted arylene, or an optionally substituted heteroarylene; Each R 21 are independently selected from H, optionally substituted (C1-C6) alkyl, -COR, and optionally substituted heteroaryl; Each R 22 are independently selected from H, halogen, and optionally substituted (C1-C6) alkyl.

[0034] In some embodiments, X is represented by formula (a-II). [ka] (a-II)

[0035] In some embodiments, a lysosome-targeting bifunctional molecule of the present disclosure (e.g., Formulas (I)-(Ia)) can include an ASGPR ligand moiety of formula (II): [ka] (II) During the ceremony, R 1 -Z 1selected from -*, -H, -OH, -CH3, -OCH3, and -OCH2CH=CH; R 2 -Z 1 -*, -NHCOCH3, -NHCOCF3, -NHCOCH2CF3, -OH, and optionally substituted triazole; R 6 -Z 1 -*, -OH, -OC(O)R, -C(O)NHR, and optionally substituted triazole, where R is optionally substituted (C1-C6) alkyl or optionally substituted aryl; R 1 , R 2 , and R 6 One of them is -Z 1 -*, and "*" is Z 1 represents the point of attachment to the linker (L), R 3 and R 4 are each independently H or a promoiety, or R 3 and R 4 are linked in a ring to form a promoiety, R 11 is H or a bridging moiety connecting the 5-carbon to the 1-carbon of the ring, Z 1 -Z 11 -, -Z 11 -A 1 -, -A 2 -, -NR 21 CO-, -CONR 21 -, -NR 21 SO2-, -SO2NR 21 -, -NR 21 C(=O)NR 21 - and -NR 21 C(=S)NR 21 - a linking moiety selected from -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 )2, -A 1 -and-A 2- is an optionally substituted arylene or an optionally substituted heteroarylene; Each R 21 are independently selected from H and optionally substituted (C1-C6) alkyl; Each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl.

[0036] In some embodiments of Formula (II), i) When n is 3, R6 is OH, R2 is -NHCOCH3, R3 to R4 are H, and R1 is Z1, Z1 is not O, ii) n is 2 or 3, R 6 OAc, R 2 -NHCOCH3, R 3 ~R 4 is Ac, R 1 Z 1 If Z 1 is not O, iii) n is 2 or 3, R 6 Obz, R 2 -NHCOCH3, R 3~ R 4 Bz, R 1 Z 1 In the case of Z 1 is not O, iv) n is 3, R 6 OH, R 2 -NHCOCH3, R 3 ~R 4 H, R 1 Z 1 , Z 11 If is O, L contains a backbone of at least 16 consecutive atoms up to the branch point, v) n is 3, R 6 Z 1 (Z 1 is O), R 3~ R 4 If H, then R 1 is not -CH3-OCH3 or -OCH2CH=CH, vi)R 11is a group of formula -CH2O- that bridges (i.e., cyclically joins) the 1-carbon atom on the sugar ring, and R 2 is -NHCOCH3 and R 3 ~R 4 If is H, then R 1 and R 3 is Z 1 isn't it.

[0037] In some embodiments, R 1 -Z 1 -*, -H, or (C1-C6) alkyl. In some embodiments, R 1 -Z 1 -*, -H, or n-propyl.

[0038] In some embodiments, R 2 -Z 1 -* or -NHCOCH3.

[0039] In some embodiments, R 3 and R 4 are -H, respectively.

[0040] In some embodiments, L contains 10 to 60 consecutive branched or straight chain atoms.

[0041] In some embodiments, L is of formula (IIb'): [ka] (IIb') During the ceremony, L 1 ~L 6 are each independently a linking moiety, and together they form Z 1 and Y, forming a linear or branched linker therebetween; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** is Z 1 X's L via 1 represents the point of attachment to *** represents the point of attachment to Y.

[0042] In some embodiments, L is of formula (IIb'): [ka] (IIb') During the ceremony, n is 1, 2, or 3; L 1 ~L 6 are each independently a linking moiety, and together they form Z 1 and Y, forming a linear or branched linker therebetween; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** is Z 1 X's L via 1 represents the point of attachment to *** represents the point of attachment to Y.

[0043] In some embodiments, L1-L5 are each independently -C 1~20 -Alkylene-, -NHC(O)-C 1~6 -Alkylene-, -C(O)NH-C 1~6 -Alkylene-, -NH-C 1~6 -Alkylene-, -NHC(O)NH-C 1~6 -Alkylene-, -NHC(S)NH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHC(O)-, -C 1~6 -Alkylene-C(O)NH-, -C 1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHC(O)NH-, -C 1~6 -Alkylene-NHC(S)NH-, -O(CH2) p -, -(OCH2CH2) p L includes one or more linking moieties independently selected from -, -NHC(O)-, -C(O)NH-, -NHS(O)2-, -S(O)2NH-, -C(O)-, -S(O)2-, -O-, -S-, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic carbocycle, an amino acid residue, -NH-, and -NMe-;1 ~L 5 are each independently optionally substituted with 1 to 5 halo; each p is independently 1 to 50; L 6 -C 1~20 -Alkylene-, -NR 16 C(O)-C 1~6 -Alkylene-, -C(O)NR 16 -C 1~6 -Alkylene-, -NR 16 -C 1~6 -Alkylene-, -NR 16 C(O)NR 16 -C 1~6 -Alkylene-, -NR 16 C(S)NR 16 -C 1~6 -Alkylene-, -C 1~6 -Alkylene-NR 16 C(O)-, -C 1~6 -Alkylene-C(O)NR 16 -, -C 1~6 -Alkylene-NR 16 -, -C 1~6 -Alkylene-NR 16 C(O)NR 16 -, -C 1~6 -Alkylene-NR 16 C(S)NR 16 -, -O(CH2) p -, -(OCH2CH2) p -, -NR 16 C(O)-, -C(O)NR 16 -, -NHS(O)2-, -S(O)2NH-, -C(O)-, -S(O)2-, -O-, -S-, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, amino acid residue, or -NR 16 - is a linking group comprising one or more linking moieties independently selected from Each R 16 are independently —H, optionally substituted (C1-C6) alkyl, optionally substituted aryl, optionally substituted monocyclic heteroaryl, or monocyclic heteroaryl.

[0044] In some embodiments, L1-L5 are each -C1-20-alkylene-, -NHC(O)-C 1~6 -Alkylene-, -C(O)NH-C 1~6 -Alkylene-, -NH-C 1~6 -Alkylene-, -NHC(O)NH-C 1~6 -Alkylene-, -NHC(S)NH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHC(O)-, -C 1~6 -Alkylene-C( )NH-, -C 1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHC(O)NH-, -C 1~6 -Alkylene-NHC(S)NH-, -O(CH2) p -, -(OCH2CH2) p L is independently selected from -, -NHC(O)-, -C(O)NH-, -NHS(O)2-, -S(O)2NH-, -C(O)-, -S(O)2-, -O-, -S-, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic carbocycle, an amino acid residue, -NH-, and -NMe-; 1 ~L 5 are each independently optionally substituted with 1 to 5 halo; each p is independently 1 to 50; L 6 teeth, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] R z teeth [ka] [ka] or [ka] is.

[0045] 1. Linked ASGPR Ligand Moiety In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IIa): [ka] (IIa) In the formula, R 2 , R 3 , R 4 , R 6 , and Z 1 is as defined herein. In some embodiments of Formula (IIa), R 6 is selected from —OH, —OC(O)R, and —C(O)NHR; R 2 is selected from -NHCOCH3, -NHCOCF3, and -NHCOCH2CF3.

[0046] In some embodiments of Formula (II), Z 1 is in the beta configuration and can be represented by formula (IIa-1). [ka] (IIa-1)

[0047] In some embodiments of Formula (II), Z 1 is in the alpha configuration and can be represented by formula (IIa-2). [ka] (IIa-2)

[0048] In certain embodiments of formula (IIa), (IIa-1), or (IIa-2), Z 1 Ha-Z 11 -A 1 -, where A 1 - is an optionally substituted arylene or an optionally substituted heteroarylene. 1 is an optionally substituted heteroarylene. In certain cases, the heteroarylene is a 5- or 6-membered heteroarylene. In certain cases, the heteroarylene is a 5-membered heteroarylene. In certain cases, the 5-membered heteroarylene is a triazole. In certain cases, the triazole is a 1,2,3-triazole moiety.

[0049] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IIIa) or (IIIb): [ka] [ka] During the ceremony, -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 )2-, where each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl; R 21 is H or optionally substituted (C1-C6) alkyl; -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.

[0050] In some embodiments of formula (IIIa) or (IIIb), Z 11 is -S-.

[0051] In some embodiments, Z 11 is -C(R 22 )2-. In some embodiments, Z 11 is -CH2-.

[0052] In certain embodiments, Z 11 -C(R 22 )2, where at least one R 22 is H. In certain cases, both R 22 is H. In certain embodiments, Z 11 is —O—. In certain embodiments, R 11 is -S-. In certain embodiments, Z 11 -N(R 21 ), where R 21 is H or (C1-C3) alkyl.

[0053] In certain embodiments, -A 1 - is a triazole.

[0054] In certain embodiments, Z 1 -C(R 22 )2-triazole-. In certain embodiments, Z 1 teeth, [ka] In certain embodiments, Z 1 teeth, [ka] is.

[0055] In certain embodiments of formula (IIa), (IIa-1), or (IIa-2), Z 1 is Z 11 In certain cases, Z 11 -C(R 22 )2. In certain cases, at least one R 22 is H. In certain cases, both R 22 is H and Z 11 is -CH2-. In certain cases, Z 11 is -O-. In certain cases, Z 11 is -S-. In certain other cases, Z 11 -N(R 21 ), where R 21 is H or (C1-C3) alkyl.

[0056] In certain embodiments of formula (IIa), (IIa-1), or (IIa-2), Z 1 is a monocyclic 5- or 6-membered heteroaryl or aryl. In certain cases, Z 1 teeth, [ka] In certain cases, Z 1 teeth, [ka] is.

[0057] In certain embodiments of formula (IIa), (IIa-1), or (IIa-2), Z 1 -O-, -S-, -C(R 22 )2-, -NR 21 -,-CONR 21 - and [ka] is selected from During the ceremony, X 1 is O or S, t is 0 or 1, R 21 and each R 23 is H and optionally substituted (C1-C6) alkyl (e.g., C such as methyl). (1~3) -alkyl), Each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl.

[0058] In certain embodiments of formula (IIa), (IIa-1), or (IIa-2), Z 1 is an optionally substituted (C1-C6) alkyl. Z 1 In some cases, the alkyl is methyl. 1 In some cases, the alkyl is ethyl. 1 In some cases, the alkyl is propyl. 1 In some cases, the alkyl is butyl. 1 In some cases, the alkyl is pentyl. 1 In some cases, the alkyl is hexyl.

[0059] In certain embodiments, the ASGPR binding moiety (X) of formula (IIa-1) is selected from one of the following structures: [ka] [ka] [ka] [ka] and [ka]

[0060] In some embodiments of Formula (IIa-2), Z 1 is in the beta configuration, and X is of formula (IIIb-2), [ka] In the formula, -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.

[0061] In some embodiments of formula (IIIb-2), A 1 In some embodiments of formula (IIIb-2), X is a group represented by formula (X A -4).

[0062] In some embodiments of Formula (IIa-1), Z 1 is in the beta configuration at the 1-carbon of the galactosamine ring. In some embodiments of Formula (IIa-1), Z 1 is S, and each X is a formula (X A In some embodiments of formula (IIa-1), each X is of formula (X A In some embodiments of formula (IIa-1), each X is of formula (X A In some embodiments of formula (IIa-1), each X is of formula (X AIn some embodiments of formula (IIa-1), each X is of formula (X A -5).

[0063] In certain embodiments, the compound of formula (IIa-2) is selected from one of the following structures: [ka] [ka] [ka] [ka] and [ka]

[0064] In some embodiments of formula (IIa-2), each X is represented by the formula (X B -1).

[0065] In some embodiments of formula (IIa-2), each X is represented by the formula (X B -2).

[0066] In some embodiments of formula (IIa-2), each X is represented by the formula (X B -3).

[0067] In some embodiments of formula (IIa-2), each X is represented by the formula (X B -4).

[0068] In some embodiments of Formula (IIa-2), Z 1 is in the alpha configuration, and X is of formula (IIIb-1), [ka] In the formula, -A 1- is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.

[0069] In certain embodiments of formula (IIIb-1), A 1 is an optionally substituted heteroarylene. In certain cases, the heteroarylene is a 5- or 6-membered heteroarylene. In certain cases, the heteroarylene is a 5-membered heteroarylene. In certain cases, the 5-membered heteroarylene is a triazole. In certain cases, the triazole is a 1,2,3-triazole moiety.

[0070] In certain embodiments, X in formula (IIIb-1) is selected from one of the following structures: [ka]

[0071] In some embodiments of formula (IIIb-1), each X is represented by the formula (X C -1).

[0072] In some embodiments of formula (IIIb-1), each X is represented by the formula (X C -2).

[0073] Exemplary ligand moieties that bind to ASGPR and synthons thereof that can be utilized in the compounds of the present disclosure are shown in Tables 1 to 5. In certain embodiments, the compound of formula (IIa) is a compound shown in Table 1. [Table 1]

[0074] In some embodiments of any one of X1 to X5.1, Z 1 is in the alpha configuration, so that the ASGPR binding moieties X1 to X5.1 are derived from formula (IIa-2). [ka]

[0075] Two linked ASGPR ligand moieties In some embodiments, the ASGPR binding moiety (X) is linked via position 2 of the sugar analog. In some embodiments, the ASGPR binding moiety (X) has a reduced ring carbon at position 1 relative to the galactosamine-derived sugar. In some embodiments, the ASGPR binding moiety (X) of the bifunctional molecule of the present disclosure is represented by formula (IIb): [ka] In the formula, R 1 , R 3 , R 4 , R 6 , R 11 , and Z 1 is as defined herein.

[0076] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure is represented by formula (IIb'): [ka] In the formula, R 3 ~R 4 , R 6 , and Z 1 is as defined herein.

[0077] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure is represented by formula (IVa): [ka] In the formula, R 1 , R 11 , and Z 1 is as defined herein.

[0078] In some embodiments of formula (IIb), (IIb'), or (IVa), Z 1 is an optionally substituted -(C(R 22 )2) q -heteroarylene and [ka] where q is 0 or 1.

[0079] In some embodiments of formula (IIb), (IIb'), or (IVa), Z 1 is an optionally substituted -(C(R 22 )2) q -triazole, and q is 0 or 1.

[0080] In some embodiments of formula (IIb), (IIb'), or (IVa), Z 1 teeth, [ka] In some embodiments, Z 1 teeth, [ka] is.

[0081] In some embodiments of formula (IIb), (IIb'), or (IVa), Z 1 teeth, [ka] where R 23 is H or C (1~3) - alkyl.

[0082] In some embodiments of formula (IIb), (IIb'), or (IVa), Z 1 Ha-NR 23 CO-, where R 23 is H or C (1~3) - alkyl.

[0083] In certain embodiments of formula (IIb), (IIb') or (IVa), Z 1 is an optionally substituted -(C(R 22 )2) q heteroaryl and [ka] where q is 0 or 1.

[0084] In certain embodiments of formula (IIb), (IIb') or (IVa), Z 1 is an optionally substituted -(C(R 22 )2) q -triazole, where q is 0 or 1. In certain cases, Z 1 teeth, [ka] is.

[0085] In certain cases of formula (IIb), (IIb') or (IVa), Z 1 teeth, [ka] where R 23 is H or C (1~3) - alkyl.

[0086] In certain cases of formula (IIb), (IIb') or (IVa), Z 1 Ha-NR 23 CO-, where R 23 is H or C (1~3) - alkyl.

[0087] In certain embodiments of formula (IIb), (IIb') or (IVa), Z 1 is a monocyclic 5- or 6-membered heteroarylene or arylene. In certain cases, Z 1 teeth, [ka] is.

[0088] In certain embodiments of formula (IIb), (IIb') or (IVa), Z 1-O-, -S-, -C(R 22 )2-, -NR 21 -,-CONR 21 - and [ka] is selected from During the ceremony, X 1 is O or S, t is 0 or 1, R 21 and each R 23 is H and optionally substituted (C1-C6) alkyl (e.g., C such as methyl). (1~3) -alkyl), Each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl.

[0089] In certain embodiments, the compound of formula (IIb), (IIb'), or (IVa) is selected from one of the following structures: [ka] [ka] [ka] In the formula, R 1A are independently H or (C 1~3 ) alkyl.

[0090] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IVb) or (IVc): [ka] [ka] During the ceremony, -Z 11 - is -O-, -S-, -N(R21 )-, or -C(R 22 )2, -A 1 -and-A 2 - is an optionally substituted arylene or an optionally substituted heteroarylene; Each R 21 are independently selected from H and optionally substituted (C1-C6) alkyl; Each R 22 is independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl. In some embodiments of Formula (IVb) or (IVc), R 1 is H.

[0091] In some embodiments, -Z 1 -* or -Z 1 -L- is [ka] [ka] or [ka] Includes:

[0092] In certain embodiments of Formula (IIb), R 11 is H and the compounds are those in Table 2. [Table 2]

[0093] In certain embodiments, the compound of formula (Iib) is a compound shown in Table 3. In certain embodiments, in the compound of formula (Iib), C1 (i.e., R 1 ) is alpha. In certain embodiments, in compounds of formula (Iib), C1 (i.e., R 1 ) is the beta configuration. [Table 3-1] [Table 3-2]

[0094] In certain embodiments, the compound of formula (Id') is a compound shown in Table 4. [Table 4]

[0095] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IVb-1) or (IVc-1): [ka] [ka] In the formula, R 11 is the bridging moiety connecting the 5-carbon to the 1-carbon.

[0096] In some embodiments of formula (IVb) or (IVb-1), Z 11 -C(R 22 )2. In certain cases, at least one R 22 is H. In certain cases, both R 22 is H. In certain cases, Z 11 is -O-. In certain cases, Z 11 is -S-. In certain cases, Z 11 -N(R 21 ), where R 21 is H or (C1-C3) alkyl.

[0097] In certain embodiments of formula (IVb), (IVc), (IVb-1) or (IVc-1), -A 1 -and-A 2Each - is independently an optionally substituted heteroarylene. In certain cases, the heteroarylene is a 5- or 6-membered heteroarylene. In certain cases, the heteroarylene is a 5-membered heteroarylene. In certain cases, the heteroarylene is a 6-membered heteroarylene.

[0098] In some embodiments of formula (IVb) or (IVb-1), A 1 The ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. 1 The ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. 1 The ring is a triazole. 1 The ring is pyridine. 1 The ring is a pyrimidine. 1 The ring is a thiadiazole. 1 The ring is a 5- or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. 1 The ring is further substituted with one or more substituents selected from halogen, (C1-C6) alkyl, and substituted (C1-C6) alkyl (eg, CF3).

[0099] In some embodiments of any one of formulas (IVc) or (IVc-1), A 2 The ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. 2 The ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. 2 The ring is a triazole. 2The ring is pyridine. 2 The ring is a pyrimidine. 2 The ring is a thiadiazole. 2 The ring is a 5- or 6-membered arylene or heteroarylene further substituted with one or more substituents. 2 The ring is further substituted with one or more substituents selected from halogen, (C1-C6) alkyl, and substituted (C1-C6) alkyl (eg, CF3).

[0100] In certain embodiments of formula (IVb) or (IVb-1), -Z 11 -A 1 - is a monocyclic 5- or 6-membered heteroarylene having one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka]

[0101] In certain embodiments of formula (IVc) or (IVc-1), -A 2 - is a monocyclic 5- or 6-membered heteroarylene of the structure: [ka]

[0102] Various substituents can be used to select specific -Z 11 -A 1 It is understood that the -Z group can be linked to adjacent linkers. In certain embodiments of formula (IVb) or (IVb-1), -Z 11 -A 1 - is a monocyclic 5- or 6-membered heteroarylene attached to a linking moiety as shown in one of the following structures: [ka] [ka] [ka] [ka] [ka] and [ka]

[0103] In certain embodiments of formula (IVc) or (IVc-1), -Z 11 -A 1 - is a monocyclic 5- or 6-membered heteroarylene attached to a linking moiety as shown in one of the following structures: [ka] and [ka]

[0104] In some embodiments of compounds of Formula (IIb) or (IVa)-(IVc), R 1is H, so that the compounds of formula (IIb) or (IVa)-(IVc) do not have a non-hydrogen substituent at the 1-position of the sugar ring.

[0105] In some embodiments, the compound of Formula (IIb) is any one of Formulas (IVd)-(IVg): [ka] [ka] [ka] and [ka] In the formula, A 1 and A 2 Ring, R 6 , R 4 , R 3 , R 11 and R 21 is as defined herein.

[0106] In some embodiments of any one of Formulas (IVd)-(IVg), A 1 The ring is a 5- or 6-membered arylene or heteroarylene. 1 The ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. 1 The ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. 1 The ring is a triazole. 1 The ring is pyridine. 1 The ring is a pyrimidine. 1 The ring is a thiadiazole. 1The ring is a 5- or 6-membered arylene or heteroarylene further substituted with one or more substituents. 1 The ring is further substituted with one or more substituents selected from halogen, (C1-C6) alkyl, and substituted (C1-C6) alkyl (eg, CF3).

[0107] In some embodiments of any one of Formulas (IVd)-(IVg), A 2 The ring is a 5- or 6-membered arylene or heteroarylene. 2 The ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. 2 The ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. 2 The ring is a triazole. 2 The ring is pyridine. 2 The ring is a pyrimidine. 2 The ring is a thiadiazole. 2 The ring is a 5- or 6-membered arylene or heteroarylene further substituted with one or more substituents. 2 The ring is further substituted with one or more substituents selected from halogen, (C1-C6) alkyl, and substituted (C1-C6) alkyl (eg, CF3).

[0108] In some embodiments of any one of Formulas (IVd)-(IVg), A 1 Ring or A 2 The ring does not exist.

[0109] In some embodiments of any one of Formulas (IVd)-(IVg), A 1 Ring or A 2 The ring is phenylene or substituted phenylene.

[0110] In some embodiments of Formula (IVd), A 2 The ring is a 5- or 6-membered heteroarylene. In certain cases of formula (IVd), A 2 In certain embodiments of Formula (IVd), A 2 In certain embodiments of Formula (IVd), A 2 The ring does not exist.

[0111] In some embodiments of Formula (IVe), A 1 The ring is a 5- or 6-membered heteroarylene, and R 21 is H. In certain embodiments of formula (IVe), the A ring is triazole. 1 In the particular case of formula (IVe), A 1 In the particular case of formula (IVe), A 1 In some embodiments of Formula (IVe), A 1 There is no ring, R 21 is H or optionally substituted acyl. 21 is -COCH3. In some cases, R 21 is H.

[0112] In some embodiments of Formula (IVf), A 1 The ring is a 5- or 6-membered heteroarylene. In certain cases of formula (IVf), A 1 In certain embodiments of Formula (IVf), A 1 In certain embodiments of Formula (IVf), A 1 The ring does not exist.

[0113] In some embodiments of Formula (IVg), A 2 The ring is a 5- or 6-membered heteroarylene. In certain cases of formula (IVg), A 2 In certain embodiments of Formula (IVg), A 2In certain embodiments of Formula (IVg), A 2 The ring does not exist.

[0114] In some cases, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by any one of formulas (IVh)-(IVk): [ka] [ka] [ka] and [ka] During the ceremony, R 6 , R 4 , R 3 , and R 21 is as defined herein; Y 1 ~Y 3 are each independently N or CR 25 and R 24 and R 25 are H, optionally substituted C, (1~6) -alkyl, optionally substituted fluoroalkyl, and halogen.

[0115] In certain embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by any one of formulas (IVl)-(IVm): [ka] [ka] During the ceremony, R 6 , R 4 , R 3 , and R 21 is as defined herein; Y 1 ~Y 3 are each independently N or CR 25 and Y 4 is N or CR 24 and Y 5 is S, O, or NH, R 24 and R 25 are H, optionally substituted C, (1~6) -alkyl, optionally substituted fluoroalkyl, and halogen.

[0116] In some embodiments of Formula (IVi), Y 1 ~Y 3 At least one of the is N. In some cases, Y 1 ~Y 3 At least two of Y are N. In certain cases, 1 and Y 3 is N and Y 2 is CR 25 In certain cases, Y 1 and Y 2 is N and Y 3 is CR 25 In certain cases, Y 1 and Y 2 is CR 25 and Y 3 is N.

[0117] In certain embodiments of any one of formulas (IVd)-(IVk), R 6 is H.

[0118] In some embodiments of any one of Formulas (IVd)-(IVk), R 4 and R 3 are H. In certain cases, R 4 ~R 3 At least one of R is a promoiety. 4 and R 3are linked in a circular fashion to form a promoiety (eg, as described herein).

[0119] In some embodiments, the compound of formula (IVi) is of formula (IVi-1): [ka] In the formula, R 24 and R 25 are independently selected from H, halogen, (C1-C6) alkyl, and substituted (C1-C6) alkyl (e.g., CF3).

[0120] In some embodiments of formulas (IVi) to (IVi-1), R 25 is H. In certain cases, R 25 is C (1~3) -Alkyl, or C (1~3) -fluoroalkyl. In some embodiments, the fluoroalkyl is CF. In some embodiments of formula (IVi) or (IVi-1), R 24 is H. In certain cases, R 24 is C (1~3) -Alkyl, or C (1~3) -fluoroalkyl. In some cases, the fluoroalkyl is CF3.

[0121] In some embodiments, the compound of formula (IVi-1) is represented by the formula (X D ) is. [ka]

[0122] In some embodiments, the compound of formula (IVk-1) is represented by the formula (X E ) is. [ka]

[0123] In some embodiments, the compound of formula (IVl) is of formula (IVl-1): [ka] During the ceremony, R 6 , R 4 , R 3 , and R 21 is as defined herein; Y 1 ~Y 4 are each independently N or CR 25 and Y 5 is S, O, or NH, Each R 25 is H, optionally substituted C (1~6) -alkyl, optionally substituted fluoroalkyl, and halogen.

[0124] In certain embodiments, each R 25 is H.

[0125] In certain embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by one of the following structures: [ka] and [ka]

[0126] In certain embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by one of the following structures: [ka] [ka] and [ka]

[0127] In certain embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by one of the following structures: [ka] [ka] [ka] and [ka]

[0128] In certain embodiments of Formula (IIb), R 1 , R 3 , R 4 , and R 11 is H and R 6 is OH, [ka] In the formula, Z 1 is -NH-, -CH2-, -S-, or -O-.

[0129] In certain embodiments of Formula (IIb'), R 3 , R 4 is H and R 6 is OH, [ka] In the formula, Z 1 is -NH-, -CH2-, -S-, -O-, triazole, e.g., [ka] is.

[0130] 6-linked ASGPR ligand moiety In some embodiments, the ASGPR binding moiety (X) is linked via position 6 of the sugar analog. In some embodiments, the ASGPR binding moiety (X) has a reduced ring carbon at position 1 relative to the galactosamine-derived sugar.

[0131] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IIc): [ka] In the formula, R 1 ~R 4 , and Z 1 is as defined herein.

[0132] In certain embodiments of Formula (IIc), Z 1 -O-, -S-, -CONR 21 - and optionally substituted -(C(R 22 )2) q -heteroarylene, where q is 0 or 1. In certain cases, Z 1 is -O-. In certain other cases, Z 1 is an optionally substituted -(C(R 22 )2) q -triazole, where q is 0 or 1. In certain cases, Z 1 teeth, [ka] is.

[0133] In certain embodiments of Formula (IIc), Z 1 Ha-Z 11 -A 1 -, where -A 1 - is an optionally substituted -A 1 -, or optionally substituted arylene. In certain cases, -A 1- is an optionally substituted heteroarylene. In certain cases, the heteroarylene is a 5- or 6-membered heteroarylene. In certain cases, the heteroarylene is a 5-membered heteroarylene. In certain cases, the 5-membered heteroarylene is a triazole. In certain cases, the triazole is a 1,2,3-triazole moiety. In certain cases, Z 11 -C(R 22 )2. In certain cases, at least one R 22 is H. In certain cases, both R 22 is H. In certain cases, Z 11 is -O-. In certain cases, Z 11 is -S-. In certain other cases, Z 11 -N(R 21 ), where R 21 is H or (C1-C3) alkyl. In certain cases, Z 1 -C(R 22 ) 2-triazole-. In certain cases, Z 1 teeth, [ka] is.

[0134] In certain embodiments of Formula (IIc), Z 1 is Z 11 In certain cases, Z 11 -C(R 22 )2. In certain cases, at least one R 22 is H. In certain cases, both R 22 is H and Z 11 is -CH2-. In certain cases, Z 11 is -O-. In certain cases, Z 11 is -S-. In certain other cases, Z 11 -N(R 21 ), where R 21 is H or (C1-C3) alkyl.

[0135] In certain embodiments of Formula (IIc), Z1 is a monocyclic 5- or 6-membered heteroarylene or arylene. In certain cases, Z 1 teeth, [ka] is.

[0136] In certain embodiments of Formula (IIc), Z 1 -O-, -S-, -C(R 22 )2-, -N(R 21 )-CON(R 21 )-, and [ka] is selected from During the ceremony, X 1 is O or S, t is 0 or 1, R 21 and each R 23 is H and optionally substituted (C1-C6) alkyl (e.g., C such as methyl). (1~3) -alkyl), Each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl.

[0137] In certain embodiments, the compound of formula (IIc) has the following structure: [ka]

[0138] In certain embodiments, the compound of formula (IIc) has the following structure: [ka]

[0139] In certain embodiments of Formula (IIc), R 11 is H and the compounds are those in Table 5. [Table 5]

[0140] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IId): [ka] During the ceremony, R 6 , R 4 , R 3 , and Z 1 is as defined herein; Y 6 and Y 5 are -O-, -S-, and NR, respectively. 21 - and -C(R 22 )2 are independently selected from R 21 is H, optionally substituted (C1-C6) alkyl, and —C(O)R 22 is selected from Each R 22 are independently selected from H, halogen, and optionally substituted (C1-C6) alkyl; Ring B is a 5- or 6-membered optionally substituted cyclic group. In some embodiments of Formula (IId), Y 5 is linked to the sugar ring via an alpha configuration. In some embodiments of Formula (IId), Y 5 is linked to the sugar ring via a beta configuration.

[0141] In some embodiments, the ASGPR binding moiety (X) of the compounds of the present disclosure can be represented by formula (IId'): [ka] During the ceremony, R 6 , R 4 , R 3 , and Z 1 is as defined herein; Y 5 and Y 6are -O-, -S-, and NR, respectively. 21 - and -C(R 22 )2 are independently selected from R 21 is H, optionally substituted (C1-C6) alkyl, and —C(O)R 22 is selected from Each R 22 are independently selected from H, halogen, and optionally substituted (C1-C6) alkyl; Ring B is a 5- or 6-membered optionally substituted cyclic group.

[0142] In some embodiments of Formula (IId)-(IId′), Y 5 is O. In certain cases, Y 5 is S. In certain cases, Y 5 Ha-NR 21 - In certain cases, Y 5 -C(R 22 )2, and each R 22 is H.

[0143] In some embodiments of Formula (IId)-(IId′), Y 6 Ha-NR 21 - where R 21 is H. In certain cases, Y 6 Ha-NR 21 - where R 21 -C(O)R 22 In some cases, R 22 is methyl.

[0144] In some embodiments of Formula (IId)-(IId'), the B ring is a 5- or 6-membered heterocycle. Optionally, the B ring is a 5-membered heterocycle. Optionally, the B ring is a 6-membered heterocycle.

[0145] In some embodiments of Formula (IId)-(IId′), Z 1 is Z 11 where Z 11 -O-, -S-, NR 21 - and -C(R 22)2. In some cases, Z 1 is -O-. In some cases, Z 1 is -S-. In some cases, Z 1 is NR 21 where R 21 is H. In some cases, Z 1 -C(R 22 )2, where each R 22 is H.

[0146] In some embodiments of Formula (IId)-(IId′), Z 1 is an arbitrarily substituted Z 11 -heteroarylene or optionally substituted Z 11 -arylene. In some embodiments, Z 1 is CH2-heteroarylene or CH2-arylene. In some embodiments of Formula (IId)-(IId'), Z 1 is an optionally substituted amide. In some embodiments of Formula (IId)-(IId'), Z 1 is an optionally substituted sulfonamide. In some embodiments of Formula (IId)-(IId'), Z 1 is an optionally substituted urea or an optionally substituted thiourea.

[0147] In some embodiments, the compound of Formula (IId)-(IId') has one of the following structures: [ka] or [ka]

[0148] In certain embodiments of any one of formulas (IIa), (IIb), or (IId), R 6 is OH. In certain other cases, R 6 is -OC(O)R. In certain cases, R 6is —C(O)NHR, where R is an optionally substituted alkyl. In certain cases, R terminates in an alkenyl or alkynyl group. In certain other cases, R 6 is an optionally substituted triazole. In certain cases, the triazole has the following structure: [ka]

[0149] In certain embodiments of (IIa) and (IIc), R 2 is —NHCOCH. In certain other embodiments, R 2 is -NHCOCF3. In certain other embodiments, R 2 is -NHCOCH2CF3. In certain cases, R 2 is —OH. In certain other cases, R 2 is an optionally substituted triazole. In certain cases, the triazole has the following structure: [ka]

[0150] In certain embodiments, R 6 or R 2 When is a substituted triazole, the triazole is a 1,2,3-triazole and the substituents are at the 4- or 5-position. In certain cases, the substituents on the triazole moiety include optionally substituted (C 1~6 ) alkyl, optionally substituted (C 1~6 ) alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and optionally substituted alkylheteroaryl. It will be understood that the triazole moiety can include any convenient substituent. See, for example, the triazole moieties disclosed in Mamidayala et al., J. Am. Chem. Soc. 2012, 134, 1978-1981.

[0151] Z 1 , Z 11 , and Z 11 It is understood that the -Ar linking moiety can be considered part of the X group of formula (I). In the ASGPR binding moieties (X) described herein, -Z 1 - can be connected to -L (e.g., in a linker described herein) via various bonds and linking moieties depending on the method of preparation. 1 In some embodiments, the subject compounds can be linked to a -Z moiety selected from: 1 -L 1 - includes parts, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] In the formula, each R 21is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl, and o, p, q, r, s, t, u, v, w, x, y, z, and z1 are each independently 1 to 6. In certain embodiments, o, p, q, r, s, t, u, v, w, x, y, z, and z1 are each independently 1 to 6.

[0152] In certain embodiments, Z 1 -L 1 -The base is [ka] and o is 1 or 2.

[0153] In certain embodiments, Z 1 -L 1 -The base is [ka] and each R 22 is H and p is 1 or 2.

[0154] In certain embodiments, Z 1 -L 1 -The base is [ka] where q is 1 to 3.

[0155] In certain embodiments, Z 1 -L 1 -The base is [ka] where r is 1 to 3.

[0156] In certain embodiments, Z 1 -L 1 -The base is [ka] where r is 1 to 3.

[0157] In certain embodiments, Z 1 -L 1 -The base is [ka] where s and t are each independently 1 to 3.

[0158] In certain embodiments, Z 1 -L 1 -The base is [ka] where u is 1 to 3.

[0159] In certain embodiments, Z 1 -L 1 -The base is [ka] where v and w are each independently 1 to 3.

[0160] In certain embodiments, Z 1 -L 1 -The base is [ka] where x is 0 to 3.

[0161] In certain embodiments, Z 1 -L 1 -The base is [ka] where y is 1 to 3.

[0162] In certain embodiments, Z 1 -L 1 -The base is [ka] where each R 21 is H and z is 1 to 4.

[0163] In certain embodiments, Z 1 -L 1 -The base is [ka] where each R 21 is H and z1 is 1 to 4.

[0164] In certain embodiments, Z 1 -L 1 -The base is [ka] where each R 22 is H and p is 0 to 3. In certain embodiments, Z 1 -L 1 -The base is [ka] where each R 22 is H and p is 1 to 3.

[0165] In certain embodiments, Z 1 -L 1 -The base is [ka] where q is 1 to 3.

[0166] In certain embodiments, the subject compound is selected from the following: 1 Contains the -L- group. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka]

[0167] In certain embodiments, Z 1 -L 1 -The base is [ka] where q is 1 to 3. In certain cases, q is 1. In certain cases, q is 2. In certain cases, q is 3.

[0168] In certain embodiments, Z 1 -L 1-The base is [ka] is.

[0169] In certain embodiments, Z 1 -L 1 -The base is [ka] is.

[0170] In certain embodiments, Z 1 -L 1 -The base is [ka] is.

[0171] In certain embodiments, -Z 1 -L 1 - includes optionally substituted -NH-heteroarylene-. In certain embodiments, heteroarylene is triazole. In certain cases, heteroarylene is pyridine. In certain cases, heteroarylene is pyrimidine. In certain cases, heteroarylene is thiadiazole.

[0172] In certain embodiments, -Z 1 -L 1 - includes a group selected from: [ka] [ka] [ka] [ka] [ka] [ka] and [ka] In the formula, each R 21 are independently selected from H, optionally substituted (C1-C6) alkyl, and optionally substituted acyl; R 24 and R 25 are H, optionally substituted C, (1~6) -alkyl, optionally substituted fluoroalkyl, and halogen.

[0173] In certain embodiments, -Z 1 -L 1 - includes a group selected from: [ka] and [ka] In the formula, R 24 and R 25 are H, optionally substituted C, (1~6) -alkyl, optionally substituted fluoroalkyl, and halogen; 21 is independently selected from H, optionally substituted (C1-C6) alkyl, and optionally substituted acyl. In certain cases, R 21 is H. In certain cases, R 24 is C (1~3) -Alkyl, or C (1~3) -fluoroalkyl. In some cases, the fluoroalkyl is CF3. In certain cases, R 25 is C (1~3) -Alkyl, or C (1~3) -fluoroalkyl. In some cases, the fluoroalkyl is CF3.

[0174] In certain embodiments, -Z 1 -L 1 -teeth, [ka] is.

[0175] It is understood that a variety of substituents and chemistries can be utilized to link a particular X ligand moiety to an adjacent linker (e.g., as described herein). In some embodiments, the linking moiety of the linker comprises a triazole derived from a click chemistry bond. In certain embodiments, the ASGPR ligand moiety (X) is attached to a linking moiety as shown in one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka]

[0176] In certain embodiments of Formula (IIb), R 1 , R 3 , R 4 , and R 11 is H and R 6 is OH, [ka] In the formula, Z 1 is triazole, -NH-heteroaryl (e.g., -NH- attached to a pyridine or pyrimidine), -NH-, -O-, or -CH2-, and / or Z 1 is attached to a linking moiety as shown in one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka]

[0177] In certain embodiments of Formula (Iib), R 1 , R 3 , R 4 , and R 11 is H and R 6 is OH, [ka] In the formula, Z 1 is attached to a linking moiety as shown in one of the following structures: [ka] [ka] [ka] or [ka]

[0178] M6PR joint part As summarized above, the M6PR-binding moieties (also referred to as M6PR ligand moieties) of the present disclosure can be linked to a variety of moieties of interest without affecting their specific binding to and function of cell-surface M6PR. The present inventors have demonstrated that M6PR-binding moieties having the specific structures described below enable high-affinity binding to cell-surface M6PR and, when configured via a linker according to the bifunctional compounds of the present disclosure, can exploit the function of cell-surface M6PR in biological systems, for example, for internalization and / or degradation of target molecules such as autoantibodies.

[0179] The terms "mannose-6-phosphate receptor" and "M6PR" refer to receptors of the mannose-6-phosphate receptor family. M6PR is a transmembrane glycoprotein receptor that delivers enzymes to lysosomes within cells. M6R endogenously transports proteins bearing N-glycans capped with mannose-6-phosphate (M6P) residues to lysosomes and cycles between endosomes, the cell surface, and the Golgi complex. See, e.g., Ghosh et al., Nat. Rev. Mol. Cell Biol. 2003;4:202-213. The M6PR family includes the cation-independent mannose-6-phosphate receptor (CI-M6PR). CI-M6PR is also known as insulin-like growth factor 2 receptor (IGF2R), and in humans is encoded by the IGF2R gene (see, e.g., NCBI Reference Sequence: NM_000876.3 and NCBI Gene ID: 3482). CI-M6PR binds to insulin-like growth factor 2 (IGF-2) and mannose-6-phosphate (M6P)-tagged proteins. Compounds of the present disclosure can specifically bind to cell surface M6PR, e.g., internalized CI-M6PR cell surface receptors. In certain embodiments, the surface CI-M6PR is human CI-M6PR. It is understood that the terms M6PR and CI-M6PR are used interchangeably when referring to the binding properties of M6PR-binding moieties and compounds of the present disclosure.

[0180] Compounds (e.g., as described herein) comprising such an M6PR binding moiety (X) may bind to other receptors, and may bind with lower affinity as measured, for example, by immunoassay or other assays known in the art. In certain embodiments, X, or a compound described herein comprising such X, specifically binds to cell surface CI-M6PR with an affinity that is at least 2 log, 2.5 log, 3 log, 4 log, or more than the affinity with which X or the compound binds to another cell surface receptor. In certain embodiments, X, or a compound described herein comprising X, specifically binds to CI-M6PR with an affinity (Kd In certain embodiments, such binding is characterized by an affinity (K d ) is 10 mM or less, 1 mM or less, 100 uM or less, 10 uM or less, 1 uM or less, 100 nM or less, 10 nM or less, or 1 nM or less. In this context, the terms "bind," "bind," "specifically bind," or "specifically bind to" are used interchangeably.

[0181] The M6PR-binding compounds of the present disclosure include a moiety (X) that is a D-mannopyranose analog that specifically binds to the cell surface receptor M6PR (e.g., as described herein). M6PR-binding compounds may be monovalent or multivalent (e.g., bivalent or trivalent or higher), with monovalent compounds containing a single M6PR ligand moiety and monovalent compounds containing two or more such moieties.

[0182] Alpha-linked pyranose ring The M6PR binding portion of the compounds of the present disclosure can include a linked pyranose ring represented by formula (II): [ka] (II) During the ceremony, W is a hydrophilic head group; Z 1 is selected from optionally substituted (C1-C3) alkylene and optionally substituted ethenylene; Z 2 are O, S, NR 21 and C(R 22 )2, where each R 21 is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl.

[0183] In some embodiments of Formula (II), Z 2is a linking moiety attached in the alpha configuration to the pyranose sugar ring at the anomeric or 1-position, as shown in formula (IIa) below. [ka] (IIa)

[0184] Beta-linked pyranose ring The present inventors have demonstrated that M6PR-binding compounds having an M6PR-binding moiety with the anomeric alpha configuration of formula (IIa) can provide good binding and internalization activity at the receptor, but in some cases, configuring the central pyranose sugar ring of the M6PR-binding moiety in the beta configuration at the anomeric position can confer stronger binding and internalization activity at M6PR. In some embodiments, such M6PR-binding compounds can have improved stability at the pyranose ring.

[0185] Thus, in some embodiments of formula (II), Z 2 is a linking moiety attached in a beta configuration to the sugar ring at the anomeric or 1-position, as shown in formula (IIb) below. [ka] (IIb)

[0186] M6PR binding compound Although the moiety of formula (II) can exhibit M6PR binding activity, the inventors have discovered that certain types of cyclic groups can be linked to the linking moiety Z 2 It has been demonstrated that when linked in a specific configuration adjacent to the pyranose ring of formula (II) via

[0187] Thus, in some embodiments of formula (II), the M6PR binding moiety (X) can be represented by formula (III), or a prodrug thereof, or a salt thereof: [ka] (III) During the ceremony, W is a hydrophilic head group; Z 1 is selected from optionally substituted (C1-C3) alkylene and optionally substituted ethenylene; Z 2 are O, S, NR 21 and C(R 22 )2, where each R 21 is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl; A is independently an optionally substituted cyclic group; Z 3 is independently a linking moiety.

[0188] In some embodiments of Formulas (II)-(III), W is a non-hydrolyzable hydrophilic head group.

[0189] In some embodiments of Formulas (II)-(III), Z 2 is optionally substituted ethylene. In some embodiments of Formulas (II)-(III), Z 2 is optionally substituted ethenylene.

[0190] In some embodiments of Formulas (II)-(III), Z 2 is O. In some embodiments of Formulas (II)-(III), Z 2 is S. In some embodiments of Formulas (II)-(III), Z 2 Ha-NR 21 In some embodiments of Formulas (II)-(III), Z 2 -C(R 22 )2-, where each R 22 is independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl. In some embodiments of Formulas (II)-(III), Z 2 is -CH2-.

[0191] In some embodiments of Formulas (II)-(III), A is an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycle, or an optionally substituted cycloalkyl. In some embodiments of Formulas (II)-(III), A is independently an optionally substituted aryl or heteroaryl linking moiety (e.g., an optionally substituted monocyclic or bicyclic aryl or heteroaryl).

[0192] Exemplary Z groups of formulas (II) to (III) 3 Linking moieties are described herein.

[0193] Such M6PR-binding moieties of formula (III) can be conjugated to a moiety or molecule of interest to generate bifunctional compounds that undergo efficient M6PR-mediated cellular internalization. The inventors have further demonstrated that when the moiety or molecule of interest is a target protein-binding moiety, the M6PR-binding compounds also enable M6PR-mediated internalization and / or degradation of the bound target protein.

[0194] Thus, in some embodiments of formula (XI), the M6PR binding compound is of formula (XII), or a prodrug thereof, or a salt thereof: [ka] (XII) During the ceremony, W is a hydrophilic head group; Z 1 is selected from optionally substituted (C1-C3) alkylene and optionally substituted ethenylene; Z 2 are O, S, NR 21 and C(R 22 )2, where each R 21 is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl; A is independently an optionally substituted cyclic group; Z 3 are independently linking moieties, n is 1 to 500; L is a linker, Y is the part of interest, m is 1 to 100.

[0195] In some embodiments of Formulas (XI)-(XII), m is 1 and the cell surface M6PR binding compound is of Formula (XIII), or a prodrug thereof, or a salt thereof: [ka] (XIII) During the ceremony, W is a hydrophilic head group; Z 1 is selected from optionally substituted (C1-C3) alkylene and optionally substituted ethenylene; Z 2 are O, S, NR 21 and C(R 22 )2, where each R 21 is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 are independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl; A is independently an optionally substituted cyclic group; Z 3 are independently linking moieties, n is 1 to 500; L is a linker, Y is a moiety of interest (e.g., as described herein).

[0196] In some embodiments of Formula (XIII), Y is a chemoselective ligation group. In some embodiments of Formula (XIII), n is 1. In some embodiments of Formula (XIII), Y is a chemoselective ligation group linked to "n" M6PR binding moieties (Xn-) via a single linker -L-. In some embodiments of Formula (XIII), n is 2, 3, 4, or 5. In some embodiments of Formula (XIII), n is 5 to 10. In some embodiments of Formula (XIII), n is 10 to 100, e.g., 20 to 80, or 20 to 50. In some embodiments of Formula (XIII), when n is 5 or greater, L is a polypeptide-containing linker (e.g., as described herein).

[0197] In some embodiments of Formulas (XII)-(XIII), when n is 1 and A is phenyl, i) L comprises a backbone of at least 16 consecutive atoms (e.g., at least 18 consecutive atoms, or at least 20 consecutive atoms, and in some cases up to about 200 consecutive atoms), ii) Y is a biomolecule, and / or ii) Z 3 is an amide, sulfonamide, urea, or thiourea linking moiety to the linker L.

[0198] In some embodiments of Formula (XII), Z 2 is a linking moiety linked in the alpha configuration to the sugar ring at the anomeric or 1 position, as shown in formula (IIa), so that the compound is of formula (XIIa). [ka] (XIIa)

[0199] In some embodiments of Formula (XII), Z 2 is a linking moiety linked in a beta configuration to the sugar ring at the anomeric or 1-position, as shown in formula (IIb), so that the compound is of formula (XIIb). [ka] (XIIb)

[0200] In some embodiments of Formula (XI)-(XIIb), multiple M6PR binding moieties, for example of Formula (III), are linked via multiple linkers L to different ligation sites on the moiety of interest Y. In some embodiments, when Y is a biomolecule, the compounds of Formula (XI)-(XIIb) can be referred to as conjugates.

[0201] Hydrophilic head group and linking moiety In some embodiments of Formulas (II)-(XIII), the M6PR binding moiety (X) is a linking moiety (Z 1 The present invention includes analogs of a D-mannopyranose ring, or precursors or prodrugs thereof, having a hydrophilic head group linked to the 5-position of the sugar ring via a linking moiety. The linking moiety can be 1 to 6 atoms in length, e.g., 1 to 5, 1 to 4, or 1 to 3 atoms in length, e.g., 1 or 2 atoms in length. It is understood that the length of the linking moiety can be selected in conjunction with the hydrophilic head group.

[0202] The hydrophilic head group (W) can be any suitable negatively charged group or salt thereof. In some embodiments, the hydrophilic head group is a neutral, polar, hydrophilic group. Generally, the hydrophilic head group can hydrogen bond or electrostatically interact with M6PR under aqueous or physiological conditions, similar to the hydrophilic head group of the phosphate group of M6P. The hydrophilic head group can be a bioequivalent (e.g., a structural or functional mimetic) of the 6-phosphate group of a naturally occurring mannose-6-phosphate ligand. In some embodiments, the hydrophilic head group is non-hydrolyzable, i.e., under physiological conditions, the Z of X to which the hydrophilic head group is attached is non-hydrolyzable. 1 The functional group is stable to cleavage (eg, chemically or enzymatically) from the linking moiety and / or pyranose ring.

[0203] The hydrophilic head group is generally a small group such as a heteroatom-containing functional group or a single heterocycle, optionally having a MW of less than 200, eg, less than 150, or less than 100.

[0204] In some embodiments, the hydrophilic head group is a phosphonate, or a bioisostere thereof, such as a carboxylate or malonate. In some embodiments, the hydrophilic head group is a thiophosphonate.

[0205] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group is not a phosphate, thiophosphate, or dithiophosphate because such groups have a phosphate ester bond to the compound that may be unstable under physiological conditions and prone to cleavage (e.g., by phosphatases in biological systems or chemically). For example, the 6-phosphate group of M6P exhibits undesirable stability compared to phosphonate analogs or other more stable head groups. The present disclosure provides alternative non-hydrolyzable head groups, in addition to phosphonates, that retain the binding and internalization activity of the resulting M6PR-binding compounds.

[0206] In one embodiment of any one of formulas (II)-(XIII), the hydrophilic head group W is selected from the group consisting of -OH, -CR 2 R 2 OH, -NR 3 P=O(OH)2, -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), P(=O)R 1 OH, -PH(=O)OH, -(CR 2 R 2 )-P=O(OH) 2、 -SO2OH (i.e., -SO3H), -S(O)OH, -OSO2OH, -COOH, -CN, -CONH2, -CONHR 3 , -CONR 3 R 4 , -CONH(OH), -CONH(OR 3 ), -CONHSO2R 3 , -CONHSO2NR 3 R 4 , -CH(COOH)2, -CR 1 R 2 COOH, -SO2R 3 , -SOR 3R 4 , -SO2NH2, -SO2NHR 3 , -SO2NR 3 R 4 , -SO2NHCOR 3 , -NHCOR 3 , -NHC(O)CO2H, -NHSO2NHR 3 , -NHC(O)NHS(O)2R 3 , -NHSO2R 3 , -NHSO3H, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] or salts thereof; During the ceremony, R 1 and R 2 are independently hydrogen, SR 3 , halo, or CN, R 3 and R 4 are independently H, C 1~6 Alkyl or substituted C 1~6 alkyl (e.g., —CF or —CHCF), A, B, and C are each independently CH or N; Each D is independently O or S.

[0207] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a phosphate or thiophosphate, e.g., -OP=O(OH), -SP=O(OH), -OP=O(SH)(OH), -SP=O(SH)(OH), -OP=S(OH), -OP=O(N(R 3 )2)(OH), or -OP=O(R 3 )(OH), or a salt thereof. In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is non-hydrolyzable and therefore is a phosphate or thiophosphate, e.g., -OP=O(OH), -SP=O(OH), -OP=O(SH)(OH), -SP=O(SH)(OH), -OP=S(OH), -OP=O(N(R 3 )2)(OH), or -OP=O(R 3 )(OH), or salts thereof.

[0208] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is charged and capable of forming a salt, e.g., under aqueous or physiological conditions. In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is -NR 3 P=O(OH)2, -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), P(=O)R 1 OH, -PH(=O)OH, -(CR 2 R 2 )-P=O(OH)2, -COOH, -CH(COOH)2, -CR 1 R 2 COOH, and —NHC(O)COH.

[0209] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a phosphonate or thiophosphonate (e.g., -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), or -P=S(SH)(OH), or a salt thereof). In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a phosphonate or a salt thereof. In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is -CO2H or a salt thereof. In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a malonate (e.g., -CH(COOH)2 or a salt thereof).

[0210] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is selected from -SO2OH (i.e., -SO3H), -S(O)OH, -OSO2OH, and -NHSO3H. In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a sulfonate (e.g., -SO3H or a salt thereof).

[0211] In some embodiments, the hydrophilic head group W is neutrally hydrophilic. In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is selected from the group consisting of -OH, -CR 2 R 2 OH, -CN, -CONH2, -CONHR 3 , -CONR 3 R 4 , -CONH(OH), -CONH(OR 3 ), -CONHSO2R 3 , -SO2R 3 , -SOR 3 R 4 , -SO2NH 2、 -SO2NHR 3 , -SO2NR 3 R 4 , -SO2NHCOR 3 , -NHCOR 3 , -NHSO2NHR 3 , -NHC(O)NHS(O)2R 3 , and -NHSO2R 3 is selected from.

[0212] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a heterocycle, e.g., [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] or salts thereof, wherein A, B, and C are each independently CH or N, and D is each independently O or S.

[0213] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is a 5-membered heterocycle, such as [ka] [ka] [ka] [ka] [ka] or [ka] or salts thereof.

[0214] In some embodiments of Formulas (II)-(XIII), the hydrophilic head group W is selected from optionally substituted (C1-C2) alkylene and optionally substituted ethenylene. 1 It is connected to the pyranose ring via Z. 1 Z can be selected in conjunction with W to provide the desired spacing between the 5-position of the ring and the charged or polar center of W. For example, if W is a malonate having a C-H atom connecting the two carboxylic acid groups, then Z 1 may be methylene, which together provide a desirable two-carbon spacer between the ring and the COOH group.

[0215] In some embodiments of Formulas (II)-(XIII), Z 1 is methylene or substituted methylene. In some embodiments of Formulas (II)-(XIII), Z 1 is ethyl or substituted ethyl. In some embodiments of Formulas (II)-(XIII), Z 1 is ethenylene or substituted ethenylene. In some embodiments of Formulas (II)-(XIII), Z 1 is substituted with one or more halogens, such as fluoro.

[0216] In some embodiments of formula (III), the M6PR binding moiety (X) is represented by one of formulas (IV-1) to (IV-3): [ka] [ka] [ka] In the formula, R a , Rb , R c , and R d are independently H or F.

[0217] In some embodiments of formulas (IV-1) to (IV-3), Z 2 is O.

[0218] In some embodiments of formulas (IV-1) to (IV-3), Z 2 is S.

[0219] In some embodiments of formulas (IV-1) to (IV-3), Z 2 Ha-NR 21 -It is.

[0220] In some embodiments of formulas (IV-1) to (IV-3), Z 2 -C(R 22 )2-, where each R 22 is independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl. In some embodiments of formulas (IV-1) to (IV-3), Z 2 is -CH2-.

[0221] In some embodiments of formulas (IV-1) to (IV-3), R a , R b , R c and R d are H respectively.

[0222] In some embodiments of formula (IV-1), R a is H and R b is F. In some embodiments of formula (IV-1), R a and R b are F respectively.

[0223] In some embodiments of formula (IV-2), R c is H. In some embodiments of formula (IV-2), R c is F.

[0224] In some embodiments of formula (IV-3), R d is H. In some embodiments of formula (IV-3), R d is F.

[0225] In some embodiments of Formula (IV-1) and (IV-3), W is selected from -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), and -COOH, or a salt thereof. In some embodiments of Formula (IV-1) and (IV-3), W is -P=O(OH)2 or a salt thereof. In some embodiments of Formula (IV-1) and (IV-3), W is COOH or a salt thereof.

[0226] In some embodiments of formula (IV-1), R a and R b are each F, and W is -P=O(OH)2 or a salt thereof. In some embodiments of formula (IV-1), R a and R b are each H, and W is -P=O(OH)2 or a salt thereof. In some embodiments of formula (IV-1), R a is F and R b is H and W is -P=O(OH)2 or a salt thereof.

[0227] In some embodiments of formulas (IV-1) to (IV-3), Z 2 is linked in an alpha configuration to the anomeric position of the pyranose ring. In such cases, the M6PR binding moieties (X) of (IV-1) to (IV-3) can be designated by formulas (IV-A1) to (IV-A3), respectively.

[0228] In some embodiments of formulas (IV-A1) to (IV-A3), Z 2 is S. In some embodiments of formulas (IV-A1) to (IV-A3), Z 2 is O. In some embodiments of formulas (IV-A1) to (IV-A3), Z 2 In some embodiments of formulas (IV-A1) to (IV-A3), Z2 is -CF2-.

[0229] In some embodiments of Formula (IV-A1) and (IV-A3), W is selected from -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), and -COOH, or a salt thereof. In some embodiments of Formula (IV-A1) and (IV-A3), W is -P=O(OH)2 or a salt thereof. In some embodiments of Formula (IV-A1) and (IV-A3), W is COOH or a salt thereof.

[0230] In some embodiments of formula (IV-A1), R a and R b are each F and W is -P=O(OH)2 or a salt thereof. In some embodiments of formula (IV-A1), R a and R b are each H and W is -P=O(OH)2 or a salt thereof. In some embodiments of formula (IV-A1), R a is F and R b is H and W is -P=O(OH)2 or a salt thereof.

[0231] In some embodiments of formulas (IV-1) to (IV-3), Z 2 is linked in a beta configuration to the anomeric position of the pyranose ring. The inventors have demonstrated that compounds comprising an M6PR-binding moiety with a β-glycosidic configuration can have at least equivalent binding activity and / or cellular uptake activity compared to conjugates with a corresponding α-glycosidic configuration. In some embodiments, such M6PR-binding moieties with a β-glycosidic configuration can have improved stability compared to reference compounds with a β-glycosidic configuration. Thus, in some embodiments of formula (IV), the M6PR-binding moiety (X) is represented by one of formulas (IV-B1) to (IV-B3): [ka] [ka] [ka] In the formula, R a , R b , R c , and R d are independently H or F.

[0232] In some embodiments of formulas (IV-B1) to (IV-B3), Z 2 is S. In some embodiments of formulas (IV-B1) to (IV-B3), Z 2 is O. In some embodiments of formulas (IV-B1) to (IV-B3), Z 2 In some embodiments of formulas (IV-B1) to (IV-B3), Z 2 is -CF2-.

[0233] In some embodiments of Formula (IV-B1) and (IV-B3), W is selected from -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), and -COOH, or a salt thereof.

[0234] In some embodiments of Formula (IV-B1) and (IV-B3), W is selected from -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), and -COOH, or a salt thereof. In some embodiments of Formula (IV-B1) and (IV-B3), W is -P=O(OH)2 or a salt thereof. In some embodiments of Formula (IV-B1) and (IV-B3), W is COOH or a salt thereof.

[0235] In some embodiments of formula (IV-B1), R a and R b are each F and W is -P=O(OH)2 or a salt thereof. In some embodiments of formula (IV-B1), R a and R bare each H and W is -P=O(OH)2 or a salt thereof. In some embodiments of formula (IV-B1), R a is F and R b is H and W is -P=O(OH)2 or a salt thereof.

[0236] The present inventors have demonstrated that conjugates containing M6PR-binding moieties with a β-S-glycosidic configuration can have at least equivalent or superior binding and / or cellular uptake activity compared to conjugates with the corresponding α-S-glycosidic or α-O-glycosidic configurations (see Figure 19).

[0237] Thus, in some embodiments of formulas (IV-B1) to (IV-B3), the M6PR binding moiety (X) is represented by one of formulas (IV-BS1) to (IV-BS3): [ka] [ka] [ka] In the formula, R a , R b , R c , and R d are independently H or F.

[0238] In some embodiments of formulas (IV-BS1) to (IV-BS3), R a , R b , R c and R d are H respectively.

[0239] In some embodiments of Formula (IV-BS1), R a is H and R b is F. In some embodiments of formula (IV-BS1), R a and R b are F respectively.

[0240] In some embodiments of Formula (IV-BS2), R c is H. In some embodiments of formula (IV-B2), R c is F.

[0241] In some embodiments of Formula (IV-BS3), R d is H. In some embodiments of formula (IV-BS3), R d is F.

[0242] In some embodiments of formulas (IV-BS1) to (IV-BS3), Z 2 is S. In some embodiments of formulas (IV-BS1) to (IV-BS3), Z 2 is O. In some embodiments of formulas (IV-BS1) to (IV-BS3), Z 2 In some embodiments of formulas (IV-BS1) to (IV-BS3), Z 2 is -CF2-.

[0243] In some embodiments of Formula (IV-BS1) and (IV-BS3), W is selected from -P=O(OH)2, -P=S(OH)2, -P=O(SH)(OH), -P=S(SH)(OH), and -COOH, or a salt thereof. In some embodiments of Formula (IV-BS1) and (IV-BS3), W is -P=O(OH)2 or a salt thereof. In some embodiments of Formula (IV-BS1) and (IV-BS3), W is COOH or a salt thereof.

[0244] In some embodiments of Formula (IV-BS1), R a and R b are each F and W is -P=O(OH)2 or a salt thereof. In some embodiments of Formula (IV-BS1), R a and R b are each H and W is -P=O(OH)2 or a salt thereof. In some embodiments of Formula (IV-BS1), R a is F and R bis H and W is -P=O(OH)2 or a salt thereof.

[0245] In some embodiments, the mannose ring or analog thereof of the M6PR binding moiety has a Z bonded to the anomeric or 1-position of the sugar ring. 2 Linking moieties can be attached to groups to be incorporated into compounds of the present disclosure.

[0246] In some embodiments, the M6PR binding moiety is a Z bonded to the cyclic group A. 3 In the compound of formula (III), Z is incorporated into the compound of the present disclosure by attaching a linker to the group. 2 It is understood that the cyclic group attached to can be considered part of the M6PR binding moiety (X) and can provide desirable binding properties to M6PR.

[0247] cyclic group A The A cyclic group of formulas (III)-(XIII) can be a monocyclic or bicyclic group. The bicyclic group in question can be a fused bicyclic group or a bicyclic group comprising two monocyclic rings linked via a covalent bond. The A cyclic group of formulas (III)-(XIII) can be an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycle (e.g., a saturated heterocycle), or an optionally substituted cycloalkyl.

[0248] The A cyclic group in Formulas (III)-(XIII) can be a monocyclic aryl group or a monocyclic heteroaryl group. In some embodiments of Formulas (III)-(XIII), A is a 5-membered monocyclic heteroaryl group. In some embodiments of Formulas (III)-(XIII), A is a 6-membered monocyclic aryl or heteroaryl group. In some embodiments of Formulas (III)-(XIII), A can be a polycyclic aryl or polycyclic heteroaryl group, such as a bicyclic aryl or bicyclic heteroaryl group. In some embodiments of Formulas (III)-(XIII), A is a fused bicyclic group. In some embodiments of Formulas (III)-(XIII), A is a bicyclic group comprising two aryl and / or heteroaryl monocyclic rings linked via a covalent bond. In some embodiments of Formulas (III)-(XIII), A is a bicyclic aryl or bicyclic heteroaryl group having two 6-membered rings. In some embodiments of Formulas (III)-(XIII), A is a bicyclic aryl or bicyclic heteroaryl group having one 6-membered ring covalently linked to or fused to a 5-membered ring.

[0249] In some embodiments of Formulas (III)-(XIII), A is selected from optionally substituted phenyl, optionally substituted pyridyl, optionally substituted biphenyl, optionally substituted naphthalene, optionally substituted quinoline, optionally substituted triazole, and optionally substituted phenylene-triazole.

[0250] In some embodiments of Formulas (III)-(XIII), A is not phenyl (also called phenylene in the context of Formula (III), eg, 1,4-phenylene).

[0251] In some embodiments of Formulas (III)-(XIII), A is substituted with at least one OH substituent. In some embodiments of Formulas (III)-(XIII), A is substituted with one, two, or more OH groups. In some embodiments of Formulas (III)-(XIII), A is substituted with at least one optionally substituted (C1-C6) alkyl.

[0252] In some embodiments of Formulas (III)-(XIII), A is optionally substituted 1,4-phenylene, optionally substituted 1,3-phenylene, or optionally substituted 2,5-pyridylene.

[0253] In some embodiments of Formulas (III)-(XIII), A is selected from: [ka] [ka] and [ka] During the ceremony, R 11 ~R 14 is H, halogen, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C1-C6) alkoxy, COOH, NO2, CN, NH2, -N(R 25 )2, -OCOR 25 , -COOR 25 , -CONHR 25 , and -NHCOR 25 are independently selected from R 25 are independently selected from H and optionally substituted (C1-C6) alkyl.

[0254] In some embodiments of Formulas (III)-(XIII), A is an optionally substituted fused bicyclic aryl or an optionally substituted fused bicyclic heteroaryl.

[0255] In some embodiments of Formulas (III)-(XIII), A is an optionally substituted naphthalene or an optionally substituted quinoline.

[0256] In some embodiments of Formulas (III)-(XIII), A is selected from: [ka] [ka] [ka] [ka] and [ka] During the ceremony, R 11 and R 13 ~R 14 is H, halogen, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C1-C6) alkoxy, COOH, NO2, CN, NH2, -N(R 25 )2, -OCOR 25 , -COOR 25 , -CONHR 25 , and -NHCOR 25 are independently selected from s is 0 to 3, Each R 25 are independently selected from H and optionally substituted (C1-C6) alkyl.

[0257] In some embodiments of Formulas (III)-(XIII), A is selected from: [ka] [ka] [ka] and [ka]

[0258] In some embodiments of Formulas (III)-(XIII), A is an optionally substituted bicyclic aryl or an optionally substituted bicyclic heteroaryl of the following formula, or a salt thereof: [ka] During the ceremony, Cy is independently a monocyclic aryl or a monocyclic heteroaryl; R 11 ~R 15 is H, halogen, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C1-C6) alkoxy, COOH, NO2, CN, NH2, -N(R 25 )2, -OCOR 25 , -COOR 25 , -CONHR 25 , and -NHCOR 25 are independently selected from s is 0 to 4, Each R 25 are independently selected from H and optionally substituted (C1-C6) alkyl.

[0259] In some embodiments, when Cy is optionally substituted phenyl, A is an optionally substituted biphenyl of the formula: [ka]

[0260] In some embodiments of Formulas (III)-(XIII), A is selected from: [ka] and [ka]

[0261] In some embodiments, when Cy is triazole, A is selected from: [ka] and [ka]

[0262] In some embodiments, R 11 ~R 15 At least one of is OH (eg, at least two are OH).

[0263] In some embodiments, R 11 ~R 15 are H respectively.

[0264] Connecting part Z 3 Connecting part Z 3 can be any convenient linking moiety that connects the linker L to the cyclic ring A. In some embodiments of Formulas (III)-(XIII), Z 3 has a backbone of 3 atoms or less. In some embodiments of formulas (III)-(XIII), Z 3 is a covalent bond, -O-, -NR 23 -, -NR 23 CO-, -CONR 23 -, -NR 23 CO2-, -OCONR 23 , -NR 23 C(=X 1 )NR 23 -, -CR 24 =N-, -CR 24 =NX 2 , -N(R 23 )SO2- and -SO2N(R 23 )-, where X 1 and X 2 are O, S and NR 23 Selected from R23 and R 24 is H, C (1-3) -Alkyl (e.g., methyl) and substituted C (1-3) -alkyl.

[0265] In some embodiments of Formulas (III)-(XIII), Z 3 is the covalent bond connecting A to L.

[0266] In some embodiments of Formulas (III)-(XIII), Z 3 is an optionally substituted amide, urea or thiourea.

[0267] In some embodiments of Formulas (III)-(XIII), Z 3 is [ka] During the ceremony, X 1 is O or S, t is 0 or 1, Each R 23 is H, C (1~3) -Alkyl (e.g., methyl or ethyl) and substituted C (1~3) -alkyl. 3 In some embodiments, X 1 is O. Z 3 In some embodiments, X 1 is S. Z 3 In some embodiments of 1 is O, and as a result, Z 3 is an amide. Z 3 In some embodiments, t is 1, such that Z 3 is urea or thiourea.

[0268] In some embodiments of Formulas (III)-(XIII), Z 3 -N(R 23 )SO2- or -SO2N(R 23In some embodiments of Formulas (III)-(XIII), Z 3 is -NHSO2- or -SO2NH-.

[0269] In some embodiments of Formulas (III)-(XIII), Z 3 -N(R 23 )CO- or -CON(R 23 In some embodiments of Formulas (III)-(XIII), Z 3 is -NHCO- or -CONH-.

[0270] In some embodiments of Formulas (III)-(XIII), Z 3 is -NHC(=X 1 )NH-, where X 1 is O or S. In some embodiments, X 1 is O (i.e., Z 3 is —NHC(═O)NH—). In some embodiments, X 1 is S.

[0271] In some embodiments of Formulas (III)-(XIII), Z 3 is an optionally substituted triazole. 3 When is an optionally substituted triazole, it can be synthetically derived from click chemistry coupling of an azide-containing precursor with an alkyne-containing precursor of the compound.

[0272] In some embodiments, Z 3 is a cyclic group A and / or a linking moiety Z 1 are selected in combination to provide X with desirable M6PR binding and internalization properties.

[0273] In some embodiments of Formulas (III)-(XIII), -AZ 3 - is selected from: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka]

[0274] In some embodiments of Formulas (III)-(XIII), -AZ 3 - is selected from: [ka] [ka] and [ka]

[0275] In some embodiments of Formulas (III)-(XIII), -AZ 3 - is selected from: [ka] [ka] [ka] and [ka]

[0276] In some embodiments of Formulas (II)-(XIb), -AZ 3 is selected from: [ka] [ka] [ka] [ka] and [ka]

[0277] In some embodiments of Formulas (III)-(XIII), -AZ 3 - is selected from: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka]

[0278] In some embodiments of Formulas (III)-(XIII), Z 2 is O.

[0279] In some embodiments of Formulas (III)-(XIII), Z 2 is S.

[0280] In some embodiments of Formulas (III)-(XIII), Z 2 Ha-NR 21 -It is.

[0281] In some embodiments of Formulas (III)-(XIII), Z 2 -C(R22 )2-, where each R 22 is independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl. In some embodiments, Z 2 is -CH2-. In some embodiments, Z 2 is -CHF-. In some embodiments, Z 2 is -CF2-.

[0282] In some embodiments of Formulas (III)-(XIII), Z 2 -A-Z 3 - is the following: [ka] During the ceremony, Z 21 is O, S, or -C(R 22 )2- and R 16 is OH or CH3, w is 0 to 4 (eg, w is 0, 1, or 2).

[0283] In some embodiments, Z 21 is S or O. In some embodiments, Z 21 is -CH2-. In some embodiments, Z 21 is -CHF-. In some embodiments, Z 21 is -CF-. In some embodiments, R 16 is OH and w is 1. In some embodiments, R 16 is CH3 and w is 1. In some embodiments, w is 0.

[0284] In some embodiments of Formulas (III)-(XIII), -Z 2 -A-Z 3 - is as follows. [ka]

[0285] In some embodiments of Formulas (III)-(XII), -Z 2 -A-Z 3 - is as follows. [ka]

[0286] In some embodiments of Formulas (III)-(XIII), -Z 2 -A-Z 3 - is as follows. [ka]

[0287] In some embodiments of Formulas (III)-(XIII), -Z 2 -A-Z 3 - is as follows. [ka]

[0288] In some embodiments of Formulas (III)-(XIII), -Z 2 -A-Z 3 - is as follows. [ka]

[0289] In some embodiments of Formulas (III)-(XIII), -Z 2 -A-Z 3 - is as follows. [ka]

[0290] Prodrug Aspects of the present disclosure include prodrugs of any of the ASGPR and M6PR binding moieties described herein, which are incorporated into the compounds and conjugates of the present disclosure.

[0291] The term "prodrug" refers to an agent that is converted into a drug in vivo by some physiological or chemical process (e.g., a prodrug is converted into the desired drug form when brought to physiological pH).

[0292] Prodrug forms of any of the ASGPR or M6PR binding moieties described herein may be useful because they may provide certain therapeutic benefits, for example, as a result of increasing the half-life of the resulting compound or conjugate in the body or reducing the required active dose.

[0293] Prodrugs may be useful in some situations because they may be easier to administer than the parent drug. They may, for example, be bioavailable by oral administration whereas the parent drug is not. Prodrugs may also have improved solubility in pharmacological compositions over the parent drug.

[0294] Prodrug derivatives of ASGPR or M6PR binding moieties generally contain a promoiety substituent at an appropriate labile site on the compound, where the promoiety is a group that is removed by enzymatic or chemical reaction when the prodrug is converted to a drug in vivo.

[0295] In some embodiments, the promoiety is a group attached to a hydroxyl group of a compound or drug via an ester bond.

[0296] In some embodiments, prodrug derivatives of one or more hydroxyl groups on the sugar ring may be incorporated into the compound. For example, an ester promoiety can be incorporated into one or more of the 3- and / or 4-hydroxyl groups on the sugar (e.g., as described herein). In some embodiments, the 3- and 4-hydroxyl groups on the sugar are cyclically linked to form a promoiety (e.g., as described herein).

[0297] Linker The terms "linker," "linking moiety," and "linking group" are used interchangeably and refer to a linking moiety that covalently bonds two or more moieties or compounds, such as a ligand and another moiety of interest. In some cases, the linker is divalent and connects two moieties. In certain cases, the linker is a trivalent or higher polyvalent branched linking group. In some cases, the linker connecting two or more moieties has a linear or branched backbone that is 500 atoms or less (such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length, for example, as measured between the two or more moieties. The linking moiety can be a covalent bond connecting two groups, or a straight or branched chain of 1 to 500 atoms in length, e.g., about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 carbon atoms in length, and the linker can be straight, branched, cyclic, or single atom. In certain cases, 1, 2, 3, 4, 5, or more, 10, or more carbon atoms in the linker backbone can be optionally substituted with heteroatoms, e.g., sulfur, nitrogen, or oxygen heteroatoms. In certain examples, when the linker contains a PEG group, every third atom in that segment of the linker backbone is substituted with oxygen. The bonds between the backbone atoms can be saturated or unsaturated, and typically, there are no more than 1, 2, or 3 unsaturated bonds in the linker backbone. The linker may include one or more substituents, such as alkyl, aryl, or alkenyl groups. Linkers may include, but are not limited to, one or more of oligo(ethylene glycol), ether, thioether, disulfide, amide, carbonate, carbamate, tertiary amine, alkyl (e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc.) that may be linear or branched. The linker backbone may include a cyclic group, such as an aryl, heterocycle, cycloalkyl group, or heterocyclic group, where two or more atoms, such as 2, 3, or 4 atoms, of the cyclic group are included in the backbone.

[0298] In some embodiments, a "linker" or linking moiety is derived from a molecule having two reactive ends, one reactive end for attachment to a moiety of interest (Y), such as a biomolecule (e.g., an antibody), and the other for attachment to a moiety (designated X) that binds to an ASGPR cell surface receptor. When Y is a polypeptide, the polypeptide-binding reactive end of the linker is a site that can be attached to the polypeptide via a cysteine ​​thiol or lysine amine group on the polypeptide, as the case may be, and thus can be a thiol-reactive group such as maleimide or dibromomaleimide, or a thiol-reactive group as defined herein, or an amine-reactive group such as an active ester (e.g., a perfluorophenyl ester or a tetrafluorophenyl ester), or an amine-reactive group as defined herein.

[0299] In certain embodiments of the formulas described herein, the linker L comprises one or more straight-chain or branched-chain carbon moieties and / or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CHCHO-), as well as combinations thereof. In certain embodiments, these linkers optionally have an amide bond, a urea or thiourea bond, a carbamate bond, an ester bond, an amino bond, an ether bond, a thioether bond, a sulfhydryl bond, a heteroaryl bond, or other heterofunctional bond. In certain embodiments, the linker comprises one or more carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. In certain embodiments, the linker comprises one or more ether bonds, thioether bonds, amine bonds, amide bonds, carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, and combinations thereof. In certain embodiments, the linker comprises a linear structure. In certain embodiments, the linker comprises a branched structure. In certain embodiments, the linker comprises a cyclic structure. In certain cases, the linker comprises one or more heteroaryl ring structures, for example, a triazole, such as a 1,2,3-triazole.

[0300] In certain embodiments, the length of L is about 10 Å to about 20 Å. In certain embodiments, the length of L is about 15 Å to about 20 Å. In certain embodiments, the length of L is about 15 Å. In certain embodiments, the length of L is about 16 Å. In certain embodiments, the length of L is about 17 Å.

[0301] In certain embodiments, L is a linker of about 5 Å to about 500 Å. In certain embodiments, L is about 10 Å to about 400 Å. In certain embodiments, L is about 10 Å to about 300 Å. In certain embodiments, L is about 10 Å to about 200 Å. In certain embodiments, L is about 10 Å to about 100 Å. In certain embodiments, L is about 10 Å to about 20 Å, about 20 Å to about 30 Å, about 30 Å to about 40 Å, about 40 Å to about 50 Å, about 50 Å to about 60 Å, about 60 Å to about 70 Å, about 70 Å to about 80 Å, about 80 Å to about 90 Å, or about 90 Å to about 100 Å. In certain embodiments, L is a linker of about 5 Å to about 500 Å and comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker of about 10 Å to about 500 Å and comprises an optionally substituted arylene linked to X, or an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker of about 10 Å to about 400 Å and comprises an optionally substituted arylene linked to X, or an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker of about 10 Å to about 200 Å and comprises an optionally substituted arylene linked to X, or an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.

[0302] In certain embodiments, the linker L is a linker between X and Y (or Z 1 ) separated by a chain of 4 to 500 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z 1) separated by a chain of 4 to 50 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z). 1 ) are separated by a chain of 6 to 50 contiguous atoms, a chain of 11 to 50 contiguous atoms, a chain of 16 to 50 contiguous atoms, a chain of 21 to 50 contiguous atoms, a chain of 26 to 50 contiguous atoms, a chain of 31 to 50 contiguous atoms, a chain of 36 to 50 contiguous atoms, a chain of 41 to 50 contiguous atoms, or a chain of 46 to 50 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z). 1 ) separated by a chain of 6 to 50 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z). 1 ) by a chain of 11 to 50 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z 1 ) separated by a chain of 16 to 50 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z). 1 ) separated by a chain of 21 to 50 contiguous atoms. In certain embodiments, the linker L separates X and Y (or Z 1 ) by a chain of 26 to 50 consecutive atoms. In certain embodiments, the linker L separates X and Y (or Z 1 ) by a chain of 31 to 50 consecutive atoms. In certain embodiments, the linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, the linker L separates X and Y (or Z). 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, the linker L separates X and Y (or Z 1 ) are separated by a chain of 46 to 50 consecutive atoms.

[0303] In certain embodiments, the linker L is a linker between X and Y (or Z 1) are separated by a chain of 4 or 5 consecutive atoms, a chain of 6 to 10 consecutive atoms, a chain of 11 to 15 consecutive atoms, a chain of 16 to 20 consecutive atoms, a chain of 21 to 25 consecutive atoms, a chain of 26 to 30 consecutive atoms, a chain of 31 to 35 consecutive atoms, a chain of 36 to 40 consecutive atoms, a chain of 41 to 45 consecutive atoms, or a chain of 46 to 50 consecutive atoms.

[0304] In certain embodiments, the linker L is a linker between X and Y (or Z 1 ) and comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an optionally substituted alkylene linked to X, or a heteroatom linked to X. In certain embodiments, the linker L is a chain of 5 to 500 contiguous atoms separating X and Y (or Z). 1 ) and comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an optionally substituted alkylene linked to X, or a heteroatom linked to X. In certain embodiments, the linker L is a chain of 7 to 500 contiguous atoms separating X and Y (or Z). 1 ) and comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an optionally substituted alkylene linked to X, or a heteroatom linked to X. In certain embodiments, the linker L is a chain of 10 to 500 contiguous atoms separating X and Y (or Z). 1 ) and comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an optionally substituted alkylene linked to X, or a heteroatom linked to X.

[0305] In certain embodiments, the linker L is a linker between X and Y (or Z 1 ) and comprises an alkylene, heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, the linker L is a chain of 5 to 500 contiguous atoms separating X and Y (or Z). 1) and comprises an alkylene, heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, the linker L is a chain of 7 to 500 contiguous atoms separating X and Y (or Z). 1 ) and comprises an alkylene, heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, the linker L is a chain of 10 to 500 contiguous atoms separating X and Y (or Z). 1 ) and a chain of 15 to 400 contiguous atoms separating the heteroatom, heteroaryl, or heteroaryl groups, which includes an alkylene, heteroatom, or optionally substituted heteroarylene group linked to X.

[0306] In certain embodiments, the linker L is a linker between X and Y (or Z 1 ) and comprises an optionally substituted triazole linked to X. In certain embodiments, the linker L is a chain of 5 to 500 contiguous atoms separating X and Y (or Z). 1 ) and comprises an optionally substituted triazole linked to X. In certain embodiments, the linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z). 1 ) and comprises an optionally substituted triazole linked to X. In certain embodiments, the linker L is a chain of 10 to 500 contiguous atoms separating X and Y (or Z). 1 ) and an optionally substituted triazole linked to X.

[0307] In certain embodiments, the linker L is a chain of 16 to 400 contiguous atoms separating X and Y (or Z) and comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an optionally substituted alkylene linked to X, or a heteroatom linked to X.

[0308] The linker is Z of the ASGPR binding moiety (X). 1It is understood that the linker may be considered to be directly linked to the Z group (e.g., as described herein). In some embodiments of any of Formulas (Ia)-(Ip), the linker is 1 Alternatively, -Z may be considered to be directly linked to the group. 1 -L 1 The - group (e.g., as described herein) can be considered part of the linking moiety connecting L to Y. The present disclosure is intended to include all such arrangements of ASGPR binding moieties (X) and linkers (L).

[0309] In some embodiments of Formula (I)-(Ia), L is a linker of formula (VI): [ka] (II) During the ceremony, L 1 and L 3 are independently linkers, and L 2 is the branched junction, and L 1 ~L 3 together form a linear or branched linker between X and Y, a, b, and c are independently 0 or 1; ** is Z 1 X's L via 1 represents the point of attachment to *** represents the point of attachment to Y; where: If n is 1, a is 1 and b is 0; If n>1, a is 1 and b is 1.

[0310] In some embodiments of Formula (I), L is a linker of Formula (XI): [ka] (XI) During the ceremony, L 1 and L 3 are each independently a linear linking moiety, and L 2is the branched junction, and L 1 ~L 3 together form a linear or branched linker between X and YB, a, b, and c are independently 0 or 1; * is Z 1 via L 1 represents the point of attachment of X to ** represents the point of attachment of the linker L to YB; where: When n is 1, b is 0 and at least one of a and c is 1; When n is 2 or 3, a, b, and c are each 1.

[0311] In some embodiments of the linker of formula (XI), n is 1, a is 1, b is 0, and c is 1, such that the linker L is of formula (XIa). [ka] (XIa)

[0312] In some embodiments of the linker of formula (XI), n is 1, a is 1, b is 0, and c is 0, such that the linker L is of the formula: [ka]

[0313] In certain embodiments, the linear linker of formula (XIa) is Z 1 In certain embodiments of Formula (XIa), the linear linker has a backbone of 10 or more consecutive atoms, e.g., 12 or more consecutive atoms, 14 or more consecutive atoms, 16 or more consecutive atoms, and in some cases up to 100 consecutive atoms, covalently linking X to YB via 1 ) and YB are separated by a chain of 20 to 50 consecutive atoms. In certain embodiments of Formula (Xa), the linear linker L is a linker between X (or Z 1 ) and YB are separated by a chain of 30 to 60 consecutive atoms.

[0314] In some embodiments of the linker of formula (XI), n is 2, a is 1, b is 1, and c is 1, such that the linker L is of formula (XIb). [ka]

[0315] In some embodiments of the linker of formula (XI), n is 3, a is 1, b is 1, and c is 1, such that the linker L is of formula (XIc). [ka]

[0316] In some embodiments of the linker of any one of formulas (XI) or (XIa)-(XIc), each L 1 is of formula (XII) [ka] During the ceremony, L 10 is the linking part, * is Z 1 via L 1 represents the point of attachment of X to L 11 ~L 19 is independently absent or a linking moiety, Each L 1 L 10 ~L 19 are respectively, -C 1~6 -Alkylene-, -C 1~12 -Alkylene-, -C 1~20 -Alkylene-, -NHCO-C 1~6 -Alkylene-, -CONH-C 1~6 -Alkylene-, -NH-C 1~6 -Alkylene-, -NHCONH-C 1~6 -Alkylene-, -NHCSNH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHCO-, -C 1~6 -Alkylene-CONH-, -C1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHCONH-, -C 1~6 -Alkylene-NHCSNH-, -O(CH2) p -, -(OCH2CH2) p -, -NHCO-, -CONH-, -NHSO2-, -SO2NH-, -NHCONH-, -NHCSNH-, -CO-, -SO2-, -O-, -S-, pyrrolidine-2,5-dione, 1,2,3-triazole, -NH-, -N(C 1~6 -N(CH3)-, -N(CH4)-, ...

[0317] In certain embodiments of Formula (XII), the linking moiety L 1 comprises a linear backbone of 6 to 40 consecutive atoms, e.g., 10 to 40, 10 to 30, 16 to 30, or 20 to 30 consecutive atoms. In certain embodiments of Formula (XII), the linking moiety L 1 comprises a linear backbone of 6 to 20 consecutive atoms, for example, 6 to 16 consecutive atoms, for example, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive atoms.

[0318] In certain embodiments, the linking moiety of formula (XII) comprises one or more repeating ethylene glycol moieties (e.g., -CH2CH2O- or -OCH2CH2-). In certain cases, the linking moiety of formula (XII) comprises 1 to 10 ethylene glycol moieties, e.g., 1, 2, 3, 4, 5, or 6 ethylene glycol moieties.

[0319] In certain embodiments, the linking moiety of formula (XII) comprises one or more triazole (e.g., 1,2,3-triazole)-containing linking moieties. It is understood that the triazoles may be derived from azide-alkyne click chemistry and thus have two possible orientations depending on the synthetic method. [ka] or [ka]

[0320] In certain embodiments, the triazole-containing linking moiety is: [ka] or [ka] In the formula, w1 and u1 are independently 0 to 12, for example, 0, 1, 2, 3, 4, 5, or 6.

[0321] In some embodiments of the linker of formula (XI), b is 1 and L 2 is of formula (XIIIa) or (XIIIb), [ka] During the ceremony, L 20 represents an amino acid residue (e.g., a residue such as Gly, Ala, beta-Ala, Lys, Orn, Asp, Glu, Ser, Cys, or a derivative thereof), -NH-CH[(CH2) q ]2O- or -NH-C[(CH2) q ]3O-, [ka] -C 1~6 -Alkylene-, -NHCO-, -CONH-, -NHSO2-, -SO2NH-, -CO-, -SO2-, -O-, -S-, pyrrolidine-2,5-dione, 1,2,3-triazole, -NH-, -Nme-, -NHC(=O)NH-, -NHC(=S)NH-, -O(CH2) p - and -(OCH2CH2) p - a branched linking moiety comprising one or more linking moieties independently selected from wherein each p is independently 1 to 50, and q is 1 to 6.

[0322] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is selected from one of (L2A) to (L2D), [ka] [ka] [ka] and [ka] During the ceremony, Z 2 and Z 3 are each independently selected from absent or -NHCO-, -CONH-, -CO-, -O-, -NH-, and -Nme-; x is 1 to 12 (e.g., 1 to 6, or 1 to 3), y is 0 to 12 (for example, 1 to 6, or 1 to 3).

[0323] In some embodiments of any one of L2A to L2D, Z 2 is -NHCO-. In some embodiments of any one of L2A-L2D, Z 2 is -CONH-. In some embodiments of any one of L2A-L2D, Z 2 is -CO-. In some embodiments of any one of L2A-L2D, Z 2 is —O—. In some embodiments of any one of L2A-L2D, Z 2 is -NH-. In some embodiments of any one of L2A-L2D, Z 2 is -Nme-. In some embodiments of any one of L2A-L2D, Z 2 does not exist.

[0324] In some embodiments of any one of L2A to L2D, Z3 is -NHCO-. In some embodiments of any one of L2A-L2D, Z 3 is -CONH-. In some embodiments of any one of L2A-L2D, Z 3 is -CO-. In some embodiments of any one of L2A-L2D, Z 3 is —O—. In some embodiments of any one of L2A-L2D, Z 3 is -NH-. In some embodiments of any one of L2A-L2D, Z 3 is -Nme-. In some embodiments of any one of L2A-L2D, Z 3 does not exist.

[0325] In some embodiments of L2A, Z 2 is -O-, y is 0, and the linking moiety is structure L2Ai. [ka]

[0326] In some embodiments of L2B, Z 2 is -O- or -CO-, and the linking moiety is structure L2Bi or L2Bii. [ka] [ka]

[0327] In some embodiments of L2C, Z 2 is -O-, -CO-, -NHCO-, or -NH-, and the linking moiety is structure L2Ci, L2Cii, L2Ciii, or L2Civ. [ka] [ka] [ka] [ka]

[0328] In some embodiments of L2D, Z 2 does not exist, and the linking portion has the structure L2Di. [ka]

[0329] In some embodiments of any one of formulas L2A through L2Di, x is 1 to 6. Optionally, x is 1 to 3. Optionally, x is 1. Optionally, x is 2. Optionally, x is 3.

[0330] In some embodiments of any one of Formulas L2A through L2Di, y is 0 to 6. In some cases, y is 0 to 3. In some cases, y is 0. In some cases, y is 1. In some cases, y is 2. In some cases, y is 3.

[0331] In certain embodiments of Formula (XI), b is 1 and the linking moiety L 2 is selected from: [ka] [ka] [ka] and [ka]

[0332] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is of formula (XIV), [ka] During the ceremony, r is 1 or 2, If n is 2, then r is 1, If n is 3, then r is 2.

[0333] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is of formula (Xva) or (XVb), [ka] [ka] During the ceremony, r is 1 or 2, If n is 2, then r is 1, If n is 3, then r is 2.

[0334] In some embodiments, L 2 is of formula (XIIIa) or (XIIIb), and L 2 comprises two or more amino acid residues (e.g., three or more or four or more amino acid residues, linear or dendrimer). In some embodiments, L 2 comprises four or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., the side chain, amino, and carboxylic acid are each linked to adjacent moieties).

[0335] In some embodiments of the linker of any one of Formula (XI) or (Xa)-(Xc), each L 3 is of formula (XVI) [ka] During the ceremony, L 30 ~L 39 is independently absent or a linking moiety, Z is a residue moiety resulting from the covalent bond between the chemoselective ligation group of the linker and the compatibility group of YB; L 30 ~L 39 are respectively, -C 1~20 -Alkylene-, -NHCO-C 1~6 -Alkylene-, -CONH-C 1~6 -Alkylene-, -NHC 1~6 -Alkylene-, -NHCONH-C 1~6 -Alkylene-, -NHCSNH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHCO-, -C 1~6 -Alkylene-CONH-, -C 1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHCONH-, -C 1~6 -Alkylene-NHCSNH-, -O(CH2) p -, -(OCH2CH2) p and independently selected from -, -NHCO-, -CONH-, -NHSO2-, -SON2NH-, -NHCONH-, -NHCSNH-, -CO-, -SO2-, -O-, -S-, pyrrolidine-2,5-dione, 1,2,3-triazole, -NH-, and -Nme-, where each p is independently 1 to 50.

[0336] In certain embodiments, the linking moiety of formula (XVI) comprises a linear backbone of 6 to 40 consecutive atoms, for example, 10 to 40, 10 to 30, or 20 to 30 consecutive atoms.

[0337] In certain embodiments, the linking moiety of formula (XVI) includes repeating ethylene glycol moieties (e.g., -CHCHO- or -OCHCH-). In certain cases, the linking moiety of formula (XVI) includes 2 to 20 ethylene glycol moieties, e.g., 2 to 15, 2 to 10, 3 to 20, 3 to 15, 3 to 10, 4 to 15, 5 to 15, or 5 to 10 ethylene glycol moieties. In some cases, the linking moiety of formula (XVI) includes more than one ethylene glycol moiety, e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or even more ethylene glycol moieties.

[0338] In certain embodiments, the linking moiety of formula (XVI) comprises one or more triazole linking moieties. In some cases, the linker comprises one or more 1,2,3-triazole linking moieties. In certain cases, the one or more 1,2,3-triazole linking moieties are selected from one of the following structures: [ka] [ka] [ka] [ka] [ka] In the formula, w1, u1, and q1 are independently 1 to 25 (for example, 1 to 12, for example, 1 to 6).

[0339] In certain embodiments, the linking moiety L 3 is (C 10 ~C 20 -Alkylene (e.g., C 12 -alkylene), or -(OCH2CH2) p -, where p is 1 to 25 (e.g., 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25, or 20 to 24).

[0340] In some embodiments, the linker L is of Formula XVII: [ka] During the ceremony, a is 0 to 12 (e.g., 2 to 6, or 2, or 3), b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2, d is 1 to 6 (e.g., 1, 2, or 3); e is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); Z is a residue moiety resulting from the covalent bond between a chemoselective ligation group (eg, as described herein) of a linker precursor and a compatible group of YB.

[0341] In some embodiments of Formula XVII, Z is a residue moiety resulting from the covalent bond (e.g., via a thioether bond) between a thiol-reactive chemoselective ligation group and one or more cysteine ​​residues of YB. In some embodiments, the thiol-reactive chemoselective ligation group includes maleimide, bromomaleimide, haloacetamide, vinyl sulfone, or thiolactone. In some embodiments, the thiol-reactive group is selected from one of the following structures: [ka] [ka] and [ka] During the ceremony, u is 1 to 11 (e.g., 1 to 5), v is 1 to 11 (e.g., 1 to 5), X is H or Br.

[0342] In some embodiments of Formula XVII, Z is a residue moiety resulting from the covalent attachment (e.g., via an amide bond) of an amine-reactive chemoselective ligation group to one or more lysine residues of Y. In some embodiments, the amine-reactive chemoselective ligation group includes an active ester (e.g., N-hydroxysuccinimidyl (NHS) ester, sulfo-NHS ester, pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester, etc.).

[0343] In some embodiments, the linker L comprises one of (XVIIIa)-(XVIIIc): [ka] [ka] or [ka] During the ceremony, a is 0 to 12 (e.g., 2 to 6, or 2, or 3), b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2, d is 1 to 6 (e.g., 1, 2, or 3); e is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (for example, 1, 2, or 3).

[0344] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2 to 6, for example, 2 to 3. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6.

[0345] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), b is 1 to 4, for example, 1 to 3. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3.

[0346] In any one embodiment of Formula (XVII) or (XVIIIa)-(XVIIIc), c is 1 to 4, for example, 1 to 3. In some embodiments, c is 1. In some embodiments, c is 2. In some embodiments, c is 3.

[0347] In embodiments of any one of Formulas (XVII) or (XVIIIa) through (XVIIIc), r is 1. In some embodiments, r is 2.

[0348] In any one embodiment of Formula (XVII) or (XVIIIa)-(XVIIIc), d is 1 to 4, for example, 1 to 3. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3.

[0349] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), e is 1 to 5, for example, 1 to 3. In some embodiments, e is 1. In some embodiments, e is 2. In some embodiments, e is 3. In some embodiments, e is 4. In some embodiments, e is 5.

[0350] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), f is 1 to 4, for example, 1 to 3. In some embodiments, f is 1. In some embodiments, f is 2. In some embodiments, f is 3.

[0351] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 1-4, b is 1-4, c is 1-3, r is 1, d is 1-3, e is 1-6, and f is 1-3.

[0352] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 1-4, b is 1-4, c is 1-3, r is 2, d is 1-3, e is 1-6, and f is 1-3.

[0353] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2, b is 1, c is 2, r is 1, d is 2, e is 3, and f is 2.

[0354] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2, b is 1, c is 2, r is 2, d is 2, e is 3, and f is 2.

[0355] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 4, b is 1, c is 2, r is 1, d is 2, e is 3, and f is 2.

[0356] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 4, b is 1, c is 2, r is 2, d is 2, e is 3, and f is 2.

[0357] In one embodiment of any one of formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2, b is 2, c is 2, r is 1, d is 2, e is 3, and f is 2.

[0358] In one embodiment of any one of formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2, b is 2, c is 2, r is 2, d is 2, e is 3, and f is 2.

[0359] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 0, b is 3, c is 2, r is 2, d is 2, e is 3, and f is 2.

[0360] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2, b is 4, c is 2, r is 2, d is 2, e is 3, and f is 2.

[0361] In some embodiments of any one of Formulas (XVII) or (XVIIIa)-(XVIIIc), a is 2, b is 4, c is 2, r is 1, d is 2, e is 3, and f is 2.

[0362] In some embodiments, the linker L is L A Including, [ka] During the ceremony, Z 4 is selected from -NHC(O)NH-, -NHC(O)-, -C(O)NH-, -O-, -NH-; a is 0 to 12 (e.g., 2 to 6, or 2, or 3), b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); d is 1 to 6 (e.g., 1, 2, or 3); e is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (for example, 1, 2, or 3).

[0363] L A In some embodiments, Z 4 is -NHC(O)NH-. In some cases, Z 4 is -NHC(O)-. In some cases, Z 4 is -C(O)NH-. In some cases, Z 4 is -O-. In some cases, Z 4 is -NH-.

[0364] L A In some embodiments, a is 1 to 4, b is 1 to 4, c is 1 to 3, d is 1 to 3, e is 1 to 6, and f is 1 to 3. In some embodiments, a is 4, b is 1, c is 2, d is 2, e is 5, and f is 2.

[0365] In some embodiments, Z 4 is -NHC(O)NH-, a is 1 to 4, b is 1 to 4, c is 1 to 3, r is 1, d is 1 to 3, e is 1 to 6, and f is 1 to 3. In some embodiments, Z 4 is -NHC(O)-, a is 1 to 4, b is 1 to 4, c is 1 to 3, r is 1, d is 1 to 3, e is 1 to 6, and f is 1 to 3.

[0366] In some embodiments, the linker L is L B Including, [ka] During the ceremony, a is 0 to 12 (e.g., 2 to 6, or 2, or 3), b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2, d is 1 to 6 (e.g., 1, 2, or 3); e is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (for example, 1, 2, or 3).

[0367] L B In some embodiments, a is 1-4, b is 1-4, c is 1-3, r is 1, d is 1-3, e is 1-6, and f is 1-3. In some embodiments, a is 4, b is 1, c is 2, r is 1, d is 2, e is 5, and f is 2. In some embodiments, a is 2, b is 1, c is 2, r is 1, d is 2, e is 3, and f is 2. In some embodiments, a is 4, b is 1, c is 2, r is 1, d is 2, e is 3, and f is 2. In some embodiments, a is 1, b is 2, c is 2, r is 1, d is 2, e is 3, and f is 2. In some embodiments, a is 0, b is 3, c is 2, r is 1, d is 2, e is 3, and f is 2.

[0368] L B In some embodiments, a is 1-4, b is 1-4, c is 1-3, r is 2, d is 1-3, e is 1-6, and f is 1-3. In some embodiments, a is 2, b is 1, c is 2, r is 2, d is 2, e is 3, and f is 2. In some embodiments, a is 4, b is 1, c is 2, r is 2, d is 2, e is 3, and f is 2. In some embodiments, a is 1, b is 2, c is 2, r is 2, d is 2, e is 3, and f is 2. In some embodiments, a is 0, b is 3, c is 2, r is 2, d is 2, e is 3, and f is 2.

[0369] In some embodiments, the linker L is L C Including, [ka] During the ceremony, a is 0 to 12 (e.g., 1 to 6, 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1 to 4, e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1 to 3, e.g., 1, 2, or 3), r is 1 or 2, d is 1 to 6 (e.g., 1 to 3, e.g., 1, 2, or 3), e is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (eg, 1 to 3, for example, 1, 2, or 3).

[0370] L c In some embodiments, a is 1 to 4, b is 1 to 4, c is 1 to 3, r is 1, d is 1 to 3, e is 1 to 6, and f is 1 to 3. In some embodiments, a is 2, b is 4, c is 2, r is 1, d is 2, e is 5, and f is 2.

[0371] L c In some embodiments, a is 1 to 4, b is 1 to 4, c is 1 to 3, r is 2, d is 1 to 3, e is 1 to 6, and f is 1 to 3. In some embodiments, a is 2, b is 4, c is 2, r is 2, d is 2, e is 5, and f is 2.

[0372] In certain embodiments of the ASGPR binding moiety (X) described herein, -Z 1 - is (e.g., in a linker described herein) -L 1 In some embodiments, the subject compounds comprise a linking moiety selected from the following: -Z 1 -L 1 - includes parts, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] In the formula, each R 21 is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 is H, halogen (e.g., F) and optionally substituted (C 1~ C6) alkyl, and o, p, q, r, s, t, u, v, w, x, y, z, and z1 are each independently 1 to 6.

[0373] In certain embodiments, Z 1 -L 1 -The base is [ka] and o is 1 or 2.

[0374] In certain embodiments, Z 1 -L 1 -The base is [ka] and each R 22 is H and p is 1 or 2.

[0375] In certain embodiments, Z 1 -L 1 -The base is [ka] where q is 1 to 3.

[0376] In certain embodiments, Z 1 -L 1 -The base is [ka] where r is 1 to 3.

[0377] In certain embodiments, Z 1 -L 1 -The base is [ka] where r is 1 to 3.

[0378] In certain embodiments, Z 1 -L 1 -The base is [ka] where s and t are each independently 1 to 3.

[0379] In certain embodiments, Z 1 -L 1 -The base is [ka] where u is 1 to 3.

[0380] In certain embodiments, Z 1 -L 1 -The base is [ka] where v and w are each independently 1 to 3.

[0381] In certain embodiments, Z 1 -L 1 -The base is [ka] where x is 0 to 3.

[0382] In certain embodiments, Z 1 -L 1 -The base is [ka] where y is 1 to 3.

[0383] In certain embodiments, Z 1 -L 1 -The base is [ka] where each R 21 is H and z is 1 to 4.

[0384] In certain embodiments, Z 1 -L 1 -The base is [ka] where each R 21 is H and z1 is 1 to 4.

[0385] In certain embodiments, Z 1-L 1 -The base is [ka] where each R 22 is H and p is 1 to 3.

[0386] In certain embodiments, Z 1 -L 1 -The base is [ka] where q is 1 to 3.

[0387] In some embodiments, the subject compounds comprise a linking moiety selected from: -Z 1 -L- moiety, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] In the formula, R 21 is independently selected from H and optionally substituted (C-C) alkyl (e.g., methyl), and each R 22 is independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl (e.g., methyl). In certain embodiments, R 21 is H. In certain embodiments, each R 22 is H.

[0388] In certain embodiments, -Z 1 -L 1 -The base is [ka] where q is 1 to 3. In certain cases, q is 1. In certain cases, q is 2. In certain cases, q is 3.

[0389] In certain embodiments, -Z 1 -L 1 -The base is [ka] is.

[0390] In certain embodiments, -Z 1 -L 1- includes optionally substituted -NH-heteroarylene-. In certain embodiments, heteroarylene is triazole. In certain cases, heteroarylene is pyridine. In certain cases, heteroarylene is pyrimidine. In certain cases, heteroarylene is thiadiazole.

[0391] In certain embodiments, -Z 1 -L 1 - includes a group selected from: [ka] and [ka] In the formula, R 24 and R 25 are H, optionally substituted C, (1~6) -alkyl, optionally substituted fluoroalkyl, and halogen; 21 is independently selected from H, optionally substituted (C1-C6) alkyl, and optionally substituted alkanoyl. In certain cases, R 21 is H. In certain cases, R 24 is C (1~3) -Alkyl, or C (1~3) -fluoroalkyl. In some cases, the fluoroalkyl is CF3. In certain cases, R 25 is C (1~3) -Alkyl, or C (1~3) -fluoroalkyl. In some cases, the fluoroalkyl is CF3.

[0392] In some embodiments, the linker comprises a polypeptide scaffold, in which some or all of the side groups of amino acid residues of the polypeptide scaffold have been modified for attachment to X-linked moieties (e.g., as described herein). It is understood that the X-linked moieties (e.g., as described herein) can be attached to amino acid residues of the polypeptide-containing linker (e.g., Asp, Lys, Orn, Glu, and Ser) via convenient conjugation chemistries. In some embodiments, the linker comprises a polylysine polypeptide. In some embodiments, the linker comprises a polyornithine polypeptide. In some embodiments, the linker comprises a polyserine polypeptide. In some embodiments, the linker comprises a polyaspartic acid polypeptide. The polypeptide backbone of the linker can be a randomly polymerized polymer having an average length, or a polymer of a defined length, prepared, for example, by a controlled, stepwise method. In some cases, the polypeptide linker has a length of 10 to 100 amino acid residues, e.g., 20 to 90, or 20 to 50 amino acid residues. In some embodiments, the N-terminus or C-terminus of the polypeptide linker is modified to include a linking moiety to an additional X-linked moiety (e.g., as described herein). In some embodiments, the N-terminus or C-terminus of the polypeptide linker segment is modified with one or more linking moieties suitable for attachment to a protein construct (YB) comprising a polypeptide that specifically binds to a target autoantibody (e.g., as described herein).

[0393] In certain embodiments of the linker of formula (II), L 1 ~L 3 are each independently -C 1~20 -Alkylene-, -NHCO-C 1~6 -Alkylene-, -CONH-C 1~6 -Alkylene-, -NHC 1~6 -Alkylene-, -NHCONH-C 1~6 -Alkylene-, -NHCSNH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHCO-, -C 1~6 -Alkylene-CONH-, -C1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHCONH-, -C 1~6 -Alkylene-NHCSNH-, -O(CH2) p -, -(OCH2CH2) p and one or more linking moieties independently selected from -, -NHCO-, -CONH-, -NHSO2-, -SONH-, -CO-, -SO2-, -O-, -S-, monocyclic heteroaryl (e.g., 1,2,3-triazole), monocyclic aryl (e.g., phenyl, e.g., 1,4-linked phenyl or 1,3-linked phenyl), monocyclic heterocycle (e.g., a pyrrolidine-2,5-dione, piperazine, or piperidine ring as described herein), amino acid residue (natural or unnatural amino acid residue), -NH-, and -Nme-, where each p is independently 1 to 50.

[0394] In certain embodiments of the linker of formula (II), L 1 ~L 3 and (II) each contain repeating ethylene glycol moieties (e.g., -CH2CHO- or -OCH2CH2-). In certain cases, the linker of formula (II) contains 1 to 25 ethylene glycol moieties, e.g., 3 to 25, 5 to 25, 7 to 25, 10 to 25, 15 to 25, 17 to 25, 20 to 25, or 22 to 25 ethylene glycol moieties. In some cases, the linker of formula (II) contains 3 or more ethylene glycol moieties, e.g., 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, or even more ethylene glycol moieties.

[0395] In certain embodiments of the linker of formula (II), L 1 ~L 3 Any of the above may contain one or more triazole linking moieties. In some cases, the linker contains one or more 1,2,3-triazole linking moieties. In certain cases, the one or more 1,2,3-triazole moieties are selected from one of the following structures: [ka] [ka] [ka] [ka] [ka] In the formula, w1, u1, and q1 are independently 1 to 25 (for example, 1 to 12, for example, 1 to 6).

[0396] In certain embodiments of the linker of formula (II), n is 1, such that b is 0, and the linker is of formula (IIa): [ka] During the ceremony, L 1 and L 3 are independently a linker (e.g., as described herein), and L 1 ~L 3 together form a linear linker between X and Y, a is 1, c is 0 or 1, ** is Z 1 X's L via 1 represents the point of attachment to *** represents the point of attachment to Y.

[0397] In certain embodiments of the linker of formula (IIa), the linear linker is Z 1 In certain embodiments of Formula (IIa), the linear linker has a backbone of 20 or more consecutive atoms, e.g., 25 or more consecutive atoms, or 30 or more consecutive atoms, and in some cases up to 100 consecutive atoms, covalently linking X to Y (or Z) via 1 ) separated by a chain of 20 to 50 contiguous atoms. In certain embodiments, the linear linker L separates X and Y (or Z). 1) are separated by a chain of 21 to 50 consecutive atoms, a chain of 22 to 50 consecutive atoms, a chain of 23 to 50 consecutive atoms, a chain of 24 to 50 consecutive atoms, a chain of 25 to 50 consecutive atoms, a chain of 26 to 50 consecutive atoms, a chain of 27 to 50 consecutive atoms, a chain of 28 to 50 consecutive atoms, or a chain of 29 to 50 consecutive atoms. In certain embodiments of Formula (IIa), the linear linker is a linker between X and Y (or Z). 1 ) separated by a chain of 30 to 60 contiguous atoms. In certain embodiments, the linear linker is a linker that separates X and Y (or Z 1 ) separated by a chain of 31 to 60 contiguous atoms. In certain embodiments, the linear linker is 1 ) separated by a chain of 32 to 60 contiguous atoms. In certain embodiments, the linear linker is 1 ) separated by a chain of 33 to 60 contiguous atoms. In certain embodiments, the linear linker is 1 ) separated by a chain of 34 to 60 contiguous atoms. In certain embodiments, the linear linker is 1 ) separated by a chain of 35 to 50 contiguous atoms. In certain embodiments, the linear linker is 1 ) separated by a chain of 36 to 50 consecutive atoms. In certain embodiments, the linear linker is a linker between 4X and Y (or Z 1 ) separated by a chain of 1 to 50 contiguous atoms. In certain embodiments, the linear linker is a linker that separates X and Y (or Z 1 ) are separated by a chain of 46 to 50 consecutive atoms.

[0398] In certain other embodiments of Formula (II), n is 2 or greater, such that L 1 ~L 3 together form a branched linker between X and Y.

[0399] In certain embodiments of Formula (II), n is 2 and L 2 is selected from the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] In the formula, x and y each independently represent an integer of 1 to 10.

[0400] In certain embodiments of Formula (II), L 1 ~L 2 comprises a backbone of 14 or more consecutive atoms between X and the branching atom, e.g., 14 to 50, 14 to 40, 14 to 35, or 14 to 30 consecutive atoms between X and the branching atom.

[0401] In certain embodiments of Formula (II) or (IIa), L 3 comprises a backbone of 10 to 80 consecutive atoms, e.g., 12 to 70, 12 to 60, or 12 to 50 consecutive atoms.

[0402] In certain embodiments of Formula (II) or (IIa), L 3 is (C 10 ~C 20 -Alkylene (e.g., C 12 -alkylene), or -(OCH2CH2) p-, where p is 1 to 25, e.g., 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25, or 20 to 24.

[0403] In certain embodiments, L is of formula (IIb): [ka] During the ceremony, L 1 ~L 5 are each independently a linking moiety, and together they form Z 1 and Y, forming a linear or branched linker therebetween; a, b, c, d, and e are each independently 0, 1, or 2; ** is Z 1 X's L via 1 represents the point of attachment to *** represents the point of attachment to Y; where: If n is 1, a is 1 and c is 0; If n>1, a is 1 and c is 1.

[0404] In certain embodiments of the linker of formula (IIb), L 1 ~L 5 are each independently -C 1~20 -Alkylene-, -NHCO-C 1~6 -Alkylene-, -CONH-C 1~6 -Alkylene-, -NHC 1~6 -Alkylene-, -NHCONH-C 1~6 -Alkylene-, -NHCSNH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHCO-, -C 1~6 -Alkylene-CONH-, -C 1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHCONH-, -C 1~6 -Alkylene-NHCSNH-, -O(CH2) p -, -(OCH2CH2) pand one or more linking moieties independently selected from -, -NHCO-, -CONH-, -NHSO2-, -SONH-, -CO-, -SO2-, -O-, -S-, monocyclic heteroaryl (e.g., 1,2,3-triazole), monocyclic aryl (e.g., phenyl, e.g., 1,4-linked phenyl or 1,3-linked phenyl), monocyclic heterocycle (e.g., a pyrrolidine-2,5-dione, piperazine, or piperidine ring as described herein), amino acid residue (natural or unnatural amino acid residue), -NH-, and -Nme-, where each p is independently 1 to 50.

[0405] In certain embodiments of Formula (IIb), -(L 1 ) a - includes an optionally substituted alkyl or ethylene glycol linking moiety. In certain cases, L 1 is an optionally substituted -C 1~6 In certain cases, L 1 contains an ethylene glycol linking moiety.

[0406] In certain embodiments of Formula (IIb), L 1 -C 1~6 -Alkylene-, -(CH2CH2O) t -, -C 1~6 -Alkylene-NR 4 CO-, -C 1~6 -alkyleneCONH-, or OCH2, where t is 1 to 20; R 4 is independently selected from H and optionally substituted (C1-C6) alkyl. In certain cases, L 1 Ha-C 1~6 -Alkylene-, e.g. -C 1~3 -alkylene-. In certain cases, L 1 Ha-(CH2CH2O) t -, where t is 1 to 20, e.g., 1 to 15, 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In certain cases, L 1 Ha-C 1~6 -Alkylene-NR 4 CO-. In certain cases, L 1 Ha-C1~6 -alkyleneCONH-. In certain cases, L 1 or OCH 2 is.

[0407] In some embodiments of Formula (IIb), one or more L 1 are independently -CH2O-, -(CH2CH2O) t -, -NR 4 CO-, -C 1~6 -alkylene-, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and In the formula, R 13 is H, halogen, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C1-C6) alkoxy, COOH, NO2, CN, NH2, -N(R 21 )2, -OCOR 21 , -COOR 21 , -CONHR 21 , and -NHCOR 21 is selected from Each r is independently 0 to 20, and L 1 Any of the moieties are optionally further substituted.

[0408] In certain embodiments of Formula (IIb), L 2 is -NR 4’ CO-C 1~6 -Alkylene-, -CONR 4’ -C 1~6 alkylene, [ka] [ka] [ka] [ka] [ka] -OCH2- and -(OCH2CH2) q -, wherein q is 1 to 10, u is 0 to 10, w is 1 to 10, and R 4’ is independently selected from H and optionally substituted (C1-C6) alkyl. In certain cases, L 2 Ha-NR 4’ CO-C 1~6 -alkylene-. In certain cases, L 2 -CONR 4’ -C 1~6 - alkylene.

[0409] In certain cases, L 2 teeth, [ka] where w is 1 and u is 0 or 1.

[0410] In certain cases, L 2 teeth, [ka] where w is 1 and u is 0 or 1.

[0411] In certain cases, L 2 teeth, [ka] wherein w is 1, u is 0 or 1, and q is 1.

[0412] In certain cases, L 2 teeth, [ka] where u is 0 or 1.

[0413] In certain cases, L 2 teeth, [ka] is.

[0414] In certain cases, L 2 is —OCH—. In certain other embodiments, L 2 is (OCH2CH2) q - and q is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In certain cases, q is 2 to 8, for example, 2 to 6, 4 to 6, or 2 to 4.

[0415] In certain embodiments of Formula (IIb), L 4 does not exist or -C 1~6 -Alkylene-, -(CH2CH2O) t -, -C 1~6 -Alkylene-NHCO-, -C 1~6 -alkyleneCONH-, or OCH2, where t is 1 to 20. In certain cases, L 4 does not exist. In certain cases, L 4 Ha-C1~6 -alkylene-. In certain cases, L 4 Ha-(CH2CH2O) t -, where t is 1 to 20, e.g., 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 3. In certain cases, L 4 Ha-C 1~6 -alkylene-NHCO-. In certain cases, L 4 Ha-C 1~6 -alkyleneCONH-. In certain cases, L 4 is OCH2.

[0416] In some embodiments of the subject compounds, n is 1 and L in formula (IIb) 3 does not exist.

[0417] In some embodiments of the subject compounds, n is 2 or greater, and L of formula (IIb) 3 is the branched junction.

[0418] Thus, in some embodiments of formula (IIb), L 3 is a branched linking moiety, e.g., a bivalent or trivalent linking moiety. For example, L 3 The linking moiety can be one of the following general formulas: [ka]

[0419] In some embodiments of Formula (IIb), the branched linking moiety may be of higher valency and may be represented by one of the following general formulas: [ka] etc. In the formula, any two L 3 The groups can be directly linked or linked via any linear linking moiety (eg, as described herein).

[0420] In some embodiments of Formula (IIb), the branched linking moiety comprises one, two, or more L 3 The aryl groups may include linking moieties, which when linked together can provide multiple branching points for covalent attachment of ligands, and may be represented by the general formula: [ka] In the formula, t is 0 to 500, for example, 0 to 100, 0 to 20, or 0 to 10.

[0421] In some embodiments, a branched linking moiety (e.g., L 3 ) include one or more of amino acid residues (e.g., Asp, Lys, Orn, Glu), N-substituted amides (-N(-)C(=O)-), tertiary aminos, polyols (e.g., O-substituted glycerol), and the like.

[0422] In some embodiments of Formula (IIb), one or more L 3 teeth, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] a branched moiety selected from wherein each x and y is independently 1 to 10, for example, 1 to 6, 1 to 3, for example, 1 or 2. In some cases, each x is 1, 2, or 3, for example, 2.

[0423] In some embodiments of Formula (IIb), L 5 is -CH2O-, -(CH2CH2O) t -, -NR 4 CO-, -C 1~6 -alkylene-, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] is selected from During the ceremony, R 13 is H, halogen, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C1-C6) alkoxy, COOH, NO2, CN, NH2, -N(R 21 )2, -OCOR21 , -COOR 21 , -CONHR 21 , and -NHCOR 21 is selected from Each r is independently 0 to 20, and L 5 Any of the moieties are optionally further substituted.

[0424] In certain cases, L 5 is -CH2O-. In certain cases, L 5 Ha-(CH2CH2O) t -, where t is 1 to 20, e.g., 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In certain cases, L 5 Ha-NR 4 CO—, where R 4 is H or optionally substituted (C1-C6) alkyl. In certain cases, L 5 Ha-C 1~6 -alkylene-.

[0425] In certain cases, L 5 teeth, [ka] wherein r is 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5.

[0426] In certain cases, L 5 teeth, [ka] wherein each r is independently 0 to 20, e.g., 0 to 15, 0 to 10, 0 to 8, or 0 to 5; R 13 is H or optionally substituted (C1-C6) alkyl.

[0427] In certain cases, L 5 teeth, [ka] wherein r is 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5; 13 is H or optionally substituted (C1-C6) alkyl.

[0428] In certain cases, L 5 teeth, [ka] wherein r is 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5; 13 is H or optionally substituted (C1-C6) alkyl.

[0429] In certain cases, L 5 teeth, [ka] wherein r is 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5; 13 is H or optionally substituted (C1-C6) alkyl.

[0430] In certain cases, L 5 teeth, [ka] wherein each r is independently 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5.

[0431] In certain cases, L 5 teeth, [ka] wherein each r is independently 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5.

[0432] In certain cases, L 5 teeth, [ka] wherein each r is independently 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5.

[0433] In certain cases, L 5 teeth, [ka] wherein each r is independently 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5.

[0434] In certain cases, L 5 teeth, [ka] wherein r is 0 to 20, for example, 0 to 15, 0 to 10, 0 to 8, or 0 to 5.

[0435] In some embodiments of Formula (IIb), L 5 comprises one or more of an amino acid residue (e.g., Asp, Lys, Orn, Glu), an amino acid analog, an N-substituted amide (-N(-)C(=O)-), a tertiary amino, a polyol (e.g., O-substituted glycerol), and the like. Amino acid analogs include, but are not limited to, unnatural amino acids and other modifications known in the art. Amino acids include L-amino acids, D-amino acids, or both, which may include any of a variety of amino acid modifications or analogs known in the art.

[0436] In some embodiments of Formula (IIb), L 1 ~L 5 contains one or more of the following units: [ka] In the formula, R ais (C1-C6) alkyl or substituted (C1-C6) alkyl, for example, (C1-C6) alkyl optionally substituted with an amine, a tertiary amine, an optionally substituted alkoxy, an optionally substituted carboxyl, an optionally substituted aryl, or an optionally substituted heteroaryl. a It is understood that can be linked to the M6PR binding moiety.

[0437] In some embodiments, the linker comprises a polypeptide scaffold modified (e.g., as described herein) to allow some or all of the side groups of the amino acid residues to be attached to the X-linking moiety. It is understood that the X-linking moiety (e.g., as described herein) can be attached to an amino acid residue (such as Asp, Lys, Orn, Glu, and Ser) of the polypeptide-containing linker via any convenient conjugation chemistry. In some embodiments, the linker comprises a polylysine polypeptide. In some embodiments, the linker comprises a polyornithine polypeptide. In some embodiments, the linker comprises a polyserine polypeptide. In some embodiments, the linker comprises a polyaspartic acid polypeptide. The polypeptide can be a randomly polymerized polymer having an average length, or a polymer of a defined length, prepared, for example, by a controlled, stepwise method. In some cases, the polypeptide linker segment has a length of 10 to 100 amino acid residues, e.g., 20 to 90, or 20 to 50 amino acid residues. In some embodiments, the N-terminus or C-terminus of the polypeptide linker segment is modified (e.g., as described herein) to include a linking unit to an additional M6PR-binding moiety. In some embodiments, the N- or C-terminus of the polypeptide linker segment is modified with one or more linking units suitable for attachment to the Yi moiety of interest (e.g., as described herein).

[0438] In certain embodiments of Formula (IIb), a is 1. In certain cases, at least one of b, c, d, and e is not 0. In certain cases, b is 1 or 2. In certain cases, c is 1 or 2. In certain cases, e is 1 or 2. In certain cases, b, d, and e are independently 1 or 2. In certain cases, a, b, d, and e are each 1 and c is 0.

[0439] In certain embodiments of Formula (II), (IIa), or (IIb), the linker comprises 20 to 100 consecutive atoms, e.g., 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, or 20 to 30 consecutive atoms. In certain cases, the linker comprises 25 to 100 consecutive atoms, e.g., 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, or 95 to 100 consecutive atoms.

[0440] In certain embodiments of Formula (II), (IIa), or (IIb), the linker comprises 25 or more consecutive atoms, e.g., 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more consecutive atoms. In certain embodiments of Formula (II), (IIa), or (IIb), the linker comprises 30 or more consecutive atoms, e.g., 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more consecutive atoms.

[0441] The present inventors have demonstrated that compounds of the present disclosure having a specific configuration with a linker of desired valency and length can specifically bind to both receptors and targets simultaneously with high affinity, and exhibit high target uptake activity.Therefore, the conjugates of the present disclosure can enable the sequestration of target proteins in cellular lysosomes and the degradation of target proteins.

[0442] Exemplary Linkers and Linking Moieties Exemplary linkers and linking moieties (eg, those that link an ASGPR ligand (X) to a moiety of interest (Y)) that can be used to prepare compounds of the present disclosure are shown in Tables 6-8.

[0443] In certain embodiments, the linker is a linear linker or linking moiety as shown in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0444] Table 7 includes various linker component synthetic precursors (eg, linear and branched linker precursors) that can be utilized to prepare the subject compounds. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8]

[0445] In certain embodiments, the linker is a branched linker or linking moiety as shown in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0446] Table 9 shows exemplary synthetic precursors of linker components used to prepare compounds of the present disclosure, for example, via conjugation chemistry. It is understood that various homologs of the structures shown in Table 9, providing linkers of various lengths, are also encompassed by the present disclosure. It is understood that alternative chemoselective ligation groups and other chemical functionalities can also be incorporated as needed to prepare desired linkers. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8]

[0447] Chemoselective Ligation Groups A chemoselective ligation group is a group having a reactive functional group or functional groups that can be bonded to a compatible group on a second moiety. For example, the chemoselective ligation group (or a precursor thereof) may be one of a pair of groups associated with coupling chemistries such as azide-alkyne click chemistry, copper-free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Picteth-Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol-haloacetamide, or alkyne hydrothiolation), amine-activated ester coupling, tyrosine-specific coupling chemistry (e.g., eY-CLICK), methionine-specific coupling chemistry (e.g., oxaziridine-based or ReACT chemistry), reductive amination, dialkylsquarate chemistry, etc.

[0448] Chemoselective ligation groups that can be utilized in joining two moieties include, but are not limited to, amino (e.g., the N-terminal amino group or lysine side chain group of a polypeptide), azide, arylazide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, or PFP ester or thioester), haloacetamide (e.g., iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, hydrazide, maleimide, vinyl sulfone, 2-sulfonylpyridine, cyano-alkyne, thiol (e.g., cysteine ​​residue), disulfide or protected thiol, isocyanate, isothiocyanate, aldehyde, ketone, alkoxyamine, hydrazide, aminooxy, phosphine, HIPS hydrazinyl-indolyl group, or aza-HIPS hydrazinyl-pyrrolo-pyridinyl group, tetrazine, cyclooctene, squarate, and the like.

[0449] In some cases, the chemoselective ligation group and the compatible chemical group can spontaneously bond to the compatible chemical group when the two groups, the chemoselective ligation group and the compatible chemical group, are brought into contact under appropriate conditions (e.g., copper-free click chemistry conditions). In some cases, the chemoselective ligation group can bond to the compatible chemical group when the two groups, the chemoselective ligation group and the compatible chemical group, are brought into contact in the presence of a catalyst or other reagent (e.g., copper-catalyzed click chemistry conditions).

[0450] In some embodiments, the chemoselective ligation group is a photoactive ligation group. For example, upon irradiation with ultraviolet light, a diazirine group can form a reactive carbene that can insert into the C-H, N-H, and O-H bonds of a second moiety.

[0451] In some cases, Y is a reactive functionality or a precursor of a functional group that can be attached to a compatible group on a second moiety. For example, a carboxylic acid is a precursor of an active ester chemoselective ligation group.

[0452] In certain embodiments of Formula (I), Y is a reactive moiety that can form a covalent bond with a polypeptide (e.g., with an amino acid side chain of the polypeptide that has a compatible reactive group). The reactive moiety can be referred to as a chemoselective ligation group.

[0453] In certain embodiments of Formula (I), Y is a thio-reactive chemoselective ligation group (e.g., as described in Table 10). In some cases, Y can generate a residue moiety, Z, resulting from the covalent attachment of a thiol-reactive chemoselective ligation group to one or more cysteine ​​residues of a protein (e.g., an Ab).

[0454] In certain embodiments of Formula (I), Y is a Cys-reactive chemoselective ligation group (e.g., a maleimide derivative described in Table 10). In some cases, the Cys-reactive chemoselective ligation group comprises a maleimide group. In some embodiments, the chemoselective ligation group includes a maleimide group in Table 10, e.g., mal-1 through mal-7.

[0455] In certain embodiments of Formula (I), Y is an amino-reactive chemoselective ligation group (e.g., as described in Table 10). In some cases, Y can generate a residue moiety, Z, resulting from the covalent attachment of the amine-reactive chemoselective ligation group to one or more cysteine ​​residues of a protein (e.g., an Ab).

[0456] In certain embodiments of Formula (I), Y is a Lys-reactive chemoselective ligation group (e.g., an active ester described in Table 10). In some embodiments, the Lys-reactive chemoselective ligation group is a PFP ester.

[0457] Exemplary chemoselective ligation groups, and their synthetic precursors, that can be adapted for use in the compounds of the present disclosure are shown in Table 10. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0458] In Table 10, [ka] can represent the linking moiety or the point of attachment of Y to the linked X moiety.

[0459] Table 11 shows exemplary residue moieties, where "***" indicates the point of attachment of Y. [Table 11]

[0460] Exemplary ASGPR-binding compounds with chemoselective ligation groups for preparing conjugates The present disclosure includes compounds of formula (I), which can be prepared from precursor ligand-linker compounds including: (1) one or more specific ASGPR ligands (X) (e.g., as described herein, e.g., ligands X1-X20 in Tables 1-5), or a specific ASGPR ligand (X) (e.g., as described herein), (2) a linker comprising one or more linking moieties (e.g., as described herein, e.g., any one or more of the linking moieties in Tables 6-8), and (3) A chemoselective ligation group (Y) (e.g., as described herein, e.g., any one of the groups in Table 10).

[0461] Table 12 shows various monovalent ASGPR ligand-linker compounds for use in preparing conjugates of the present disclosure. [Table 12]

[0462] Table 13 shows various multivalent ASGPR ligand-linker compounds for use in the conjugates of the present disclosure. [Table 13-1] [Table 13-2]

[0463] Tables 14-17 show several exemplary ASGPR-binding compounds of the present disclosure that include a chemoselective ligation group or a precursor thereof. It is understood that the present disclosure includes conjugates of each of the exemplary compounds in Tables 14-17, where Y (e.g., as described herein) is a different chemoselective ligation group. Included are conjugates in which the chemoselective ligation group is attached to a different Y (e.g., an antibody or antibody fragment against a target protein).

[0464] The chemoselective ligation group of such compounds can be utilized to link to another Y, moiety of interest (e.g., as described below). It is understood that any of these compounds can also be prepared de novo (e.g., as described below) to include an alternative Y, moiety of interest rather than a chemoselective ligation group. In some embodiments, such compounds are referred to as conjugates, e.g., biomolecular conjugates that specifically bind to a target protein. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] Table 15-6 Table 15-7 Table 15-8 Table 15-9 Table 15-10 Table 15-11 Table 15-12 Table 15-13 Table 15-14 Table 15-15 Table 15-16 Table 15-17 Table 15-18 Table 15-19 Table 15-20 Table 15-21 Table 15-22 [Table 15-23] [Table 15-24] [Table 15-25] [Table 15-26] [Table 16-1] [Table 16-2] [Table 17]

[0465] The present disclosure is intended to encompass any one stereoisomer of the compounds described herein. In some cases, the compounds include an enantiomer of DN-acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc.

[0466] Other exemplary compounds Table 18 shows exemplary ASGPR binding compounds of the present disclosure that include a binding moiety or precursor thereof. [Table 18]

[0467] Table 19 shows exemplary trivalent ASGPR binding intermediate compounds of the present disclosure that include the X group of formula (Ie). [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6] [Table 19-7] [Table 19-8] [Table 19-9] [Table 19-10]

[0468] Table 20 shows exemplary monovalent ASGPR-binding intermediate compounds of the present disclosure of formula (Ib), including a promoiety and an X group. [Table 20]

[0469] Table 21 shows exemplary ASGPR-binding intermediate compounds of the present disclosure of formula (In) that include an X group. [Table 21]

[0470] Table 21A shows exemplary ASGPR binding intermediate compounds. [Table 21A-1] [Table 21A-2] [Table 21A-3] Table 21A-4 Table 21A-5 Table 21A-6 Table 21A-7

Table 21A-8

Table 21A-9

Table 21A-10

Table 21A-12

Table 21A-13

Table 21A-14

Table 21A-15

Table 21A-16

Table 21A-17

Table 21A-19

[0471] Exemplary CI-M6PR Binding Compounds with Chemoselective Ligation Groups for Preparing Conjugates Exemplary M6PR binding moieties X of Formulas (I)-(XIII) that can be utilized to prepare compounds and conjugates of the present disclosure are shown in Table 22. [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 22-5] [Table 22-6] [Table 22-7]

[0472] Exemplary synthons or synthetic precursors that can be utilized to incorporate a desired M6PR binding moiety of interest in preparing compounds of the present disclosure are shown in Table 23. It is understood that alternative synthons are possible, including homologs and analogs of those shown in Table 23, depending on the M6PR binding moiety and the linker selected. The synthons in Table 23 include a linking moiety Z 3 It is understood that the structural precursors of Table 23 can include structural elements that become part of the linker (L) in the compounds and conjugates of the present disclosure. Based on the exemplary synthetic precursors of Table 23, it is understood that synthons corresponding to any of the M6PR binding moieties of Table 22 can be utilized to prepare compounds of the present disclosure. [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7] [Table 23-8] [Table 23-9]

[0473] Other M6PR binding moieties of interest and their synthons or synthetic precursors are shown in Table 24. X101-X103 represent compounds with a phosphate or thiophosphate head group. X109-X110 represent exemplary compounds of formula (V). In some embodiments, such M6PR binding moieties are used in reference compounds for evaluation of compounds of formula (XII). [Table 24-1] [Table 24-2]

[0474] Conjugates Bifunctional molecules of the present disclosure can be referred to as conjugates, for example, when the target-binding moiety of interest is a polypeptide (e.g., in the form of B or YB) (see, e.g., Formula (Ia) as described herein). Such conjugates can be prepared by combining the chemoselective ligation group of any one of the ligand moiety-linker compounds described herein with a compatible reactive group on molecule B or YB. The compatible group on molecule B or YB can be introduced by modification prior to conjugation or can be a group present in the molecule. Alternatively, such conjugates can be prepared de novo, for example, by modifying the starting B or YB molecule of interest to introduce a linker to which, for example, an ASGPR ligand moiety (X) can be attached. In some cases, the linking moiety between XL and B or YB incorporates a residue that is the product of chemoselective ligation chemistry (e.g., Z in Formula (Ia)).

[0475] As summarized above, in some embodiments of Formula (I), the bifunctional molecule is a conjugate of Formula (Ia): [ka] During the ceremony, Z is a residue moiety resulting from the covalent bond between the chemoselective ligation group of the linker and the compatibility group of YB; n is 1, 2, or 3; m is bonded to YB (X n -L) moieties, where m is in the range of about 1 to about 80 (e.g., m is 1 to 20, 1 to 10, 1 to 8, 2 to 8, 3 to 6, or 4 to 5, or m is 1 to 3, e.g., 1, 2, or 3).

[0476] In certain embodiments of Formula (Ia), Z is a residue moiety resulting from the covalent bond between a thiol-reactive chemoselective ligation group and one or more cysteine ​​residues of YB, or Z is a residue moiety resulting from the covalent bond between an amine-reactive chemoselective ligation group and one or more lysine residues of YB.

[0477] In certain embodiments of formula (Ia), YB is a chimeric fusion protein comprising a carrier polypeptide and a polypeptide that specifically binds to a target autoantibody.

[0478] In certain embodiments of Formulas (I)-(Ia), Z is a residue moiety resulting from the covalent attachment of a chemoselective ligation moiety of Table 4.

[0479] In certain embodiments of the conjugates described herein, L is attached to a lysine residue of the polypeptide via an amide bond (Z). In certain embodiments of the conjugates described herein, L is attached to a cysteine ​​residue of the polypeptide via a thioether bond (Z).

[0480] In certain embodiments, conjugation to the polypeptide may be via site-specific conjugation. Site-specific conjugation may result in, for example, uniform loading and minimization of conjugate subpopulations, which may alter antigen binding or pharmacokinetics. For example, in certain embodiments, conjugation may involve engineering cysteine ​​substitutions at positions on the polypeptide or antibody, such as positions on the heavy and / or light chains of an antibody, that provide reactive thiol groups and do not interfere with folding and assembly of the polypeptide or alter polypeptide binding.

[0481] In another non-limiting approach, selenocysteine ​​is co-translationally inserted into a polypeptide or antibody sequence by recoding the stop codon UGA from the terminus to a selenocysteine ​​insertion, which allows site-specific covalent attachment at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids (see, e.g., Hofer et al., Proc. Natl. Acad. Sci. USA 2008;105:12451-56, and Hofer et al., Biochemistry 2009;48(50):12047-57). Further non-limiting techniques that allow for site-specific conjugation to a polypeptide or antibody include engineering unnatural amino acids, such as p-acetylphenylalanine (p-acetyl-Phe), p-azidomethyl-N-phenylalanine (p-azidomethyl-Phe), and azidolysine (azido-Lys), at specific conjugation sites, as well as engineering unique functional tags, such as LPXTG, LLQGA, sialic acid, and GlcNAc, for enzyme-mediated conjugation. See Jackson, Org. Process Res. Dev. 2016;20:852-866 and Tsuchikama and An, Protein Cell 2018;9(1):33-46, the contents of each of which are incorporated herein by reference in their entireties. See also US2019 / 0060481 A1 and US2016 / 0060354 A1, the contents of each of which are incorporated herein by reference in their entireties. All such methods are contemplated for use in connection with making the conjugates described herein.

[0482] The loading of a ligand-linker moiety (e.g., Xn-L-) on a polypeptide or protein construct (e.g., YB of a conjugate of Formula (I)) refers to the number of ligand-linker moieties attached per molecule of the polypeptide or protein construct (discrete loading or average loading). Loading is related to the concept of drug-to-antibody ratio ("DAR") in antibody-drug conjugates. As used herein, loading can be referred to as linker-to-polypeptide ratio ("LPR"). The loading of a ligand-linker moiety (Xn-L-) on a polypeptide bait-HSA protein construct (YB) of Formula (I) described herein can be represented by the symbol "m". The symbol "m" in Formula (I) can refer to the average number of "Xn-L-" units per polypeptide conjugate molecule, or "m" can refer to the discrete number of "Xn-L-" units per polypeptide conjugate molecule.

[0483] The number of "X" moieties per each "Xn-L-" unit is represented by "n" in Formula (I). As used herein, the terms "valency" and "multivalent" refer to the number of "X" moieties per unit (i.e., "n"). Thus, a divalent ligand-linker moiety has n=2, and a trivalent ligand-linker moiety has n=3. It is understood that loading or LPR does not necessarily equal the total number of "X" moieties per conjugate molecule, but rather is a combination of n and m. For example, if there is one "X" moiety per unit (n=1, valency is "1") and one "Xn-L-" unit (m=1) per conjugate, there is 1 x 1 = 1 "X" moiety per conjugate. However, if there are two "X" moieties per unit (n=2, valency is "2") and about four "Xn-L-" units (m=4) per conjugate, there are about 2 x 4 = about 8 "X" moieties per conjugate. Thus, for the conjugates described herein, the total number of "X" moieties per conjugate molecule is nxm.

[0484] The LPR (loading) can range from 1 to 20 ligand-linker moieties per protein conjugate. The conjugates provided herein can include a collection of polypeptides conjugated with a range of ligand-linker moieties, e.g., 1 to 20 ligand-linker moieties. The number of ligand-linker moieties per polypeptide in a preparation of conjugates from a conjugation reaction can be characterized by conventional means, such as mass spectrometry. It is understood that the definition of "m" as a discrete or average loading value is determined in part by the nature of the chemoselective ligation chemistry and the groups utilized in preparing the conjugates. For example, stoichiometric thiol-maleimide conjugation chemistry can provide individual conjugates through site-specific conjugation to specific target cysteine ​​residues of interest. Alternatively, if amine-specific conjugation chemistry is used, conjugation to one or up to a few accessible lysine residues in the protein can occur, and the resulting conjugate preparation can contain several different species of conjugates, with loading expressed as an average number. In some embodiments, the quantitative distribution of LPR (loading) with respect to "m" can also be determined. In some cases, separation, purification, and characterization of homogeneous conjugates where "m" is a discrete value can be achieved by means such as electrophoresis.

[0485] In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 80. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 20. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 18. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 15. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 12. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 10. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 9. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 8. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 7. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 6. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 5. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 4. In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to 3. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 12. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 10. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 9. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 8. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 7. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 6. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 5. In certain embodiments, the LPR of the conjugates provided herein ranges from 2 to 4. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 12. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 10.In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 9. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 8. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 7. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 6. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 5. In certain embodiments, the LPR of the conjugates provided herein ranges from 3 to 4.

[0486] In certain embodiments, the LPR of the conjugates provided herein ranges from 1 to about 8, from about 2 to about 6, from about 3 to about 5, from about 3 to about 4, or from about 4 to about 6. In certain embodiments, the LPR of the conjugates ranges from about 3.1 to about 3.9, from about 3.2 to about 3.8, from about 3.2 to about 3.7, from about 3.2 to about 3.6, from about 3.3 to about 3.8, or from about 3.3 to about 3.7. In certain embodiments, the LPR of the conjugates ranges from about 4.1 to about 4.9, from about 4.2 to about 4.8, from about 4.2 to about 4.7, from about 4.2 to about 4.6, from about 4.3 to about 4.8, or from about 4.3 to about 4.7.

[0487] In certain embodiments, the LPR of the conjugates provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more. In some embodiments, the LPR of the conjugates provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the LPR of the conjugates provided herein is about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, or about 4.9.

[0488] In some embodiments, the LPR of the conjugates provided herein is in the range of 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, or 2 to 13. In some embodiments, the LPR of the conjugates provided herein is in the range of 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, or 3 to 13. In some embodiments, the LPR of the conjugates provided herein is about 1. In some embodiments, the LPR of the conjugates provided herein is about 2. In some embodiments, the LPR of the conjugates provided herein is about 3. In some embodiments, the LPR of the conjugates provided herein is about 4. In some embodiments, the LPR of the conjugates provided herein is about 5. In some embodiments, the LPR of the conjugates provided herein is about 6. In some embodiments, the LPR of the conjugates provided herein is about 7. In some embodiments, the LPR of the conjugates provided herein is about 8. In some embodiments, the LPR of the conjugates provided herein is about 9. In some embodiments, the LPR of the conjugates provided herein is about 10.

[0489] In certain embodiments, fewer than the theoretical maximum number of units are conjugated to a polypeptide during a conjugation reaction. For example, a polypeptide may contain lysine residues that do not react with a compound or linker reagent. For example, polypeptides generally do not contain many free and reactive cysteine ​​thiol groups that can be linked to a ligand-linker moiety; in fact, many cysteine ​​thiol residues in polypeptides or proteins exist as disulfide bridges. In certain embodiments, a polypeptide can be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups. In certain embodiments, a polypeptide is subjected to denaturing conditions to expose reactive nucleophilic groups, such as lysine or cysteine. In some embodiments, a ligand-linker moiety is conjugated via a lysine residue on the polypeptide. In some embodiments, a ligand-linker moiety is conjugated via a cysteine ​​residue on the polypeptide.

[0490] The loading (LPR) of the conjugate can be controlled in various ways, for example, by (i) limiting the molar excess of compound or conjugation reagent relative to the polypeptide, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reducing conditions for cysteine ​​thiol modification, or (iv) recombinantly manipulating the amino acid sequence of the polypeptide to alter the number and position of cysteine ​​residues to control the number and / or position of linker-drug bonds (such as in the case of thiomab prepared as disclosed in WO2006 / 034488).

[0491] It should be understood that the preparation of the conjugates described herein may result in a mixture of conjugates in which one or more ligand-linker moieties are distributed on the polypeptide. Individual conjugate molecules may be identified in the mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (including such methods known in the art). In certain embodiments, homogeneous conjugates having a single LPR (loading) value may be isolated from the conjugated mixture by electrophoresis or chromatography.

[0492] In certain embodiments of the conjugate of Formula (I), m is 1 to 20, e.g., 2 to 10, 2 to 8, or 2 to 6. In certain cases, m is 10 or less. In certain cases, m is 2 to 8. In certain cases, m is 2 to 6. In certain cases, m is an average loading of about 4.

[0493] Polypeptides that bind to extracellular target molecules As summarized above, bifunctional molecules of the present disclosure can include an extracellular target molecule binding moiety that specifically binds to a target molecule. In some embodiments, the extracellular target molecule binding moiety B is bound or linked to a carrier polypeptide Y.

[0494] "Extracellular target molecule" refers to a soluble molecule that is outside the cell membrane of any cell in proximity to the soluble molecule. The extracellular target molecule can be any extracellular target molecule that is desirable for targeted degradation via the endosomal / lysosomal pathway. In certain embodiments, the extracellular target molecule binding moiety (B) specifically binds to the target autoantibody.

[0495] In some embodiments, the target-binding polypeptide is a protein antigen that contains the binding site (e.g., epitope) of a disease-causing autoantibody, hi some embodiments, the target-binding polypeptide is the primary immunogenic or epitope-containing region of the protein to which the autoantibody primarily binds in vivo.

[0496] antibody In some embodiments, the extracellular target molecule is an antibody, e.g., an antibody (Ab) that specifically binds to a cell surface molecule or a different extracellular molecule. The target antibody can be an extracellular autoantibody associated with a disease or condition of interest.

[0497] In some embodiments, the extracellular target molecule is human immunoglobulin A (IgA). In some embodiments, IgA is a specific antibody that plays an important role in mucosal immune function.

[0498] In the blood, IgA interacts with an Fc receptor called CD89 expressed on immune effector cells, triggering an inflammatory response. Abnormal IgA expression is involved in many autoimmune and immune-mediated diseases. In some embodiments, the target is human immunoglobulin G (IgG). The Fc region of IgG contains a conserved N-glycosylation site at asparagine 297 in the constant region of the heavy chain. Various N-glycans can be attached to this site. N-glycan IgG composition is associated with several autoimmune, infectious, and metabolic diseases. Furthermore, overexpression of IgG4 is associated with IgG4-related diseases.

[0499] In some embodiments, the target is human immunoglobulin E (IgE), a type of immunoglobulin that plays an important role in type I hypersensitivity, which can occur in a variety of allergic diseases and conditions.

[0500] In some embodiments, the extracellular target molecule is an autoantibody. Autoantibodies are commonly associated with autoimmune diseases. Non-limiting examples of extracellular target molecules include rheumatoid factor (RF), antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA, aPR3, MPO), anti-double-stranded DNA (dsDNA), anticentromere antibodies (ACA), anti-histone antibodies, cyclic citrullinated peptide antibodies (CCP), soluble nuclear antigen antibodies (e.g., anti-SS-A (Ro) and anti-SS-B (La), anti-RNP, anti-Jo-1, anti-Sm, Scl-70), cardiolipin antibodies, beta-2 glycoprotein 1 antibodies, antiphospholipid antibodies (APA), lupus anticoagulant (LA), diabetes-associated autoantibodies, anti-tissue transglutaminase (anti-tTC), and anti-gliadin antibodies (AGA). , intrinsic factor antibodies, parietal cell antibodies, thyroid autoantibodies (e.g., TPO antibodies, TSH receptor antibodies), smooth muscle antibodies (SMAs), antimitochondrial antibodies (AMAs), liver-kidney microsomal type 1 antibodies (LKM-1), anti-glomerular basement membrane (GBM), acetylcholine receptor (aChR) antibodies, N-methyl-D-aspartic acid (NMDAR) antibodies, myasthenia gravis antibodies (anti-aChR, anti-MuSK), antibodies associated with autoimmune nephropathy (e.g., anti-IgA and anti-PLA2R), antibodies associated with pregnancy-induced preeclampsia (anti-AT1R antibodies), encephalitis antibodies (anti-NMDAR antibodies), and pemphigus antibodies (e.g., anti-BP180 and anti-BP230). In some embodiments, the target molecule is a TSH receptor autoantibody. In some embodiments, the target molecule is a MuSK antibody. In some embodiments, the target molecule is an aChR antibody. In some embodiments, the target molecule is a BP180 antibody. In some embodiments, the target molecule is a PR3 antibody.

[0501] In some embodiments, the extracellular target molecule is a neutralizing antibody or an anti-drug antibody. A "neutralizing antibody" refers to an antibody that protects cells from pathogens (e.g., viral antigens or proteins) or infectious particles. Non-limiting examples of pathogens or infectious particles include coronaviruses, SARS-CoV-1, SARS-CoV-2, cytomegalovirus (CMV), hepatitis B virus (HBV), rabies virus, measles virus, respiratory syncytial virus (RSV), and diphtheria antitoxin. In one example, a neutralizing antibody exerts its effect by directly binding to viral particles and blocking their subsequent interaction with receptors or by inhibiting post-entry events such as viral uncoating and replication. In another example, a neutralizing antibody can neutralize the toxic effects of bacterial toxins (diphtheria antitoxin).

[0502] In some embodiments, the extracellular target molecule is an anti-drug antibody. The anti-drug antibody may bind to a drug or a therapeutic protein product. In some embodiments, the anti-drug antibody is an antibody that specifically binds to the idiotope of another antibody (typically an antibody drug). The idiotope corresponds to a region within the Fv region that binds to the paratope of a different antibody. The anti-drug antibody may be used in the treatment or management of cancer.

[0503] Extracellular target molecule binding moiety (B or YB) In some embodiments, the extracellular target molecule binding moiety comprises a polypeptide B that specifically binds to an extracellular target molecule (e.g., autoAb-A, autoAb-B, or autoAb-C), and optionally a carrier polypeptide (Y). A diagram of an extracellular target molecule binding moiety YB is shown. FIG. 2 shows exemplary extracellular target molecule binding moieties described herein. In some embodiments, the carrier polypeptide Y comprises a protein domain. In some embodiments, polypeptide B and carrier polypeptide Y are directly fused. In some embodiments, polypeptide B and carrier polypeptide Y are indirectly fused via a spacer domain. In some embodiments, the extracellular target molecule binding moiety (YB) is a chimeric protein comprising a polypeptide encoded by a nucleic acid molecule for polypeptide B and a carrier polypeptide Y. In some embodiments, carrier polypeptide Y is human serum albumin (HSA), which is attached to polypeptide B at a cysteine ​​residue on HSA to form the extracellular target molecule polypeptide binding moiety.

[0504] In some embodiments, the carrier polypeptide (Y) is an Fc fragment, an Fc monomer, an Fc dimer, or a fragment thereof (e.g., a synthetic peptide) generated from the heavy chain constant region of an immunoglobulin. An exemplary Fc mono-carrier polypeptide Y is shown. In some embodiments, the carrier polypeptide (Y) Fc is conjugated to a single variable domain on a heavy chain (VHH) antibody, nanobody, domain, or fragment thereof to form a VHH-antigen fusion. In some embodiments, the carrier polypeptide (Y) Fc is conjugated to an antigen to form an Fc-antigen fusion. In various embodiments, the Fc fragment is conjugated to polypeptide B at a cysteine ​​residue on the Fc fragment.

[0505] Carrier polypeptide (Y) Several strategies for imparting desirable pharmacokinetic properties to peptides or proteins (e.g., avoiding rapid renal clearance and / or optimizing Fc receptor-mediated recycling), e.g., carrier attachment, can be adapted for use in the bifunctional molecules of the present disclosure that comprise target-binding polypeptides.

[0506] The target-binding polypeptide can be linked to a carrier polypeptide Y, which confers one or more desirable properties to the resulting bifunctional molecule (e.g., increased in vivo stability and / or half-life) and / or provides a binding site for a ligand-linker moiety, for example, without destroying the autoantibody binding properties of the target-binding polypeptide.

[0507] In some embodiments, the target-binding polypeptide is synthetically linked to the carrier polypeptide Y. In some embodiments of Formula (I), the carrier polypeptide Y is attached to the polypeptide (B) via a bifunctional linker.

[0508] In some embodiments, the target-binding polypeptide is linked to a carrier polypeptide Y as part of a protein construct, such as a chimeric fusion protein. In some embodiments, polypeptide B and carrier polypeptide Y are fused directly to each other, for example via an N-terminal to C-terminal fusion or a C-terminal to N-terminal fusion. In some embodiments, polypeptide B and carrier polypeptide Y are fused indirectly via a spacer domain. In some embodiments, YB of formula (I) is a chimeric protein having a polypeptide encoded by nucleic acid molecules encoding both polypeptide B and carrier polypeptide Y.

[0509] Carrier polypeptide (Y) can be a serum protein or a domain of a serum protein. In some embodiments, the serum protein is albumin. In some embodiments, the serum protein is an immunoglobulin. In some embodiments, carrier polypeptide (Y) is an engineered serum protein or domain thereof. Carrier polypeptide (Y) can be prepared synthetically or recombinantly.

[0510] 2 shows an exemplary protein (YB) comprising a target-binding polypeptide (e.g., an autoantibody "bait") linked to a carrier polypeptide (e.g., an HSA carrier protein). In some embodiments, the YB is referred to as a protein construct, and the engineered chimeric protein is suitable for preparation using recombinant protein techniques.

[0511] In some embodiments, the carrier polypeptide (Y) is human serum albumin (HSA) or a fragment thereof (eg, a synthetic peptide).

[0512] In some embodiments, the carrier polypeptide (Y) is an HSA domain or a fragment thereof (eg, a synthetic peptide).

[0513] In some embodiments, the carrier polypeptide (Y) is an albumin binding domain or a fragment thereof (eg, a synthetic peptide).

[0514] In some embodiments, the carrier polypeptide (Y) is Fc (monomer) or a fragment thereof (eg, a synthetic peptide).

[0515] In some embodiments, the carrier polypeptide (Y) is an Fc (dimer) or a fragment thereof (eg, a synthetic peptide).

[0516] In some embodiments, the carrier polypeptide (Y) is linked to the extracellular target molecule (B) via a linker, which may comprise a non-peptide linking moiety.

[0517] albumin The carrier polypeptide can be a serum albumin protein or a domain or subdomain thereof, or a fragment thereof. In some embodiments, the carrier polypeptide is human serum albumin (HSA).

[0518] Albumin generally has a long plasma half-life (e.g., approximately 3 weeks) and can provide a scaffold to which biologically active molecules can be attached or fused. HSA has a long serum half-life in humans, which is due in part to its interaction with the neonatal Fc receptor (FcRn). HSA (molecular weight 66.5 kDa) contains three structurally similar, flexible domains: I (residues 1-195), II (196-383), and III (384-585). Each domain (D) is composed of two subdomains, A and B, which share a common structural motif (e.g., DIA [5-105], DIB [119-195], DIIA [196-292], DIIB [314-383], DIIIA [384-491], and DIIIB [510-582]; see, for example, SEQ ID NO: 2 in WO2017 / 029407).

[0519] In some embodiments, the carrier protein is an HSA variant with enhanced FcRn binding. FcRn binds to the C-terminus of HSA DIII and protects albumin from intracellular degradation. For example, a single amino acid substitution in HSA DIII (K537P) improves the binding affinity to FcRn by 12-fold, which leads to a longer half-life. Various amino acid residues of albumin located in domain I or domain II also affect the interaction between HSA and FcRn (for example, WO2013 / 135896 describes albumin variants with one or more modifications in domain I and one or more modifications in domain III, and WO2015 / 036579 describes albumin variants with one or more modifications in domain II).

[0520] Any convenient albumin protein can be the parent of an albumin variant that can be used as a carrier polypeptide, examples of which include human serum albumin (HSA) such as AAA98797, P02768-1, SEQ ID NO: 25 (mature HSA), or SEQ ID NO: 24 (immature HSA).

[0521] In some embodiments, other albumins are utilized as carrier polypeptides. Such other albumins include primate serum albumin (e.g., chimpanzee serum albumin, XP_517233.2), gorilla serum albumin, or macaque serum albumin (e.g., NP_001 182578), rodent serum albumin (e.g., hamster serum albumin, A6YF56), guinea pig serum albumin (e.g., Q6WDN9-1), mouse serum albumin (e.g., AAH49971, P07724-1 version 3) and rat serum albumin (e.g., AAH85359, P02770-1 version 2), bovine serum albumin (e.g., bovine serum albumin P02769-1), horse serum albumin (e.g., P35747-1) or donkey serum albumin (e.g., Q5XLE4-1), rabbit serum albumin (e.g., P49065-1), Examples of suitable amino acid sequence identity include, but are not limited to, goat serum albumin (e.g., ACF10391), sheep serum albumin (e.g., P14639-1), dog serum albumin (e.g., P49822-1), chicken serum albumin (e.g., P19121-1 version 2), and porcine serum albumin (e.g., P08835-1 version 2), or polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or at least 99.8% amino acid sequence identity to such albumins. Non-mammalian albumins of interest include ovalbumin (eg, P01012.pro: chicken ovalbumin, 073860.Pro: turkey ovalbumin).

[0522] In some embodiments, albumin, a fragment thereof, or a bindable albumin variant, or the albumin portion of a fusion polypeptide or conjugate comprising albumin or a fragment thereof, has a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24 (e.g., P02768-1) below, where Domain I is shown in bold, Domain II is underlined, and Domain III is italicized. [ka]

[0523] In some embodiments, albumin, a fragment thereof, or a bindable albumin variant, or the albumin portion of a fusion polypeptide or conjugate comprising albumin or a fragment thereof, has a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25 below. (SEQ ID NO: 25).

[0524] In some embodiments, the albumin, fragment thereof, or bindable albumin variant or variant thereof of the bifunctional molecule has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of HSA as set forth in SEQ ID NO: 7. Preferably, the albumin maintains at least one of the key properties of albumin or a tertiary structure similar to that of albumin, such as HSA. A functional fragment of albumin may have at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 100% sequence identity to the sequence of HSA domain III set forth in SEQ ID NO: 26, or the sequence of HSA domain II and domain III set forth in SEQ ID NO: 27, or a molecule consisting of or comprising two copies of domain III (e.g., SEQ ID NO: 28), or a molecule consisting of or comprising three copies of domain III (e.g., SEQ ID NO: 29), or a molecule consisting of or comprising domain I and two copies of domain III (e.g., SEQ ID NO: 30). [Table 25-1] [Table 25-2] [Table 25-3]

[0525] The human serum albumin (HSA) polypeptide chain has 35 cysteine ​​residues, which form 17 disulfide bonds and one unpaired (free) cysteine ​​at position 34, located at the DI of the mature protein. Albumin, a fragment thereof, or a bindable albumin variant according to the invention, or the albumin portion of a fusion polypeptide or conjugate comprising albumin or a fragment thereof, may, when folded, have some of the native disulfide bonds of the polypeptide of SEQ ID NO: 25, for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and preferably all 17.

[0526] Albumin carrier polypeptides that can be adapted for use in the bifunctional molecules of the present disclosure are described in WO2017 / 029407, WO2013 / 135896, and WO2015 / 036579, the disclosures of which are incorporated herein by reference in their entireties.

[0527] Attachment of molecules to the free thiol on cysteine-34 of HSA can provide site-selective attachment. To increase loading capacity, recombinant HSA (rHSA) variants can be engineered to contain additional free, conjugable cysteines.

[0528] The term "thio-albumin" is used to describe an albumin variant containing one or more (e.g., several) unpaired cysteine ​​residues, particularly an albumin variant in which the one or more (e.g., several) unpaired cysteine ​​residues are not present in naturally occurring variants of albumin. Thio-albumin refers to a conjugable albumin. An rHSA variant is a thio-albumin or a conjugable albumin. An rHSA can have one or more free conjugable cysteines introduced into DI, DII, and / or DIII. In some embodiments, an rHSA variant has at least one free conjugable cysteine ​​introduced into DI or DII. In some embodiments, an rHSA variant has at least one free conjugable cysteine ​​introduced into DI and DIII. In some embodiments, an rHSA variant has a conjugation site at Cys34 and at least one or more free conjugable cysteines introduced into DI, DII, and / or DIII.

[0529] In some embodiments, the carrier polypeptide is an rHSA variant having a conjugation site at Cys34 and having one or more conjugable cysteines introduced at a position selected from K93, A226, E230, I271, E294, E358, L24, F49, V54, D56, A92, Q94, E97, H128, F156, E227, D237, K240, D259, K262, N267, Q268, L275, L284, K317, A322, E333, D340, E354, K359, A362, E382, and L398 of SEQ ID NO:25. A free, bindable cysteine ​​can be obtained by substituting an amino acid other than cysteine ​​at any one of the positions corresponding to or equivalent to any of the residues selected from K93, A226, E230, I271, E294, E358, L24, F49, V54, D56, A92, Q94, E97, H128, F156, E227, D237, K240, D259, K262, N267, Q268, L275, L284, K317, A322, E333, D340, E354, K359, A362, E382, and L398 of SEQ ID NO:25. Alternatively, a cysteine ​​can be introduced by inserting a cysteine ​​at a position adjacent to the N- or C-side of an amino acid corresponding to a position equivalent to any of residues selected from K93, A226, E230, I271, E294, E358, L24, F49, V54, D56, A92, Q94, E97, H128, F156, E227, D237, K240, D259, K262, N267, Q268, L275, L284, K317, A322, E333, D340, E354, K359, A362, E382, and L398 of SEQ ID NO: 25. In some embodiments, at least one or more free, bindable cysteines are introduced at one or more positions corresponding to or equivalent to any of residues selected from C34, V54, H128, K240, and K262.

[0530] In some embodiments, the carrier polypeptide is an rHSA variant that exhibits up to 95% monomer stability compared to a wild-type, non-binding control (e.g., as demonstrated by biolayer interferometry) and maintains hFcRn binding. In some embodiments, introducing a free, bindable cysteine ​​into the rHSA variant does not interfere with FcRN binding. In some embodiments, the introduction of a free, bindable cysteine ​​maintains albumin half-life in the circulation.

[0531] Immunoglobulin In some embodiments, the carrier polypeptide is an Fc fragment, monomer, dimer, domain, or fragment thereof. The Fc fragment comprises the CH2 and CH3 domains and a portion of the hinge region, which are held together by one or more disulfide and non-covalent interactions. The Fc fragment and the Fc5μ fragment are produced by fragmentation of IgG and IgM, respectively. The Fc fragment is derived from the heavy chain constant region of an immunoglobulin. In some embodiments, the Fc fragment is derived from any subclass of IgG immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4). In a preferred embodiment, the Fc fragment is derived from human IgG1.

[0532] The term "Fc dimer" refers to an Fc fragment containing two CH2-CH3 chains. This dimer is typically approximately 54 kDa. An "Fc monomer" is generally half the size of the dimer (e.g., approximately 27 kDa). Methods for producing Fc dimers and monomers are described in Wang et al., "Engineering soluble monomeric IgG1 Fc with significantly decreased non-specific binding." Front. Immunol. (2017) 8:1545, and Ying et al., "Soluble monomeric IgG1 Fc." J. Biol Chem (2012) 287(23):19399-408, each of which is incorporated herein by reference in its entirety.

[0533] In some embodiments, the Fc fragment has a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31 (human IgG1 hinge Fc) below. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31).

[0534] In some embodiments, the Fc fragment is a variant of a human IgG Fc region, hi some embodiments, the Fc fragment is a variant of a human IgG Fc region comprising one or more amino acid substitutions in the CH2 and / or CH3 domains.

[0535] In some embodiments, carrier polypeptide (Y) is an Fc fragment, Fc monomer, Fc dimer, or fragments thereof generated from the heavy chain constant region of an immunoglobulin. In some embodiments, carrier polypeptide (Y)Fc is conjugated to an antibody fragment (e.g., a nanobody, scFv, VHH) comprising an antigen-binding region to form an engineered antibody. In some embodiments, carrier polypeptide (Y)Fc is conjugated to an antigen to form an Fc-antigen fusion. In various embodiments, the Fc fragment is conjugated to polypeptide B at a cysteine ​​residue on the Fc fragment.

[0536] In certain embodiments, the carrier polypeptide is an Fc fragment, monomer, dimer, domain, or fragment thereof.

[0537] The Fc fragment contains the CH2 and CH3 regions and part of the hinge region, which are held together by one or more disulfide and non-covalent interactions. The Fc fragment and the Fc5μ fragment are produced by fragmentation of IgG and IgM, respectively. The term Fc is derived from the ability of these antibody fragments to crystallize. Fc fragments are usually generated from the heavy chain constant region of immunoglobulins.

[0538] Polypeptide (B) In some embodiments, polypeptide (B) specifically binds to a target antibody (e.g., an extracellular antibody associated with a disease or condition of interest). The target antibody can be an autoantibody. The target antibody can be a neutralizing antibody or an anti-drug antibody. In some embodiments, polypeptide (B) comprises an antigen of the target antibody, or a fragment thereof (e.g., a synthetic peptide). In such instances, the antigen can be an autoantigen. According to various embodiments, polypeptide (B) comprises a protein domain, e.g., an antibody, an antigen, or a fragment thereof.

[0539] autoantibodies In certain embodiments, polypeptide B specifically binds to one or more extracellular target molecules associated with an autoimmune disorder or disease. In some embodiments, polypeptide B is an autoantibody as described herein. Non-limiting examples of autoantibodies include rheumatoid factor (RF), antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), anti-double-stranded DNA (anti-dsDNA), anticentromere antibodies (ACA), anti-histone antibodies, cyclic citrullinated peptide antibodies (CCP), soluble nuclear antigen antibodies (e.g., anti-SS-A (Ro) and anti-SS-B (La), anti-RNP, anti-Jo-1, anti-Sm, Scl-70), cardiolipin antibodies, beta-2 glycoprotein 1 antibodies, anti-phospholipid antibodies, and the like. These include lipid antibodies (APA), lupus anticoagulant (LA), diabetes-associated autoantibodies, anti-tissue transglutaminase (anti-tTC), anti-gliadin antibodies (AGA), intrinsic factor antibodies, parietal cell antibodies, thyroid autoantibodies (e.g., anti-TPO, TSH receptor antibodies), smooth muscle antibodies (SMAs), antimitochondrial antibodies (AMAs), liver-kidney microsomal type 1 antibodies (anti-LKM-1), anti-glomerular basement membrane (GBM), or acetylcholine receptor (aChR) antibodies.

[0540] In various embodiments, Polypeptide B binds to autoantibodies associated with one or more autoimmune diseases, including, but not limited to, Addison's disease, celiac disease (gluten-sensitive enteropathy), such as sprue, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis or psoriatic arthritis, and autoimmune vasculitis. https: / / medlineplus.gov / ency / article / 000431.htm In some embodiments, the autoimmune disease is associated with mucosal immunity or the gut microbiota. Such diseases are described in Campbell AW. Autoimmunity and the gut. Autoimmune Diseases. 2014, Article ID 152428, incorporated herein by reference in its entirety.

[0541] Pharmaceutical Composition In another embodiment, provided herein is a pharmaceutical composition comprising one or more conjugates disclosed herein and a pharmaceutically acceptable carrier.

[0542] In certain embodiments, the pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more conjugates provided herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier.

[0543] Pharmaceutical carriers suitable for administration of the conjugates provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

[0544] The conjugates described herein can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients.

[0545] In certain embodiments, the conjugates are formulated into one or more suitable pharmaceutical preparations such as solutions, suspensions, powders, sustained release formulations or elixirs in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparations and dry powder inhalants.

[0546] In the compositions provided herein, the conjugates described herein may be mixed with a suitable pharmaceutical carrier. The concentration of the conjugate in the composition may be, for example, effective to deliver an amount that, upon administration, treats, prevents, or ameliorates a condition or disorder described herein, or a symptom thereof.

[0547] In certain embodiments, the pharmaceutical compositions provided herein are formulated for single-dose administration.To formulate the composition, a weight fraction of the conjugate is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration such that the pathology being treated is alleviated, prevented, or one or more symptoms are ameliorated.

[0548] The concentration of the conjugate in the pharmaceutical compositions provided herein depends, for example, on the physicochemical properties of the conjugate, the administration schedule, the dosage, as well as other factors known to those of skill in the art.

[0549] The pharmaceutical compositions described herein are provided for administration to a subject, e.g., a human or an animal (e.g., a mammal), in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions containing an appropriate amount of the compound or a pharmaceutically acceptable derivative thereof. Pharmaceutical compositions are also provided for administration to humans and animals in unit dosage forms, including oral or nasal solutions or suspensions and oil-water emulsions containing an appropriate amount of the conjugate or a pharmaceutically acceptable derivative thereof. In certain embodiments, the conjugate is formulated and administered in unit dosage or multiple dosage forms. As used herein, unit dosage form refers to a physically discrete unit suitable for human or animal (e.g., mammalian) subjects and individually packaged as known in the art. Each unit dosage contains a predetermined amount of the conjugate sufficient to produce the desired therapeutic effect, together with the required pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules, syringes, and individually packaged capsules. Unit dosage forms can be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dose forms packaged in a single container for administration in separate unit dose forms. Examples of multiple dose forms include vials, bottles of capsules, or bottles. Thus, in certain embodiments, a multiple dose form is a plurality of unit doses that are not separated in packaging.

[0550] In certain embodiments, the conjugates described herein are in a liquid pharmaceutical formulation. Pharmaceutically administrable liquid formulations can be prepared, for example, by dissolving, dispersing, or otherwise mixing the conjugate and any pharmaceutical auxiliary agents in a carrier, such as water, saline, aqueous dextrose, glycerol, glycols, etc., to form a solution or suspension. In certain embodiments, the pharmaceutical compositions provided herein to be administered can also contain small amounts of non-toxic auxiliary substances, such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents.

[0551] Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy (2012) 22nd ed., Pharmaceutical Press, Philadelphia, PA. Dosage forms or compositions containing in the range of 0.005% to 100% antibody, with the remainder consisting of non-toxic carrier, can be prepared.

[0552] In certain embodiments, parenteral administration is characterized by subcutaneous, intramuscular, or intravenous injection, and is also contemplated by the present invention. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Other routes of administration can include enteral administration, intracerebral administration, intranasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.

[0553] Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be mixed with a solvent immediately before use (including hypodermic tablets), sterile suspensions ready for injection, sterile dry insoluble products ready to be mixed with a solvent immediately before use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0554] For intravenous administration, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0555] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.

[0556] Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0557] In certain embodiments, intravenous or intraarterial infusion of a sterile aqueous solution containing a conjugate described herein is an effective method of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing a conjugate described herein, injected as needed to achieve the desired pharmacological effect.

[0558] In certain embodiments, the pharmaceutical formulations are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures, or they can be reconstituted and formulated as solids or gels.

[0559] The lyophilized powder is prepared by dissolving the conjugate provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterilized. The suitable solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The suitable solvent may also contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art (in certain embodiments, at approximately neutral pH). The solution is then sterile filtered, followed by lyophilization under standard conditions known to those of skill in the art, yielding an exemplary formulation. In certain embodiments, the resulting solution is dispensed into vials for lyophilization. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0560] The lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier.

[0561] In certain embodiments, the conjugates provided herein can be formulated for topical administration or application, for example, in the form of gels, creams, and lotions for topical application to the skin and mucous membranes (e.g., the eyes), ophthalmic application, or intravesical or intrathecal application. Topical administration is contemplated for transdermal delivery, administration to the eyes or mucous membranes, or inhalation therapy. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0562] Use and Method When the ligand portion of the bifunctional molecule binds to ASGPR, it can induce internalization and lysosomal degradation of the bound target autoantibody. In some embodiments, the bifunctional molecule is a conjugate of a protein comprising a polypeptide that specifically binds to an autoantibody and a linked ligand portion. The bifunctional molecule of the present disclosure is useful for reducing the level of extracellular target molecule autoantibodies in a biological system or sample. The biological system can be a human subject.

[0563] Therefore, the method of using the conjugate described herein can remove autoantibodies from the extracellular space (extracellular environment) of cells in a biological system by sequestering target proteins in lysosomes of cells and degrading target autoantibodies.Removal of target proteins can refer to the reduction or depletion of the amount of target proteins from the extracellular space or extracellular environment.In some embodiments, the biological system or sample is a cell sample.

[0564] The term "sample" refers to an aliquot or portion taken from a source and / or provided for analysis or processing. In some embodiments, a sample is derived from a biological source such as a tissue, cell, or component (e.g., bodily fluids including, but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). In some embodiments, a sample may be or comprise a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells, or components, or a fraction or portion thereof, including, but not limited to, plasma, serum, spinal fluid, lymph, external sections of skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, or organs. In some embodiments, a sample is or comprises a medium, such as a nutrient broth or gel, which may contain cellular components such as proteins. In some embodiments, a "primary" sample is an aliquot of a source. In some embodiments, the primary sample is subjected to one or more processing (eg, separation, purification, etc.) steps to prepare the sample for analysis or other uses.

[0565] Treatment of Disease Provided herein are methods for treating diseases or disorders caused by targeted autoantibodies in human subjects. Accordingly, the present disclosure provides methods relating to using the bifunctional molecules, conjugates, and compositions of the present disclosure for therapeutic treatment by depleting pathogenic autoantibodies through degradation via the lysosomal pathway.

[0566] In some embodiments, the method for treating a disease comprises administering to a subject, e.g., a human subject in need of treatment, an effective amount of a bifunctional molecule or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the bifunctional molecule (e.g., as described herein).

[0567] In some embodiments, the patient treated according to the methods of the present disclosure is a patient identified as being positive for the target autoantibody.

[0568] The terms "administer," "administration," or "administering" refer to the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or patient (e.g., a human), such as by mucosal delivery, topical delivery, intradermal delivery, parenteral delivery, intravenous delivery, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0569] In some embodiments, administration is oral.

[0570] In some embodiments, administration is by subcutaneous injection.

[0571] In some embodiments, administration is by intravenous infusion.

[0572] The term "treating" refers to partially or completely alleviating, ameliorating, improving, alleviating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., myasthenia gravis). Treatment may be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing a condition associated with the disease, disorder, and / or condition.

[0573] A therapeutic or preventive effect is evident when there is a significant, often statistically significant, improvement in one or more parameters of the disease state, or when there is no worsening or onset of symptoms that would normally be expected. By way of example, a favorable change of at least 10%, and at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease can indicate effective treatment. The efficacy of a given compound or composition can also be determined using an experimental animal model for a given disease known in the art. When an experimental animal model is used, the effectiveness of the treatment is demonstrated when a statistically significant modulation of a marker or symptom is observed.

[0574] The term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., a conjugate or pharmaceutical composition provided herein) sufficient to treat, diagnose, prevent, delay the onset of, reduce the severity and / or duration of, and / or ameliorate a given condition, disorder, or disease, and / or its associated symptoms. These terms also encompass the amount necessary to attenuate, delay, or ameliorate the progression or progression of a given disease, attenuate, delay, or ameliorate the recurrence, progression, or onset of a given disease, and / or improve or enhance the preventive or therapeutic effect(s) of, or serve as a bridge to, another therapy. In some embodiments, "effective amount," as used herein, also refers to the amount of a conjugate described herein to achieve a particular result.

[0575] The term "therapeutic dose" refers to one or more doses of a therapeutic agent administered in the course of addressing or alleviating a therapeutic indication. A therapeutic dose may be adjusted to maintain a desired concentration or activity level of the therapeutic agent in a bodily fluid or biological system.

[0576] The bifunctional molecules of the present disclosure and the additional therapeutic agent(s) and / or treatment for MG can be administered in combination. Such combinations can be in the same composition, or the additional therapeutic agent or treatment can be administered as part of a separate composition or by another method described herein.

[0577] The terms "subject" and "patient" are used interchangeably. A subject can be a mammal, e.g., a human, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein. In some embodiments, the subject is a human. As used herein, the terms "patient," "subject," and "individual" are used interchangeably. The term "screening" refers to an examination or evaluation performed for the purposes of selection or filtering. Patients may be screened to select individuals in need of treatment. In some embodiments, subjects are screened to select individuals most likely to respond favorably to treatment.

[0578] definition It is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0579] 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 to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.

[0580] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound but also a combination of two or more compounds; reference to "a substituent" includes not only a single substituent but also two or more substituents, etc.

[0581] In describing and claiming the present invention, certain terms will be used in accordance with the definitions set forth below. It will be understood that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical moieties may be included within more than one term definition.

[0582] As used herein, the words "for example," "for instance," "such as," or "including" are intended to introduce examples that further clarify a more general subject matter. These examples are provided solely as an aid in understanding the disclosure and are not intended to be limiting in any way.

[0583] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0584] The terms "protein" and "polypeptide" are used interchangeably. A protein may contain moieties other than amino acids (e.g., it may be a glycoprotein, etc.) and / or may be processed or modified in other ways. One of skill in the art will understand that a "protein" may be an entire protein chain (with or without a signal sequence) produced by a cell, or a protein portion thereof. One of skill in the art will understand that a protein may sometimes include multiple protein chains, linked, for example, noncovalently or covalently, by one or more disulfide bonds, or associated by other means. In certain embodiments, a polypeptide can exist as a single chain or as two or more associated chains, and may exist as multimers, such as dimers, trimers, etc. The term also encompasses amino acid polymers that are modified, either naturally or by intervention (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification). Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, but not limited to, unnatural amino acids), as well as other modifications known in the art. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0585] Unless otherwise indicated, the term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, or 3 standard deviations. In certain embodiments, the term "about" or "approximately" means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.1%, or 0.05% of a given value or range. In certain embodiments, when an integer is required, the term "about" means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0586] As used herein, the words "for example," "for instance," "such as," or "including" are intended to introduce examples that further clarify a more general subject matter. These examples are provided solely as an aid in understanding the disclosure and are not intended to be limiting in any way.

[0587] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0588] The term "autoantibody" or "autoantibodies" refers to an antibody that recognizes, binds to, or otherwise interacts with an antigen normally present in a subject, or in the subject's tissues or cells. Autoantibodies are abnormal antibodies produced by pathogenic B cells when they target an individual's own tissues. The term "autoimmunity" refers to the presence of antibodies (produced by B lymphocytes) and T lymphocytes that target normal components of a person (self-antigens). These components are called autoantigens or self-antigens and typically consist of proteins (or proteins complexed with nucleic acids). Antibodies and T lymphocytes that recognize self-antigens are called "autoantibodies" and "autoreactive T cells."

[0589] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv fragments, and VHH fragments.

[0590] An "antigen" is a moiety or molecule that contains an epitope to which an antibody can specifically bind. An antigen is therefore also specifically bound by an antibody.

[0591] "Epitope" is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be a linear epitope of consecutive amino acids or can include amino acids from two or more non-contiguous regions of the antigen.

[0592] "Pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" mean excipients, diluents, carriers, and adjuvants that are useful in preparing pharmaceutical compositions and that are generally safe, non-toxic, and not biologically or otherwise undesirable, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and human pharmaceutical use. As used in this specification and claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more such excipients, diluents, carriers, and adjuvants.

[0593] "Pharmaceutical composition" is meant to encompass compositions suitable for administration to a subject, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that could cause an undesirable reaction in the subject (e.g., the compound(s) in the pharmaceutical composition are of pharmaceutical grade). Pharmaceutical compositions can be designed for administration to a subject or patient in need thereof via several different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, and subcutaneous.

[0594] "Pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0595] "Pharmaceutically acceptable salts" refers to salts suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). These salts can be prepared in situ during the final isolation and purification of the conjugate compound, or can be prepared separately by reacting the free base functional group or groups of the compound with a suitable organic acid.

[0596] Examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts or salts of inorganic acids with amino groups.

[0597] Compounds are described using standard nomenclature. Compounds of any formula described herein may be in the form of a racemate, an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of diastereomers, a tautomer, an N-oxide, an isomer (such as a rotational isomer), or the like, as if each were specifically described, unless otherwise excluded by context.

[0598] As used herein, the phrases "having the formula" or "having the structure" are not intended to be limiting and are used in the same manner as the term "comprising" is commonly used. As used herein, the term "independently selected from" is used to indicate that the listed elements, such as R groups, can be the same or different.

[0599] A dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent, for example, -(C=O)NH2 is attached through the carbon of the carbonyl (C=O) group.

[0600] The present disclosure includes compounds (e.g., as described herein) with at least one desired isotopic substitution of an atom at greater than the natural abundance, i.e., enriched, of that isotope. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but different number of neutrons.

[0601] Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, and 125 In one non-limiting embodiment, the isotope-labeled compounds are used in metabolic studies (e.g., 14 C), reaction kinetic studies (e.g., 2 H or 3 H), positron emission tomography (PET) or single photon emission computed tomography (SPECT) or other detection or imaging techniques (including drug or substrate tissue distribution assays), or for the treatment of patients. 18 F-labeled compounds may be particularly preferred for PET or SPECT studies. Isotopically labeled compounds of the present disclosure and prodrugs thereof can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents by carrying out the procedures disclosed in the schemes or examples and preparations described below.

[0602] Isotopic substitution, e.g., deuterium substitution, may be partial or complete. Partial deuterium substitution means that at least one hydrogen is replaced with deuterium. In certain embodiments, the isotope is enriched in the isotope at any position of interest by 90%, 95%, or 99% or more. In one non-limiting embodiment, deuterium is enriched at the desired position by 90%, 95%, or 99%.

[0603] In some embodiments, a deuterium atom can be substituted for a hydrogen atom in any compound of the formula described herein. In one non-limiting embodiment, the substitution of a hydrogen atom for a deuterium atom is performed by R 1 , R 2 , R 3 , R 4 , R 6 , R 11 , R 21 , R 22 , R 23 , R 24 , R 25 , R, R', and R'', etc. For example, if any of these groups is methyl, ethyl, or methoxy, or contains, for example, methyl, ethyl, or methoxy, the alkyl residue may be deuterated (such as, in non-limiting embodiments, CDH2, CD2H, CD3, CH2CD3, CD2CD3, CHDCH2D, CH2CD3, CHDCHD2, OCDH2, OCD2H, or OCD3). In certain other embodiments, when two substituents are joined to form a ring, the unsubstituted carbon may be deuterated.

[0604] "Aliphatic" refers to a saturated or unsaturated, straight-chain, branched, or cyclic hydrocarbon. As used herein, "aliphatic" is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, and thus encompasses each of these definitions. In one embodiment, "aliphatic" is used to refer to an aliphatic group having 1 to 20 carbon atoms. The aliphatic chain can be, for example, monounsaturated, diunsaturated, triunsaturated, or polyunsaturated, or alkynyl. Unsaturated aliphatic groups can be in the cis or trans configuration. In one embodiment, an aliphatic group contains 1 to about 12 carbon atoms, more typically 1 to about 6 carbon atoms, or 1 to about 4 carbon atoms. In one embodiment, an aliphatic group contains 1 to about 8 carbon atoms. In certain embodiments, an aliphatic group is C1-C2, C1-C3, C1-C4, C1-C5, or C1-C6. As used herein, a specified range refers to an aliphatic group, with each member of the range being described as a separate species. For example, as used herein, the term "C1-C6 aliphatic" is intended to refer to a linear or branched alkyl, alkenyl, or alkynyl group having 1, 2, 3, 4, 5, or 6 carbon atoms, each of which is described as a separate species. For example, as used herein, the term "C1-C4 aliphatic" is intended to refer to a linear or branched alkyl, alkenyl, or alkynyl group having 1, 2, 3, or 4 carbon atoms, each of which is described as a separate species. In one embodiment, an aliphatic group is substituted with one or more functional groups that form a stable moiety.

[0605] An "alkyl" is a branched or straight-chain saturated aliphatic hydrocarbon group. In one non-limiting embodiment, an alkyl group contains 1 to about 12 carbon atoms, more typically 1 to about 6 carbon atoms, or 1 to about 4 carbon atoms. In one non-limiting embodiment, an alkyl group contains 1 to about 8 carbon atoms. In certain embodiments, an alkyl is C1-C2, C1-C3, C1-C4, C1-C5, or C1-C6. As used herein, a specified range refers to an alkyl group, with each member of the range being described as a separate species. For example, as used herein, the term "C1-C6 alkyl" is intended to refer to a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms, each of which is described as a separate species, and thus each subset is considered separately disclosed. For example, as used herein, the term "C1-C4 alkyl" is intended to refer to a straight-chain or branched alkyl group having 1, 2, 3, or 4 carbon atoms, each of which is described as a separate species. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane. In alternative embodiments, alkyl groups are optionally substituted. The term "alkyl" also encompasses cycloalkyl groups or carbocyclic groups. For example, when a term containing "alk" is used, "cycloalkyl" or "carbocyclic" can be considered part of the definition unless clearly excluded by context. For example, but not limited to, terms such as alkyl, alkoxy, and haloalkyl can all be considered to include cyclic forms of alkyl unless clearly excluded by context.

[0606] "Alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon double bonds that may occur at any stable point along the chain. As used herein, a specified range refers to an alkenyl group, with each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, allyl, propenyl, butenyl, and 4-methylbutenyl. The term "alkenyl" also encompasses "cis" and "trans" alkenyl configurations, or "E" and "Z" alkenyl configurations. In alternative embodiments, alkenyl groups are optionally substituted. The term "alkenyl" also encompasses cycloalkyl or carbocyclic groups having at least one point of unsaturation.

[0607] "Alkynyl" refers to a branched or straight-chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds that may occur at any stable point along the chain. As used herein, a specified range refers to an alkynyl group, with each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, and 5-hexynyl. In alternative embodiments, the alkynyl group is optionally substituted. The term "alkynyl" also encompasses cycloalkyl or carbocyclic groups having at least one triple bond.

[0608] "Alkylene" is a divalent saturated hydrocarbon. Alkylene can be, for example, 1, 2, 3, 4, 5, 6, 7-8 carbon moieties, 1-6 carbon moieties, or a specified number of carbon moieties, such as C1-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C6 alkylene, or C1-C6 alkylene.

[0609] An "alkenylene" is a divalent hydrocarbon having at least one carbon-carbon double bond. The alkenylene can be, for example, a 2-8 carbon moiety, a 2-6 carbon moiety, or a specified number of carbon moieties, such as C2-C4 alkenylene.

[0610] An "alkynylene" is a divalent hydrocarbon having at least one carbon-carbon triple bond. The alkynylene can be, for example, a 2-8 carbon moiety, a 2-6 carbon moiety, or a specified number of carbon moieties, such as C2-C4 alkynylene.

[0611] The term "amino" refers to the group -NRR', where R and R' are independently hydrogen or non-hydrogen substituents, including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.

[0612] "Chain" refers to a linear chain from which all other chains (long or short, or both) may be considered pendants. When two or more chains may equally be considered the main chain, "chain" refers to the chain that leads to the simplest representation of the molecule.

[0613] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having a single ring or multiple rings, including fused, bridged, and spirocyclic systems. Examples of suitable cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures, such as adamantanyl.

[0614] "Halo" and "halogen" refer to fluorine, chlorine, bromine, or iodine.

[0615] "Haloalkyl" refers to a branched or straight-chain alkyl group substituted with one or more halo atoms as defined above (up to the maximum allowable number of halo atoms). Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. "Perhaloalkyl" refers to an alkyl group in which all hydrogen atoms have been replaced with halogen atoms. Examples include, but are not limited to, trifluoromethyl and pentafluoroethyl.

[0616] "Haloalkoxy" refers to a haloalkyl group, as defined herein, attached through an oxygen bridge (oxygen of an alcohol radical).

[0617] The term "heteroaliphatic" refers to an aliphatic moiety that contains at least one heteroatom in the chain, such as an amine, carbonyl, carboxy, oxo, thio, phosphate, phosphonate, nitrogen, phosphorus, silicon, or boron atom, instead of a carbon atom. In one embodiment, the only heteroatom is nitrogen. In one embodiment, the only heteroatom is oxygen. In one embodiment, the only heteroatom is sulfur. As used herein, "heteroaliphatic" is intended to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl moieties. In one embodiment, "heteroaliphatic" is used to refer to a heteroaliphatic group (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 20 carbon atoms. In one embodiment, heteroaliphatic groups are optionally substituted to form a stable moiety. Non-limiting examples of heteroaliphatic moieties are polyethylene glycol, polyalkylene glycol, amide, polyamide, polylactide, polyglycolide, thioether, ether, alkyl-heterocycle-alkyl, -O-alkyl-O-alkyl, alkyl-O-haloalkyl, and the like.

[0618] "Heterocycloalkyl" refers to an alkyl group, as defined herein, substituted with a heterocyclo group, as defined herein.

[0619] "Arylalkyl" refers to an alkyl group, as defined herein, substituted with an aryl group, as defined herein.

[0620] "Heteroarylalkyl" refers to an alkyl group, as defined herein, substituted with a heteroaryl group, as defined herein.

[0621] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group of 2 to 24 carbon atoms (e.g., ethynyl, n-propynyl, etc.) containing at least one triple bond. Generally, although not necessarily, alkynyl groups herein may contain from 2 to about 18 carbon atoms, and such groups may further contain from 2 to 12 carbon atoms. The term "lower alkynyl" contemplates an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to an alkynyl substituted with one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to an alkynyl having at least one carbon atom replaced with a heteroatom. Unless otherwise specified, the terms "alkynyl" and "lower alkynyl" include straight-chain, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0622] The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., 6, 10, or 14 pi electrons shared in a cyclic array) having 6 to 14 ring carbon atoms and 0 heteroatoms in the aromatic ring system ("C 6~14 In some embodiments, an aryl group has 6 ring carbon atoms ("C aryl", e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 "Aryl," e.g., anthracyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the bonding radical or point of attachment is on the aryl ring; in such cases, the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. One or more of the fused carbocyclyl or heterocyclyl groups may be 4- to 7-membered or 5- to 7-membered saturated or partially unsaturated carbocyclyl or heterocyclyl groups, optionally containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, phosphorus, sulfur, silicon, and boron, to form, for example, a 3,4-methylenedioxyphenyl group. In one non-limiting embodiment, the aryl group is pendant. An example of a pendant ring is a phenyl group substituted with a phenyl group. In an alternative embodiment, the aryl group is optionally substituted as described above. In certain embodiments, the aryl group is an unsubstituted C 6~14 In certain embodiments, the aryl group is a substituted C 6~14 Aryl. The aryl group may be optionally substituted with one or more functional groups including, but not limited to, halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, and heterocyclo.

[0623] The term "heterocyclyl" (or "heterocyclo") includes saturated and partially saturated heteroatom-containing ring radicals, where the heteroatoms may be selected from nitrogen, sulfur, and oxygen. Heterocycles include monocyclic 3- to 8-membered rings and 5- to 16-membered bicyclic ring systems (which may include bridged and spiro-fused bicyclic ring systems). This does not include rings containing -OO-, -OS-, or -SS- moieties. The "heterocyclyl" groups may be optionally substituted with one, two, three, four, or more substituents, including, but not limited to, hydroxyl, Boc, halo, haloalkyl, cyano, alkyl, aralkyl, oxo, alkoxy, and amino. Examples of saturated heterocyclic groups include saturated 3- to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms (e.g., pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, piperazinyl), saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g., morpholinyl), and saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl). Examples of partially saturated heterocyclyl radicals include, but are not limited to, dihydrothienyl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl.Examples of partially saturated and saturated heterocyclic groups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl, indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl, isochromanyl, chromanyl, 1,2-dihydroquinolyl, 1,2,3,4-tetrahydro-isoquinolyl, 1, Examples include, but are not limited to, 2,3,4-tetrahydroquinolyl, 2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl, 5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl, 3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl, 2,3-dihydro-1H-1λ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl.

[0624] Heterocyclo groups also include radicals in which a heterocyclic radical is fused / condensed with an aryl radical or a heteroaryl radical, such as unsaturated fused heterocyclic groups containing 1 to 5 nitrogen atoms (e.g., indoline, isoindoline), unsaturated fused heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, unsaturated fused heterocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, and saturated, partially unsaturated, and unsaturated fused heterocyclic groups containing 1 to 2 oxygen atoms or sulfur atoms.

[0625] The term "heteroaryl" refers to an aryl ring system containing one or more heteroatoms selected from O, N, and S, wherein the nitrogen and sulfur atom(s) of the ring are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Examples include unsaturated 5- to 6-membered heteromonocyclyl groups containing 1 to 4 nitrogen atoms (e.g., pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl]), unsaturated 5- to 6-membered heteromonocyclic groups containing an oxygen atom (e.g., pyranyl, 2-furyl, 3-furyl, etc.), unsaturated 5- to 6-membered heteromonocyclic groups containing a sulfur atom (e.g., 2-thienyl, 3-thienyl, etc.), and the like. and the like), unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g., oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]), and unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolyl, thiadiazolyl [e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl]).

[0626] As used herein, the terms "may," "optional," "optionally," or "may optionally" mean that the subsequently described circumstance may or may not occur, and thus the description includes cases where the circumstance occurs and cases where it does not occur. For example, the phrase "optionally substituted" means that non-hydrogen substituents may or may not be present on a given atom, and thus the description includes structures where non-hydrogen substituents are present and structures where non-hydrogen substituents are absent.

[0627] The term "optionally substituted" refers to groups herein that are C1 to C 10 Alkyl, C2-C10 Alkenyl, C2-C 10 Alkynyl, C3-C 12 Cycloalkyl, C3-C 12 Cycloalkenyl, C1-C 12 Heterocycloalkyl, C3-C 12 Heterocycloalkenyl, C1-C 10 Alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C1-C 10 Alkylamino, C1-C 10 Dialkylamino, arylamino, diarylamino, C1-C 10 Alkyl sulfone amino, aryl sulfone amino, C1-C 10 Alkylimino, arylimino, C1-C 10 Alkyl sulfonimino, aryl sulfonimino, hydroxyl, halo, thio, C1-C 10 Alkylthio, C1-C 10 Substitution with a moiety including, but not limited to, alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amidino, guanidine, ureido, cyano, nitro, azido, acyl, thioacyl, acyloxy, carboxyl, and carboxylic acid ester is indicated.

[0628] In an alternative embodiment, any suitable group may be present in the "substituted" or "optionally substituted" positions, if shown to form a stable molecule and fulfill the desired objectives of the invention, such as halogen (which may independently be F, Cl, Br, or I); cyano; hydroxyl; nitro; azido; alkanoyl (such as a C2-C6 alkanoyl group); carboxamido; alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, such as phenoxy; thioalkyl (including those with one or more thioether linkages); alkylsulfinyl; alkylsulfonyl groups (including those with one or more sulfonyl linkages); aminoalkyl groups (including groups with multiple N atoms); aryl (e.g., phenyl, biphenyl, naphthyl, etc., where each ring is either substituted or unsubstituted); and alkyl groups having, for example, 1 to 3 separate or fused rings and 6 to about 14 or 18 Examples of arylalkoxy include, but are not limited to, arylalkyl having 1 to 3 ring carbon atoms (benzyl is an exemplary arylalkyl group); arylalkoxy having, for example, 1 to 3 separate or fused rings (benzyloxy is an exemplary arylalkoxy group); or saturated or partially unsaturated heterocycles having 1 to 3 separate or fused rings with one or more N, O, or S atoms, or heteroaryls having 1 to 3 separate or fused rings with one or more N, O, or S atoms, such as coumarinyl, quinolinyl, isoquinolinyl, quinazolinyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, and pyrrolidinyl. Such groups may be further substituted, for example, with hydroxy, alkyl, alkoxy, halogen, and amino.In certain embodiments, "optionally substituted" includes halogen, hydroxyl, amino, cyano, -CHO, -COOH, -CONH2, alkyl including C1-C6 alkyl, alkenyl including C2-C6 alkenyl, alkynyl including C2-C6 alkynyl, -C1-C6 alkoxy, alkanoyl including C2-C6 alkanoyl, C1-C6 alkyl ester, (mono- and di-C1-C6 alkylamino)C0-C2 alkyl, C1-C6 haloalkynyl, and haloalkoxy, including C1-C6 haloalkoxy, hydroxyC1-C6 alkyl, ester, carbamate, urea, sulfonamide, —C1-C6 alkyl(heterocyclo), C1-C6 alkyl(heteroaryl), —C1-C6 alkyl(C3-C7 cycloalkyl), O—C1-C6 alkyl(C3-C7 cycloalkyl), B(OH)2, phosphate, phosphonate, and haloalkoxy, including C1-C6 haloalkoxy.

[0629] When the term "substituted" appears before or after a list of possible substituents, it is intended that the term apply to all members of that group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl."

[0630] Further to the disclosure herein, the term "substituted," when used to modify a particular group or radical, can also mean that one or more hydrogen atoms of that particular group or radical are each, independently of one another, replaced with the same or different substituents, as defined herein.

[0631] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0632] Unless otherwise indicated, naming of substituents not expressly defined herein is accomplished by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "hydroxyalkyl" refers to the group HO-(alkyl)-.

[0633] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not include any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. Furthermore, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0634] In certain embodiments, substituents may contribute to the optical isomerism and / or stereoisomerism of a compound.

[0635] The compounds of the present disclosure may form solvates with solvents (including water). Thus, in one non-limiting embodiment, the present disclosure includes solvated forms of the compounds. The term "solvate" refers to a molecular complex of a compound (including its salts) with one or more solvent molecules. Non-limiting examples of solvents are water, ethanol, isopropanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term "hydrate" refers to a molecular complex comprising a compound and water. Pharmaceutically acceptable solvates according to the present invention include those in which the solvent may be isotopically substituted, such as DO, d6-acetone, and d6-DMSO. The solvate may be in liquid or solid form.

[0636] Salts, solvates, hydrates, and prodrug forms of the compounds are of interest. All such forms are encompassed by the present disclosure. Thus, the compounds described herein include their salts, solvates, hydrates, prodrugs, and isomeric forms, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers thereof. In certain embodiments, the compounds can be metabolized to pharmaceutically active derivatives.

[0637] Unless otherwise specified, a reference to an atom is intended to include isotopes of that atom. For example, a reference to H is 1 H, 2 H (i.e., D) and 3 is intended to include H (i.e., T), and any reference to C is 12 C and all isotopes of carbon (e.g., 13 C).

[0638] Definitions of other terms and concepts are provided throughout the detailed description.

[0639] Additional Embodiments Aspects of the present disclosure are described in the following clauses.

[0640] Clause 1: An extracellular target binding conjugate of formula (I), or a pharmaceutically acceptable salt thereof, [ka] During the ceremony, X is a moiety that binds to a lysosomal targeting molecule; n is 1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 20, or 1 to 10, 1 to 6, or 1 to 4, e.g., 1, 2, or 3), L is a linker, m is bonded to YB (X n - the average number of (L) moieties, where m is in the range of from about 1 to about 20 (e.g., from about 1 to about 3 (e.g., 1, 2, or 3), or from about 1 to about 10, from about 1 to about 8, from about 2 to about 8, from about 3 to about 6, or from about 4 to about 5); Y is any carrier polypeptide linked to B; The extracellular target binding conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein B is a polypeptide that specifically binds to an extracellular target molecule.

[0641] Clause 2: The conjugate of clause 1, wherein B specifically binds to a target antibody (e.g., an extracellular antibody associated with a disease or condition of interest).

[0642] Clause 3: The conjugate of clause 2, wherein said target antibody is an autoantibody.

[0643] Clause 4: The conjugate of clause 2, wherein said targeting antibody is a neutralizing antibody or an anti-drug antibody.

[0644] Clause 5: The conjugate of any one of clauses 2 to 4, wherein B comprises an antigen of said target antibody, or a fragment thereof (e.g., a synthetic peptide).

[0645] Clause 6: The conjugate of any one of clauses 1 to 5, wherein B comprises a protein domain.

[0646] Clause 7: The conjugate of any one of clauses 1 to 6, wherein Y comprises a protein domain.

[0647] Clause 8: The conjugate of any one of clauses 1 to 7, wherein Y is directly fused to B.

[0648] Clause 9: The conjugate of any one of clauses 1 to 7, wherein Y is indirectly fused to B via a spacer domain.

[0649] Clause 10: The conjugate of any one of clauses 1 to 9, wherein YB is a chimeric protein.

[0650] Clause 11: The conjugate of any one of clauses 1 to 10, wherein Y is selected from human serum albumin (HSA), an HSA domain, an albumin binding domain, Fc (monomer), Fc (dimer), and fragments thereof (e.g., synthetic peptides).

[0651] Clause 12: The conjugate of clause 10 or 11, wherein YB is selected from an Fc-VHH antigen fusion, an Fc-antigen fusion, and an HSA-antigen fusion.

[0652] Clause 13: The conjugate of any one of clauses 1 to 6, wherein Y is covalently attached to B via a linker.

[0653] Clause 14: The conjugate of clause 13, wherein said linker comprises a non-peptide linking moiety.

[0654] Clause 15: The conjugate of any one of clauses 13-14, wherein Y is selected from human serum albumin (HSA), an HSA domain, an albumin binding domain, Fc (monomer), Fc (dimer), and fragments thereof (e.g., synthetic peptides).

[0655] Clause 16: The conjugate of any one of clauses 1 to 15, wherein m is 1 to 10 (e.g., 1 to 6 or 1 to 4, where m can be a discrete load or an average load (i.e., a DAR ratio)).

[0656] Clause 17: The conjugate of clause 16, wherein m is 1 to 2.

[0657] Clause 18: The conjugate according to clause 17, wherein m is 1.

[0658] Clause 19: The conjugate according to clause 17, wherein m is 2.

[0659] Clause 20: The conjugate of any one of clauses 16, wherein m is 2 to 4.

[0660] Clause 21: The conjugate according to clause 20, wherein m is 3.

[0661] Clause 22: The conjugate according to clause 20, wherein m is 4.

[0662] Clause 23: The conjugate of any one of clauses 1 to 22, wherein n is 1 to 20 (e.g., 1 to 10, 1 to 6, or 1 to 4 in the case of an M6PR ligand, or 1 to 3 in the case of, for example, a GalNAc ligand).

[0663] Clause 24: The conjugate according to clause 23, wherein n is 1 to 2.

[0664] Clause 25: The conjugate according to clause 24, wherein n is 1.

[0665] Clause 26: The conjugate according to clause 24, wherein n is 2.

[0666] Clause 27: The conjugate according to clause 23, wherein n is 3.

[0667] Clause 28: The conjugate of any one of clauses 1 to 27, wherein n is 1 and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to YB.

[0668] Clause 29: The conjugate of any one of clauses 1 to 27, wherein n is 2 and L is a branched linker covalently attaching said X moiety to YB.

[0669] Clause 30: The conjugate of any one of clauses 1 to 27, wherein n is 3 and L is a branched linker covalently attaching said X moiety to YB.

[0670] Clause 31: The conjugate of any one of clauses 1-30, wherein the linker L comprises a backbone of 20 to 60 consecutive atoms (e.g., 20 to 50, 20 to 40, 30 to 40, 30 to 60, or 40 to 60 consecutive atoms) between each X and YB.

[0671] Clause 32: L is of formula (XI), [ka] During the ceremony, L 1 and L 3 are independently linear linking moieties, and L 2 is the branched connection part, and L 1 ~L 3 together form a linear or branched linker between X and Y, a, b, and c are independently 0 or 1; ** is Z 1 X's L via 1 represents the point of attachment to 32. The conjugate of any one of clauses 1-31, wherein *** represents the point of attachment of said linker L to YB.

[0672] Article 33: n is 1, The conjugate of clause 32, wherein a is 1, b is 0, c is 1 and L is of formula (Xa). [ka]

[0673] Article 34: n is 2, The conjugate of clause 32, wherein a is 1, b is 1, c is 1 and L is of formula (Xb). [ka]

[0674] Article 35: n is 3, The conjugate of clause 32, wherein a is 1, b is 1, c is 1 and L is of formula (Xc). [ka]

[0675] Article 36: Each L 1 is of formula (XII), [ka] During the ceremony, L 10 is the connecting part, L 11 ~L 19 is independently absent or is a linking moiety, Each L1 Each linking part of -C 1~20 -Alkylene-, -NHCO-C 1~6 -Alkylene-, -CONH-C 1~6 -Alkylene-, -NHC 1~6 -Alkylene-, -NHCONH-C 1~6 -Alkylene-, -NHCSNH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHCO-, -C 1~6 -Alkylene-CONH-, -C 1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHCONH-, -C 1~6 -Alkylene-NHCSNH-, -O(CH2) p -, -(OCH2CH2) p 36. The conjugate of any one of clauses 32-35, wherein each p is independently selected from -, -NHCO-, -CONH-, -NHSO2-, -SON2NH-, -CO-, -SO2-, -O-, -S-, pyrrolidine-2,5-dione, 1,2,3-triazole, -NH-, and -nMe-, wherein each p is independently 1 to 50.

[0676] Article 37: Each L 1 37. The conjugate of any one of clauses 32-36, wherein comprises a linear backbone of 6 to 20 consecutive atoms (e.g., 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive atoms).

[0677] Article 38:L 2 is of formula (XIIIa) or (XIIIb), [ka] During the ceremony, L 20 is an amino acid residue (e.g., residues such as Gly, β-Ala, Lys, Orn, Asp, Glu, Ser, Cys, or derivatives thereof), -NH-CH[(CH2) q ]2O- or -NH-C[(CH2) q ]3O-, [ka] -C 1~6 -Alkylene-, -NHCO-, -CONH-, -NHSO2-, -SO2NH-, -CO-, -SO2-, -O-, -S-, pyrrolidine-2,5-dione, 1,2,3-triazole, -NH-, -nMe-, -NHC(=O)NH-, -NHC(=S)NH-, -O(CH2) p - and -(OCH2CH2) p - a branched linking moiety comprising one or more linking moieties independently selected from 38. The conjugate of any one of clauses 32 and 34-37, wherein each p is independently 1 to 50 and q is 1 to 6.

[0678] Article 39:L 2 comprises a linking moiety selected from: [ka] [ka] [ka] and [ka] During the ceremony, Z 2 and Z 3 are each independently selected from -NHCO-, -CONH-, -CO-, -O-, -NH-, and -nMe-; x is 1 to 12 (e.g., 1 to 6, or 1 to 3), 39. The conjugate of any one of clauses 32 and 34-38, wherein y is 0 to 12 (e.g., 1 to 6, or 1 to 3).

[0679] Article 40:L 2 40. The conjugate of clause 39, wherein [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka]

[0680] Article 41:L 2 comprises a linking moiety of formula (XIV), [ka] During the ceremony, r is 1 or 2, If n is 2, then r is 1, The conjugate according to clause 40, wherein when n is 3, r is 2.

[0681] Article 42:L 2 The conjugate according to clause 41, wherein is of formula (XVa) or (XVb): [ka] [ka]

[0682] Article 43:L 239. The conjugate of clause 38, wherein comprises two or more amino acid residues (e.g., three or more or four or more amino acid residues, linear or dendrimer).

[0683] Article 44:L 2 comprises four or more amino acid residues (e.g., a side chain, amino, and carboxylic acid are each linked to an adjacent moiety) that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys.

[0684] Article 45: Each L 3 is of formula (XVI), [ka] During the ceremony, L 30 ~L 39 is independently absent or is a linking moiety, Z is a residue moiety resulting from the covalent bond between the chemoselective ligation group of the linker and the compatibility group of YB; L 3 Each linking part of -C 1~20 -Alkylene-, -NHCO-C 1~6 -Alkylene-, -CONH-C 1~6 -Alkylene-, -NHC 1~6 -Alkylene-, -NHCONH-C 1~6 -Alkylene-, -NHCSNH-C 1~6 -Alkylene-, -C 1~6 -Alkylene-NHCO-, -C 1~6 -Alkylene-CONH-, -C 1~6 -Alkylene-NH-, -C 1~6 -Alkylene-NHCONH-, -C 1~6 -Alkylene-NHCSNH-, -O(CH2) p -, -(OCH2CH2) p45. The conjugate of any one of clauses 32-44, wherein each p is independently selected from -, -NHCO-, -CONH-, -NHSO2-, -SON2NH-, -CO-, -SO2-, -O-, -S-, pyrrolidine-2,5-dione, 1,2,3-triazole, -NH-, and -nMe-, wherein each p is independently 1 to 50.

[0685] Article 46:L 3 46. ​​The conjugate of clause 45, wherein comprises a linear backbone of 6 to 40 consecutive atoms (e.g., 10 to 30 consecutive atoms, or 20 to 30 consecutive atoms).

[0686] Clause 47: The linker L has the following structure: [ka] During the ceremony, a is 1 to 12 (e.g., 2 to 6, or 2, or 3), b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2, d is 1 to 6 (e.g., 1, 2, or 3); e is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); 47. The conjugate of clause 45 or 46, wherein Z is a residue moiety resulting from the covalent bond between the chemoselective ligation group of said linker and the compatibility group of YB.

[0687] Clause 48: The conjugate according to Clause 47, wherein said linker L comprises one of the following structures: [ka] or [ka]

[0688] Clause 49: The conjugate is of formula (Ia): [ka] During the ceremony, Z is a residue moiety resulting from the covalent bond between the chemoselective ligation group of the linker and the compatibility group of YB; n is 1, 2, or 3; m is bonded to YB (X n - the average number of L) moieties, wherein m is in the range of from about 1 to about 8.

[0689] Clause 50: The conjugate according to clause 49, wherein Z is a residue moiety resulting from the covalent bond between a thiol-reactive chemoselective ligation group and one or more cysteine ​​residues of YB.

[0690] Clause 51: The conjugate of Clause 50, wherein said thiol-reactive chemoselective ligation group comprises maleimide, bromomaleimide, haloacetamide, vinyl sulfone, or thiolactone.

[0691] Clause 52: The thiol-reactive chemoselective ligation group is selected from one of the following structures: [ka] [ka] and [ka] During the ceremony, u is 1 to 11 (e.g., 1 to 5), v is 1 to 11 (e.g., 1 to 5), The conjugate of clause 51, wherein X is H or Br.

[0692] Clause 53: m is bonded to YB (X n53. The conjugate of any one of clauses 50-52, wherein m is the average number of YB) moieties, and m corresponds to the number of solvent accessible cysteine ​​residues in YB.

[0693] Clause 54: The conjugate according to clause 53, wherein m is about 3 or less.

[0694] Clause 55: The conjugate according to clause 54, wherein m is about 1.

[0695] Clause 56: The conjugate according to clause 54, wherein m is about 2.

[0696] Clause 57: The conjugate according to clause 54, wherein n is about 3.

[0697] Clause 58: The conjugate according to Clause 49, wherein Z is a residue moiety resulting from the covalent bond (e.g., via an amide bond) between an amine-reactive chemoselective ligation group and one or more cysteine ​​residues of YB.

[0698] Clause 59: The conjugate of Clause 58, wherein the amine-reactive chemoselective ligation group comprises an active ester (e.g., N-hydroxysuccinimidyl (NHS) ester, sulfo-NHS ester, pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester, etc.).

[0699] Clause 60: m is bonded to YB (X n -L) moieties, and m is from about 3 to about 6.

[0700] Clause 61: The conjugate of clause 60, wherein m is from about 4 to about 5.

[0701] Clause 62: The conjugate of any one of clauses 1 to 61, wherein n is 1.

[0702] Clause 63: The conjugate according to any one of clauses 1 to 61, wherein n is 2.

[0703] Clause 64: The conjugate of any one of clauses 1 to 61, wherein n is 3.

[0704] Clause 65: The conjugate of any one of clauses 1 to 64, wherein the lysosomal targeting molecule is selected from asialoglycoprotein receptor (ASGPR), M6PR, folate receptor, CD63, transferrin, sortilin, IFITM3, molecules in the endosomal / lysosomal pathway, LIMP-1, and LIMP-2.

[0705] Clause 66: The conjugate of any one of clauses 1 to 65, wherein X is a moiety that binds to a cell surface asialoglycoprotein receptor (ASGPR).

[0706] Clause 67: X is of formula (II), [ka] During the ceremony, R 1 But -Z 1 selected from -*, -H, -OH, -CH3, -OCH3, and -OCH2CH=CH; R 2 But -Z 1 -*, -NHCOCH3, -NHCOCF3, -NHCOCH2CF3, -OH, and optionally substituted triazole; R 6 But -Z 1 -*, -OH, -OC(O)R, -C(O)NHR, and optionally substituted triazole, where R is optionally substituted (C1-C6) alkyl or optionally substituted aryl; R 1 , R 2 , and R 6 One of them is -Z 1 -*, where "*" is Z 1 represents the point of attachment to the linker (L), R 3 and R 4are each independently H or a promoiety, or R 3 and R 4 are linked in a ring to form a promoiety, R 11 is H or a bridging moiety connecting the 5-carbon to the 1-carbon of the ring, Z 1 But -Z 11 -, -Z 11 -A 1 -, -A 2 -, -NR 21 CO-, -CONR 21 -, -NR 21 SO2-, -SO2NR 21 -, -NR 21 C(=O)NR 21 - and -NR 21 C(=S)NR 21 - a linking moiety selected from -Z 11 -, -O-, -S-, -N(R 21 )-, or -C(R 22 )2, -A 1 -and-A 2 - is optionally substituted arylene or optionally substituted heteroarylene; Each R 21 are independently selected from H and optionally substituted (C1-C6) alkyl; Each R 22 66. The conjugate of clause 65, wherein is independently selected from H, halogen (e.g., F), and optionally substituted (C1-C6) alkyl.

[0707] Article 68:R 1 Ga-Z 1 -* and each X is independently of formula (IIa). [ka]

[0708] Clause 69: each X is independently of formula (IVa) or (IVb); [ka] [ka] During the ceremony, -Z 11 -, -O-, -S-, -N(R 21 )-, or -C(R 22 )2, -A 1 69. The conjugate of clause 68, wherein - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.

[0709] Article 70:Z 11 70. The conjugate according to clause 69, wherein is -S-.

[0710] Clause 71: The conjugate of clause 68 or 69, wherein each X is independently of formula (iIa-1). [ka]

[0711] Article 72:Z 1 is S, and each X is independently of the formula (X A 72. The conjugate according to clause 71, which is of the formula: [ka]

[0712] Clause 73: The conjugate of clause 68 or 69, wherein each X is independently of formula (IIa-2). [ka]

[0713] Article 74:Z 1 is S, and each X is independently of the formula (X B 74. The conjugate according to clause 73, which is of the formula: [ka]

[0714] Article 75:Z 11 -C(R 22 74. The conjugate of clause 68, 69, 71 or 73, wherein

[0715] Clause 76: Each X independently represents the formula (X C 76. The conjugate according to clause 75, which is of the formula: [ka]

[0716] Clause 77: each X is independently of formula (IIIf); [ka] During the ceremony, -A 1 76. The conjugate of clause 75, wherein - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.

[0717] Clause 78: Each X independently represents the formula (X C ) or (X D -2). [ka]

[0718] Article 79:R 2 Ga-Z 1 -* and each X is independently of formula (IIb). [ka]

[0719] Clause 80: The conjugate of Clause 79, wherein each X is independently of formula (IVa). [ka]

[0720] Clause 81: each X is independently of formula (IVb) or (IVc); [ka] [ka] (IVc) In the formula, -Z 11 -, -O-, -S-, -N(R 21 )-, or -C(R 22 81. The conjugate according to clause 80, wherein

[0721] Article 82:R 1 82. The conjugate of clause 80 or 81, wherein is H.

[0722] Article 83:R 11 83. The conjugate of any one of clauses 80 to 82, wherein

[0723] Clause 84: each X is independently of formula (IVb-1) or (IVc-1), [ka] [ka] In the formula, R 11 82. The conjugate of clause 80 or 81, wherein is a bridging moiety connecting the 5-carbon to the 1-carbon.

[0724] Article 85:R 11 The conjugate according to clause 84, wherein is -CH2O- or -OCH2-.

[0725] Article 86:-Z 11 - is -N(R 21 )- and -A 1-and-A 2 86. The conjugate of any one of clauses 81 to 85, wherein - is an optionally substituted monocyclic heteroarylene (e.g., triazole or pyrimidine).

[0726] Clause 87: Each X independently represents the formula (X E -1) or (X F ) and [ka] [ka] In the formula, R 21 and R 22 H, halogens, (C 1~6 87. The conjugate of clause 86, wherein each of the groups is independently selected from (C1-C6) alkyl, and substituted (C1-C6) alkyl (e.g., CF3).

[0727] Article 88:R 6 Ga-Z 1 -* and each X is independently of formula (IIc). [ka]

[0728] Clause 89: The conjugate is derived from a ligand linker compound of Table 1, wherein: X is, [ka] [ka] [ka] [ka] [ka] or [ka] and L, [ka] [ka] or [ka] and Z, [ka] [ka] or [ka] 89. The conjugate of any one of clauses 1 to 88, derived from a Z precursor which is [Table 27]

[0729] Clause 90: The conjugate of any one of clauses 1 to 64, wherein X is a moiety that binds to a cell surface mannose-6-phosphate receptor (M6PR).

[0730] Clause 91: X is of formula (IV), [ka] During the ceremony, W is a non-hydrolyzable hydrophilic head group; Z 1 is selected from optionally substituted (C1-C3) alkylene and optionally substituted ethenylene; Z 2 But O, S, NR 21 and C(R22 )2, where each R 21 is independently selected from H and optionally substituted (C-C) alkyl, and each R 22 are independently selected from H, halogen, and optionally substituted (C1-C6) alkyl; each A is independently an optionally substituted aryl or heteroaryl linking moiety; each Z 3 91. The conjugate of clause 90, wherein:

[0731] Clause 92: A method for reducing the level of an extracellular target molecule in a biological system, comprising: 92. A method comprising contacting said biological system with an effective amount of the conjugate of any one of clauses 1 to 91, wherein said compound specifically binds to said extracellular target molecule and specifically binds to a lysosomal targeting molecule of a cell within said biological system to promote cellular uptake and degradation of said extracellular target molecule.

[0732] Clause 93: The conjugate according to clause 12, wherein YB is an HSA-antigen fusion.

[0733] Clause 94: The conjugate according to clause 15, wherein Y is HSA.

[0734] Clause 95: The method of Clause 92, wherein said target antibody is an autoantibody.

[0735] Clause 96: The method of any one of clauses 92 to 95, wherein said target antibody is a TSH receptor autoantibody.

[0736] Clause 97: The method of any one of clauses 92 to 95, wherein said targeting antibody is a MuSK antibody.

[0737] Clause 98: The method of any one of clauses 92 to 95, wherein said targeting antibody is an aChR antibody.

[0738] Clause 99: The method of any one of clauses 92 to 95, wherein said target antibody is a BP180 antibody.

[0739] Clause 100: The method of any one of clauses 92 to 95, wherein said target antibody is a PR3 antibody.

[0740] Clause 101: The method of any one of clauses 92 to 95, wherein said lysosomal targeting molecule is ASGPR.

[0741] Clause 102: The method of any one of clauses 92 to 95, wherein said lysosomal targeting molecule is M6PR. [Example]

[0742] The examples in this section are offered by way of illustration, not by way of limitation.

[0743] Preparation of compounds Below are illustrative schemes and examples of how the compounds described herein can be prepared and tested. While the examples may represent only some embodiments, it should be understood that the following examples are illustrative and not limiting. All substituents are as defined above unless otherwise specified. Reagents and starting materials are readily available to one of ordinary skill in the art. Certain synthetic steps for each of the routes described can be combined in different ways or with steps from different schemes to prepare the compounds described herein.

[0744] Synthesis of compound 1209 [ka] [ka] To a solution of compound A-1 (1.0 equiv., 5.05 g, 13.0 mmol) and benzyl N-[3-(5-hydroxypentanamido)propyl]carbamate (compound 18A) (1.0 equiv., 4.00 g, 13.0 mmol) in dichloromethane (50.0 mL), trimethylsilyl trifluoromethanesulfonate (1.1 equiv., 2.52, 14.3 mmol) was added dropwise at room temperature. The reaction mixture was stirred at 40 °C for 5 h. Upon completion, the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, and concentrated under high vacuum to give the crude product. The crude product was purified by reverse-phase chromatography using 0 to 30% acetonitrile in water to give compound 18B as a yellow viscous liquid. Yield: 5.80 g, 70.12%. LCMS m / z 638.2[M+1] +

[0745] To a solution of compound 18B (1.0 equiv., 4.80 g, 7.53 mmol) in methanol (40.0 mL), 10% palladium on carbon (1.60 g) was added and stirred under a hydrogen atmosphere at room temperature for 4 hours. Upon completion, the reaction mixture was filtered through a syringe filter, and the filtrate was concentrated and dried to give the crude product. The crude product was triturated with diethyl ether to give compound 18C as a pale yellow viscous liquid. Yield: (3.4 g, 80.73%). LCMS m / z 504.37 [M+1] + .

[0746] A solution of 2,3,4,5,6-pentafluorophenyl 3-(2-{[(benzyloxy)carbonyl]amino}-3-[3-oxo-3-(2,3,4,5,6-pentafluorophenoxy)propoxy]-2-{[3-oxo-3-(2,3,4,5,6-pentafluorophenoxy)propoxy]methyl}propoxy)propanoate (18D) (1.0 equiv., 1.20 g, 1.24 mmol) and compound 18C (3.0 equiv., 1.87 g, 3.71 mmol) in N,N-dimethylformamide (30.0 mL) was stirred at room temperature for 1 h. Upon completion, the reaction mixture was concentrated and dried to give the crude product. The crude product was purified by flash column chromatography using 20% ​​methanol in dichloromethane to give compound 18E as a pale yellow viscous liquid. Yield: (1.60g, 67.05%). LCMS m / z 1926.78[M-1] - .

[0747] To a solution of compound 18E (1.0 equiv., 1.60 g, 0.830 mmol) in methanol (20 mL) and acetic acid (1.0 mL) was added 10% palladium on carbon (250 mg). The reaction mixture was stirred under a hydrogen atmosphere at room temperature for 16 hours. Upon completion, the reaction mixture was filtered through a bed of celite, and the filtrate was concentrated and dried to give compound 18F as a pale yellow viscous liquid. Yield: 1.45 g (crude). LCMS m / z 1794.05 [M+1] + .

[0748] To a solution of compound 18F (1.0 equiv., 1.45 g, 0.808 mmol) in methanol (10 mL) was added 25% sodium methanolate solution (8.0 equiv., 1.45 mL, 6.47 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was concentrated and dried to give the crude product. The crude product was diluted with acetonitrile and purified by preparative HPLC (30% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound 18G as an off-white semi-solid. Yield: (0.20 g, 17.4%). LCMS m / z 1415.77 [M+1] + .

[0749] To a solution of dodecanedioic acid (20A) (1.00 g, 4.34 mmol) in ethyl acetate (10.00 mL) was added pentafluorophenol (1.60 g, 8.68 mmol) and diisopropylmethanediimine (1.91 mL, 13.0 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 1 h. Upon completion, the reaction mixture was filtered through a Celite bed, and the filtrate was concentrated under reduced pressure to obtain the crude compound. The obtained crude compound was purified by flash column chromatography on a silica gel column using 5% ethyl acetate in hexane as the eluent to obtain compound 20B as an off-white solid. Yield: 1.00 g (40.95%). LCMS m / z 580.39 [M+18] + .

[0750] To a solution of compound 18G (45.0 mg, 0.031 mmol) in dimethyl sulfoxide (1.0 mL) was added N,N-diisopropylethylamine (0.016 mL, 0.093 mmol) and compound 20B (17.9 mg, 0.031 mmol). The reaction mixture was stirred at room temperature for 2 hours. Upon completion, the reaction mixture was purified by preparative HPLC (40-60% acetonitrile in water containing 0.1% trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to give compound 1209 as an off-white solid. Yield: 0.006 g (10.52%). LCMS m / z 1793.94 [M+1] + ,897.99[M / 2+1] + . 1H NMR(400MHz,DMSO-d6) δ 7.83(t,J=5.6Hz,3H),7.73(t,J=5.2Hz,3H),7.60(d,J=9.2Hz,3H),6.99(s,1H),4.57-4.4 7(m,6H),4.46(d,J=4.4Hz,3H),4.21(d,J=8.4Hz,3H),3.70-3.63(m,9H),3.55-3.49(m,21 H),3.32-3.28(m,4H),3.02(t,J=5.6Hz,12H),2.76(t,J=5.6Hz,2H),2.27(t,J=6.4Hz,6H) ,2.03(t,J=7.2Hz,8H),1.79(s,9H),1.70-1.67(m,2H),1.52-1.41(m,20H),1.23(bs,14H).

[0751] Synthesis of compound 1209-C [ka] Compound 1209-C was prepared similarly to compound 1209 using the synthetic route described for compound 1209, except that (3S,4S,5S,6S)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (6) was used as the starting material instead of A-1 to give the final product. LCMS, m / z 897.65 [M+2] ++ .

[0752] Synthesis of (3S,4S,5S,6S)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (6) [ka]

[0753] Synthesis of (2S,3S,4S)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (2): L-Galactose (5.9 g, 32.7 mmol) was suspended in a solution of acetic acid (15.0 mL) and acetic anhydride (21.7 mL, 7.0 equiv., 229 mmol), and 33% HBr-acetic acid solution (6.0 g) was added. The reaction mixture was stirred at room temperature for 3 hours, after which an additional 54.0 g of 33% HBr-acetic acid solution (7.7 equiv. HBr total) was added and stirred overnight. Sodium acetate (22.0 g) was then added to neutralize excess HBr, and the reaction mixture was added to a suspension containing ground CuSO4:5HO (1.92 g), zinc (6.3 g), water (60.0 mL), sodium acetate (54.8 g), and acetic acid (30.0 mL). The resulting reaction mixture was vigorously stirred for 3 hours. The solution was then filtered, and the solid was washed with ethyl acetate (200 mL) and water (200 mL). The organic layer of the filtrate was then washed with saturated aqueous sodium bicarbonate (200 mL) and brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude product was purified by silica gel column chromatography (30% ethyl acetate-hexane) to give (2S,3S,4S)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (2) as a colorless oil. Yield: 4.0 g, 45%, LCMS m / z 290.31 [M+18] + .

[0754] Synthesis of (2S,3S,4S,5S)-2-(acetoxymethyl)-5-azido-6-(nitrooxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3): (2S,3S,4S)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (2, 4.0 g, 14.7 mmol) in dry acetonitrile (80 mL) was added to a mixture of cerium ammonium nitrate (32.2 g, 58.8 mmol) and sodium azide (1.91 g, 29.4 mmol) under nitrogen at −20° C. After stirring at −20° C. for 7 hours, the reaction mixture was diluted with cold diethyl ether and water and extracted with diethyl ether. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product (2S,3S,4S,5S)-2-(acetoxymethyl)-5-azido-6-(nitrooxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3, 5.4 g) as a pale yellow syrup. The resulting crude mixture was used in the next reaction without further purification.

[0755] Synthesis of (3S,4S,5S,6S)-6-(acetoxymethyl)-3-azidotetrahydro-2H-pyran-2,4,5-triyl triacetate (4): To a solution of the crude product [(2S,3S,4S)-3,4-bis(acetyloxy)-5-azido-6-(nitrooxy)oxan-2-yl]methyl acetate (5.4 g) in acetic acid (50 mL) was added sodium acetate (3.53 g, 43.1 mmol) at room temperature under a nitrogen atmosphere. After stirring at 100 °C for 3 h, the reaction mixture was cooled to 0 °C, diluted with ethyl acetate and saturated aqueous sodium bicarbonate, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (28–30% ethyl acetate / n-hexane) afforded (3S,4S,5S,6S)-6-(acetoxymethyl)-3-azidotetrahydro-2H-pyran-2,4,5-triyl triacetate (4, 1.86 g, 34.64%) as a colorless viscous oil. Yield: 1.86g, 34.64%. LCMS m / z 374.2[M+1] + .

[0756] Synthesis of (3S,4S,5S,6S)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride (5): To a stirred solution of (3S,4S,5S,6S)-6-(acetoxymethyl)-3-azidotetrahydro-2H-pyran-2,4,5-triyl triacetate (4, 1.8 g, 4.82 mmol) in methanol (40 mL) was added 10% Pd / C (1.8 g) and 1N aqueous HCl (5.79 mL, 5.79 mmol) at 0 °C. After stirring under hydrogen at room temperature for 1 h, the reaction mixture was filtered through a Celite pad and concentrated in vacuo to give the crude product [(2S,3S,4S,5S)-3,4,6-tris(acetyloxy)-5-aminooxan-2-yl]methyl acetate hydrochloride (5) as an off-white solid. Yield: 1.7 g, 91.8%. LCMS m / z 348.1 [M+1] + .

[0757] Synthesis of (3S,4S,5S,6S)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (6): To a stirred solution of [(2S,3S,4S,5S)-3,4,6-tris(acetyloxy)-5-aminooxan-2-yl]methyl acetate hydrochloride (5, 1.7 g, 4.43 mmol) in pyridine (7.15 mL, 88.6 mmol) was added acetic anhydride (4.19 mL, 44.3 mmol) at 0° C. and stirred at room temperature for 12 hours. Volatiles were then evaporated under reduced pressure. The crude material was purified by silica gel column chromatography (6.5% methanol in dichloromethane) to give (3S,4S,5S,6S)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (6) as a colorless syrup. Yield: 1.4 g, 81.17%. LCMS m / z 448.1 [M+AcO] - .

[0758] Synthesis of Compound 1226 [ka] To a mixture of compound 1209 (1 equiv., 17.2 mg, 0.00959 mmol) and 1-(2-aminoethyl)pyrrole-2,5-dione hydrochloride (1.1 equiv., 1.86 mg, 0.00105 mmol) in NMP (0.5 mL) was added DIEA (3 equiv., 5 μL, 0.0288 mmol). The mixture was stirred at room temperature for 10 min, and acetic acid (4 μL) was added. The mixture was purified by preparative HPLC (10-40% MeCN / water containing 0.1% TFA) to give compound 1226 (10.1 mg, 64% yield) as a white solid (purity: 99%). LCMS m / z 1751.0 [M+H]+.

[0759] Synthesis of Compound Y [ka] Compound Y was prepared similarly to compound 1226 using the synthetic route described for compounds 1209 and 1226, except that (3S,4S,5S,6S)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (6) was used as the starting material instead of A-1 to give the final product. LCMS m / z 1750.9 [M+H]+.

[0760] Synthesis of XB47 (an intermediate of compound 1254 and compound 2346) [ka] To a mixture of benzyl N-[2-(2-prop-2-ynoxyethoxy)ethyl]carbamate (1.00 equiv., 816 mg, 2.94 mmol) in acetone (29 mL) was added NBS (1.34 equiv., 704 mg, 3.95 mmol) and silver nitrate (0.144 equiv., 72.0 mg, 0.424 mmol). The mixture was stirred at room temperature for 1 h and concentrated. The residue was diluted with EtOAc and washed with water (1x). The aqueous layer was extracted with EtOAc (2x). The combined organic layers were washed with brine, dried, concentrated, and purified by column chromatography (0-50% EtOAc / hexane) to give 1 (930 mg, 89% yield) as a clear oil. LCMS m / z 378.0 [M+Na]+.

[0761] To a mixture of N-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6-(benzyloxymethyl)-2-ethynyl-tetrahydropyran-3-yl]acetamide (prepared by the literature route described in Rouzier, et al., Synthesis (2019), 51(12), 2484-2488) (ISP2-718, 1.00 equiv., 201 mg, 0.403 mmol) 1 in MeCN (7.6 mL) and water (4.4 mL) was added benzyl N-[2-[2-(3-bromoprop-2-ynoxy)ethoxy]ethyl]carbamate (1, 1.20 equiv., 172 mg, 0.484 mmol). The mixture was cooled to 0 °C, and piperidine (5.00 equiv., 0.20 mL, 2.02 mmol) was added. The mixture was purged with N2, and CuCl (0.240 equiv., 9.6 mg, 0.0967 mmol) was added. The mixture was slowly warmed to room temperature and stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with 10% citric acid (1x) and brine (1x), dried, concentrated, and purified by column chromatography (0-80% EtOAc / hexanes) to give 2 as a white solid (232.2 mg, 74% yield). LCMS m / z 775.2 [M+H]+.

[0762] To a mixture of benzyl N-[2-[2-[5-[(2R,3S,4R,5R,6R)-3-acetamido-4,5-dibenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]penta-2,4-diynoxy]ethoxy]ethyl]carbamate (2, 1.00 equiv., 232 mg, 0.300 mmol) in HOAc (6 mL) was added 10% Pd / C (220 mg) and 20% Pd(OH) / C (230 mg). The mixture was stirred under hydrogen at room temperature for 2.5 h, filtered, concentrated, and purified by preparative HPLC (2-40% MeCN / 20 mM NHOH in water) to give XB47 as a white solid (83.6 mg, 74% yield). LCMS m / z 379.3 [M+H]+.

[0763] Synthesis of compound 1254 [ka] [ka] [ka] [ka] [ka] Synthesis of tert-butyl 1-azido-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (2) A solution of di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (1) (452 ​​mg, 0.894 mmol, 1.00 equiv.) and 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1a) (356 mg, 0.916 mmol, 1.02 equiv.) in 1 mL of acetonitrile was stirred under a nitrogen atmosphere and heated at 45 °C for 2 days until minimal 1 remained. The reaction mixture was diluted with dichloromethane, evaporated onto silica, and then purified by flash column chromatography, eluting with 0–100% ethyl acetate / dichloromethane. The product-containing fractions were concentrated and further dried under high vacuum at ambient temperature to give 2 as a clear thick syrup. Yield: 559 mg (78%). LCMS m / z 779.2 [M+H].

[0764] Synthesis of 1-azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oic acid (3) tert-Butyl 1-azido-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (2) (509 mg, 0.654 mmol) was added to 10 mL of a premixed solution of 30% trifluoroacetic acid in dichloromethane. The mixture was stirred at ambient temperature for 3.5 h until compound 2 was consumed. The solvent was evaporated to a residue under a stream of nitrogen and further dried under high vacuum at ambient temperature. The residue was then dissolved in 20% water / acetonitrile and lyophilized to dryness to give 3 as a thick, dark yellow syrup. Yield: 424 mg (99%). LCMS m / z 611.3 [M+H], 609.4 [M-1].

[0765] To a solution of 1-azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oic acid (3) (81.3 mg, 0.133 mmol, 1.0 equiv.) and 2,3,4,5,6-pentafluorophenol (3a) (91.8 mg, 0.499 mmol, 3.7 equiv.) in 1.5 mL of dichloromethane was added N,N'-diisopropylcarbodiimide (90 mL, 0.581 mmol, 4.4 equiv.). The mixture was stirred at ambient temperature for 1 h until compound 3 was consumed. The reaction mixture was diluted with dichloromethane, evaporated onto silica gel, and then purified by flash column chromatography, eluting with 0–100% ethyl acetate / hexanes. The product-containing fractions were concentrated and further dried under high vacuum at ambient temperature to give Int3 as a clear thick syrup. Yield: 104 mg (67%). LCMS m / z 1109.1 [M+H].

[0766] Synthesis of (4) Perfluorophenyl 1-azido-15-oxo-17,17-bis((3-oxo-3-(perfluorophenoxy)propoxy)methyl)-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (Int3, 1.00 equiv., 18.9 mg, 0.017 mmol) and N-[(2R,3R,4R,5R,6R)-2-[5- To a mixture of [2-(2-aminoethoxy)ethoxy]pentyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB47, 3.50 equiv., 22.6 mg, 0.0597 mmol) in DMSO (0.7 mL) was added DIPEA (10.0 equiv., 0.030 mL, 0.170 mmol). The mixture was stirred at room temperature for 2 h and purified by preparative HPLC (2-45% MeCN / water containing 0.1% TFA) to give 4 as a white solid. Yield: 21 mg (73%). LCMS: 1691.4 [M+H]+.

[0767] Synthesis of (5) To a mixture of 4 (1.00 equiv., 21.0 mg, 0.0124 mmol) in MeOH (3 mL) was added 10% Pd / C (8 mg). The mixture was stirred under hydrogen at room temperature for 1 h, filtered, and concentrated to give 5 as a white solid. Yield: 19.6 mg (95%). LCMS: 1665.8 [M+H]+.

[0768] Synthesis of compound 1254 To a mixture of (2,3,4,5,6-pentafluorophenyl) 4-(2,5-dioxopyrrol-1-yl)benzoate (CAS number: 138194-58-8, 1.10 equiv., 2.5 mg, 0.0066 mmol) in DMA (0.1 mL) was added dropwise 5 (1.00 equiv., 10.0 mg, 0.00600 mmol) in DMA (0.1 mL) at 0 °C, followed by the addition of DIPEA (5.00 equiv., 0.0052 mL, 0.0300 mmol). The mixture was stirred at room temperature for 20 min. The mixture was purified by preparative HPLC (2-50% MeCN / water with 0.1% TFA) to give compound 1254 as a white solid. Yield: 6.5 mg (58%). LCMS:1865.6[M+H]+,1863.6[MH]-.

[0769] Synthesis of compound 2346 [ka] [ka] [ka] [ka] Synthesis of benzyl (8-((3-(1H-imidazole-1-carboxamido)propoxy)methyl)-1,15-di(1H-imidazol-1-yl)-1,15-dioxo-6,10-dioxa-2,14-diazapentadecan-8-yl)carbamate (1) To a solution of carbonyldiimidazole (7.00 equiv., 142 mg, 0.875 mmol) in DMSO (0.4 mL) was added a mixture of benzyl (1,3-bis(3-aminopropoxy)-2-((3-aminopropoxy)methyl)propan-2-yl)carbamate; 2,2,2-trifluoroacetic acid (which can be prepared by the literature route described in Sun, Chengzao; et al. Bioorganic & Medicinal Chemistry Letters (2002), 12(16), 2213-2215) (1.00 equiv., 96.1 mg, 0.125 mmol) in DMSO (1.3 mL). The mixture was stirred at room temperature for 2 h, purified by preparative HPLC (5-90% MeCN / water) and repurified by column chromatography (0-10% MeOH / DCM) to give 1 as a clear syrup. Yield: 55 mg (62%). LCMS m / z 573.1[M-Cbz]

[0770] Synthesis of benzyl (1,39-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-20-(19-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-7-oxo-2,11,14-trioxa-6,8-diazanonadecyl)-13,27-dioxo-6,9,18,22,31,34-hexaoxa-12,14,26,28-tetraazanonatriacontan-20-yl)carbamate (2): To a mixture of benzyl (8-((3-(1H-imidazole-1-carboxamido)propoxy)methyl)-1,15-di(1H-imidazol-1-yl)-1,15-dioxo-6,10-dioxa-2,14-diazapentadecan-8-yl)carbamate (1, 1.00 equiv., 15.1 mg, 0.0213 mmol) in DMSO (0.25 mL) was added a solution of N-((2R,3R,4R,5R,6R)-2-(5-(2-(2-aminoethoxy)ethoxy)pentyl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (ITX0025438, 3.00 equiv., 24.2 mg, 0.0639 mmol). The mixture was stirred at 50° C. overnight and purified by preparative HPLC (5-12-30% MeCN / water with 0.1% TFA) to give 2 as a white solid. Yield: 32 mg (92%). LCMS 1641 [M+H].

[0771] N-[(2R,3R,4R,5R,6R)-2-[5-[2-[2-[3-[3-[3-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pentoxy]ethoxy]ethylcarbamoylamino]propoxy]-2-[3-[2-[2-[5-[(2R,3R,4R,5R,6R Synthesis of )-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pentoxy]ethoxy]ethylcarbamoylamino]propoxymethyl]-2-amino-propoxy]propylcarbamoylamino]ethoxy]ethoxy]pentyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (3): Benzyl(1,39-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-20-(19-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-7-oxo-2 To a mixture of (1,11,14-trioxa-6,8-diazanonadecyl)-13,27-dioxo-6,9,18,22,31,34-hexaoxa-12,14,26,28-tetraazanonateriacontan-20-yl)carbamate (2, 1.00 equiv., 28.4 mg, 0.0173 mmol) in MeOH (4 mL) was added 10% Pd / C (10 mg). The mixture was stirred under hydrogen at room temperature for 1 h, filtered, and concentrated to give 3 as a white solid. Yield: 25.2 mg (97%). LCMS 1505.4 [M+H].

[0772] Synthesis of perfluorophenyl 37-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-...

Claims

1. An extracellular target binding conjugate of formula (I) or a pharmaceutically acceptable salt thereof: 【Chemistry 574】 During the ceremony, X is a moiety that binds to a lysosomal targeting molecule; n is 1 to 50; L is a linker; m is bonded to Y—B (X ｎ -L) average number of moieties, where m is in the range of 1 to 20; Y is any carrier polypeptide linked to B; An extracellular target binding conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein B is a polypeptide that specifically binds to an extracellular target molecule.

2. The conjugate of claim 1 , wherein B specifically binds to a target antibody.

3. The conjugate of claim 2 , wherein the targeting antibody is an autoantibody.

4. The conjugate of claim 2 , wherein the targeting antibody is a neutralizing antibody or an anti-drug antibody.

5. 3. The conjugate of claim 2, wherein B comprises an antigen of the target antibody, or a fragment thereof.

6. The conjugate of claim 2 , wherein B comprises a protein domain.

7. 2. The conjugate of claim 1, wherein Y comprises a protein domain.

8. 2. The conjugate of claim 1, wherein Y is fused directly to B.

9. 2. The conjugate of claim 1, wherein Y is indirectly fused to B via a spacer domain.

10. The conjugate of claim 1, wherein Y-B is a chimeric protein.

11. 2. The conjugate of claim 1, wherein Y is selected from human serum albumin (HSA), an HSA domain, an albumin binding domain, Fc (monomer), Fc (dimer), and fragments thereof (e.g., synthetic peptides).

12. 11. The conjugate of claim 10, wherein YB is selected from an Fc-VHH antigen fusion, an Fc-antigen fusion, and an HSA-antigen fusion.

13. 13. The conjugate of claim 12, wherein YB is an HSA-antigen fusion.

14. 2. The conjugate of claim 1, wherein Y is covalently attached to B via a linker.

15. The conjugate of claim 14 , wherein the linker comprises a non-peptidic linking moiety.

16. 16. The conjugate of any one of claims 14 to 15, wherein Y is selected from human serum albumin (HSA), an HSA domain, an albumin binding domain, Fc (monomer), Fc (dimer), and fragments thereof (e.g., synthetic peptides).

17. the conjugate is of formula (I'), or a prodrug thereof, or a pharmaceutically acceptable salt thereof; 【Chemical 575】 During the ceremony, n is 1 to 500; m is 1 to 20; X is a ligand moiety; 10. A conjugate according to any one of the preceding claims, wherein Y is a target binding moiety.

18. 10. The conjugate of any one of the preceding claims, wherein the lysosomal targeting molecule is a cell surface receptor that allows internalization of the conjugate.

19. 10. The conjugate of any one of the preceding claims, wherein the lysosomal targeting molecule is selected from the asialoglycoprotein receptor (ASGPR), the cation-independent mannose-6-phosphate receptor (CI-M6PR, also referred to herein as M6PR), the folate receptor, LDLR, CD63, sortilin, IFITM3, molecules in the endosomal / lysosomal pathway, LIMP-1, and LIMP-2.

20. 10. A conjugate according to any one of the preceding claims, wherein X is a moiety that binds to ASGPR or M6PR.

21. 10. A conjugate according to any one of the preceding claims, wherein X is a moiety that binds to an ASGPR.

22. the conjugate is of formula (I'), or a prodrug thereof, or a pharmaceutically acceptable salt thereof; 【Chemical 576】 During the ceremony, n is 1 to 500; m is 1 to 20; L is a linker; X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II), 【Chemical 577】 During the ceremony, R １ But, -Z １ -*, -H, -OH, optionally substituted (C １ ~C ６ ) alkyl, —OCH ３ , -OCH ２ CH═CH, optionally substituted —S—(C １ ~C ６ ) selected from alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted —S-aryl, and optionally substituted —S-heteroaryl; R ２ But, -Z １ -*, -NHCOCH ３ , -NHCOCF ３ , -NHCOCH ２ CF ３ , —OH, —NHR, and optionally substituted triazole; R ６ But, -Z １ -*, -OH, -OR, optionally substituted (C １ ~C ６ ) alkyl, —OC(O)R, —C(O)NHR, —NR ｘｘ R ｙｙ , optionally substituted aryl, optionally substituted heteroaryl, —NHCOR, and —NRCOR; Each R is independently an optionally substituted (C １ ~C ６ ) alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R ｘｘ and R ｙｙ are independently H, optionally substituted (C １ ~C ６ ) alkyl, or R ｘｘ and R ｙｙ can cyclize to form an optionally substituted heterocyclyl; R １ , R ２ , and R ６ One of them is -Z １ -*, and "*" is Z １ represents the point of attachment to the linker (L), R ３ and R ４ are each independently H or a promoiety, or R ３ and R ４ are linked in a ring to form a promoiety, R １１ is H or a bridging moiety connecting the 5-carbon to the 1-carbon of the ring; Z １ But, -Z １１ -, -Z １１ -A １ -, -A ２ -, -NR ２１ CO-, -CONR ２１ -, -NR ２１ SO ２ -, -SO ２ NR ２１ -, -NR ２１ C(=O)NR ２１ - and -NR ２１ C(=S)NR ２１ - is a linking moiety selected from -Z １１ - is -O-, -S-, -N(R ２１ ) - or -C(R ２２ ) ２ and -A １ - and - A ２ - is optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; Each R ２１ is H, optionally substituted (C １ ~C ６ ) independently selected from alkyl, —COR, and optionally substituted heteroaryl; Each R ２２ is selected from H, halogen, and optionally substituted (C １ ~C ６ 22. The conjugate of claim 21 , wherein each of the groups is independently selected from: alkyl;

23. -L-Y is, 【Chemical 578】 【Chemistry 579】 【Chemical 580】 【Chemistry 581】 【Chemical Formula 582】 【Chemical 583】 【Chemical Formula 584】 【Chemical 585】 【Chemical 586】 【Chemical 587】 【Chemical 588】 【Chemical 589】 【Chemical 590】 【Chemistry 591】 【Chemical 592】 【Chem.593】 【Chem.594】 【Chemical 595】 【Chemical 596】 【Chem.597】 【Chemical 598】 【Chemical 599】 Including, In the formula, R Ｙ but 【Chemical 600】 【Chemical 601】 or 【Chemical 602】 35. The conjugate of claim 34, wherein:

24. The conjugate of claim 22 or 23, wherein X is represented by formula (a-II). 【Chemical 603】

25. R １ But, -Z １ -*, -H or (C １ ~C ６ 25. The conjugate of any one of claims 22 to 24, wherein:

26. R ２ But, -Z １ -* or -NHCOCH ３ The conjugate according to any one of claims 22 to 24, wherein

27. R ３ and R ４ The conjugate of any one of claims 22 to 26, wherein each is -H.

28. 21. The conjugate of claim 20, wherein X is a moiety that binds to M6PR.

29. X is of formula (IV), 【Chemical 604】 During the ceremony, W is a non-hydrolyzable hydrophilic head group; Z １ is optionally substituted (C １ ~C ３ ) alkylene and optionally substituted ethenylene; Z ２ But S, NR ２１ and C(R ２２ ) ２ where each R ２１ is H, and optionally substituted (C １ ~C ６ ) alkyl, and each R ２２ is H, halogen and optionally substituted (C １ ~C ６ ) alkyl; each A is independently an optionally substituted aryl or heteroaryl linking moiety; Each Z ３ 29. The conjugate of claim 28, wherein:

30. Z ２ The conjugate of claim 29, wherein is S.

31. 31. The conjugate of claim 29 or 30, wherein W is a phosphonate, thiophosphonate, carboxylic acid, or malonic acid, or a salt thereof.

32. X is: 【Chemical 605】 【Chemical 606】 【Chemical 607】 In the formula, R ａ , R ｂ , R ｃ , and R ｄ The conjugate of any one of claims 28 to 31, wherein

33. X is: 【Chemical 608】 【Chemical 609】 【Chemical 610】 In the formula, R ａ , R ｂ , R ｃ , and R ｄ The conjugate of any one of claims 28 to 31, wherein

34. 34. The conjugate of any one of claims 29 to 33, wherein A is optionally substituted aryl or optionally substituted heteroaryl, preferably A is independently selected from optionally substituted phenyl, optionally substituted pyridyl, optionally substituted biphenyl, optionally substituted naphthalene, optionally substituted triazole, and optionally substituted phenylene-triazole.

35. 10. The conjugate of any one of the preceding claims, wherein L comprises 10 to 60 consecutive branched or straight chain atoms.

36. L is of formula (IIb'), 【Chemical 611】 During the ceremony, n is 1, 2, or 3; L １ ~L ６ are each independently a linking moiety, and together they form Z １ and Y, forming a linear or branched linker between them; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** is Z １ X's L via １ represents the point of attachment to 10. A conjugate according to any one of the preceding claims, wherein *** represents the point of attachment to Y.

37. L １ ~L ５ are each independently -C １～２０ -Alkylene-, -NHC(O)-C １～６ -Alkylene-, -C(O)NH-C １～６ -Alkylene-, -NH-C １～６ -Alkylene-, -NHC(O)NH-C １～６ -Alkylene-, -NHC(S)NH-C １～６ -Alkylene-, -C １～６ -Alkylene-NHC(O)-, -C １～６ -Alkylene-C(O)NH-, -C １～６ -Alkylene-NH-, -C １～６ -Alkylene-NHC(O)NH-, -C １～６ -Alkylene-NHC(S)NH-, -O(CH ２ ) ｐ -, -(OCH ２ CH ２ ) ｐ -, -NHC(O)-, -C(O)NH-, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -S(O) ２ L includes one or more linking moieties independently selected from -, -O-, -S-, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic carbocycle, an amino acid residue, -NH-, and -NMe-; １ ~L ５ are each independently optionally substituted with 1 to 5 halo; each p is independently 1 to 50; L ６ But, -C １～２０ -Alkylene-, -NR １６ C(O)-C １～６ -Alkylene-, -C(O)NR １６ -C １～６ -Alkylene-, -NR １６ -C １～６ -Alkylene-, -NR １６ C(O)NR １６ -C １～６ -Alkylene-, -NR １６ C(S)NR １６ -C １～６ -Alkylene-, -C １～６ -Alkylene-NR １６ C(O)-, -C １～６ -Alkylene-C(O)NR １６ -, -C １～６ -Alkylene-NR １６ -, -C １～６ -Alkylene-NR １６ C(O)NR １６ -, -C １～６ -Alkylene-NR １６ C(S)NR １６ -, -O(CH ２ ) ｐ -, -(OCH ２ CH ２ ) ｐ -, -NR １６ C(O)-, -C(O)NR １６ -, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -S(O) ２ -, -O-, -S-, a monocyclic heteroaryl, a monocyclic aryl, a monocyclic heterocycle, an amino acid residue, or -NR １６ is a linking group comprising one or more linking moieties independently selected from: Each R １６ are independently —H, optionally substituted (C １ ~C ６ 37. The conjugate of claim 36, wherein: R is an alkyl, an optionally substituted aryl, an optionally substituted monocyclic heteroaryl, or a monocyclic heteroaryl.

38. L １ ~L ５ are respectively, -C １～２０ -Alkylene-, -NHC(O)-C １～６ -Alkylene-, -C(O)NH-C １～６ -Alkylene-, -NH-C １～６ -Alkylene-, -NHC(O)NH-C １～６ -Alkylene-, -NHC(S)NH-C １～６ -Alkylene-, -C １～６ -Alkylene-NHC(O)-, -C １～６ -Alkylene-C(O)NH-, -C １～６ -Alkylene-NH-, -C １～６ -Alkylene-NHC(O)NH-, -C １～６ -Alkylene-NHC(S)NH-, -O(CH ２ ) ｐ -, -(OCH ２ CH ２ ) ｐ -, -NHC(O)-, -C(O)NH-, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -S(O) ２ is independently selected from -, -O-, -S-, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic carbocycle, an amino acid residue, -NH-, and -NMe-; １ ~L ５ are each independently optionally substituted with 1 to 5 halo; each p is independently 1 to 50; L ６ but, 【Chemical 612】 【Chemical 613】 【Chemical 614】 【Chemical Formula 615】 【Chemical Formula 616】 【Chemical 617】 【Chemical 618】 【Chemical Formula 619】 【Chemical 620】 【Chemical 621】 【Chemical Formula 622】 【Chemical Formula 623】 【Chemical 624】 【Chemical 625】 【Chemical Formula 626】 【Chemical Formula 627】 【Chemical 628】 or 【Chemical Formula 629】 and R ｚ but 【Chemical 630】 【Chemistry 631】 or 【Chemical 632】 37. The conjugate of claim 36, wherein:

39. 1. A method for reducing the level of an extracellular target molecule in a biological system, comprising:

39. A method comprising contacting the biological system with an effective amount of the conjugate of any one of claims 1 to 38, wherein the compound specifically binds to the extracellular target molecule and specifically binds to a lysosomal targeting molecule of a cell within the biological system to promote cellular uptake and degradation of the extracellular target molecule.

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