Degradation of extracellular targets

Abstract

The present disclosure features bifunctional compounds for the degradation of extracellular targets, as well as compositions and related methods thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT application claiming priority to and the benefit of U.S. Provisional Patent Application No. 63 / 139,177, filed January 19, 2021, and U.S. Provisional Patent Application No. 63 / 231,829, filed August 11, 2021, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on January 13, 2022, is named PAT058899-WO-PCT_SL.txt and is 36,088 bytes in size. [Background technology]

[0003] Conventional protein-directed therapeutics can be used to treat diseases by modulating protein function or recruiting immune effectors to target sites. However, many potential therapeutic targets have molecular functions that are not fully understood or are not easily inhibited, and therefore are undruggable by conventional therapeutic approaches. For example, targeted protein degradation (TPD) is an approach to treat diseases involving proteins that cause such undruggable diseases by selectively degrading target proteins, rather than inhibiting their function as by conventional chemical inhibition. Examples of targeted protein degradation systems include: Proteolysis-inducing chimeras (PROTACs) (KM Sakamoto et al., Proc. Natl. Acad. Sci. (2001) 98: 8554-8559); dTAG (B. Nabet et al., Nat. Chem. Biol. (2018) 14: 431); Chaperone-mediated autophagy targeting (X. Fan et al., Nat. Neurosci., (2014) 17: 471-480); and SNIPER (M. Naito et al., Drug Discov. Today Technol., (2019)). However, improved strategies to induce / regulate the degradation of extracellular targets are needed. Summary of the Invention

[0004] The present disclosure features bifunctional compounds, and compositions and related methods thereof, which are particularly useful for the degradation of extracellular targets. The bifunctional compounds can be used to regulate the levels of extracellular targets (e.g., extracellular target molecules) in a subject; for example, the bifunctional compounds can mediate the reduction of the levels of disease-associated extracellular targets in a subject by binding to the target and tagging it for degradation in a degradative pathway (e.g., receptor-mediated endocytosis or lysosomal degradation). Such bifunctional compounds can offer an attractive therapeutic alternative to traditional chemical inhibition or biological neutralization methods by completely removing the extracellular target; such removal is desirable for targets whose presence causes disease (e.g., low density lipoprotein), proteins with multiple functions or active sites (e.g., antibodies), or proteins with large active sites that are difficult to inhibit with small molecules (e.g., PCSK9). Exemplary extracellular targets described herein include proteins (soluble proteins and membrane-associated proteins, e.g., antibodies, receptors, growth factors, cytokines, chemokines, enzymes, hormones, neurotransmitters, or combinations thereof), lipoproteins (e.g., low density lipoproteins, very low density lipoproteins, chylomicrons), nucleic acids (e.g., oligonucleotides, DNA, RNA), toxins, liposomes, viral particles, and cells (e.g., prokaryotic cells, eukaryotic cells).

[0005] The bifunctional compounds of the present disclosure include a first portion capable of binding to an extracellular target and a second portion capable of binding to a membrane-bound receptor associated with a degradative pathway. In one aspect, the bifunctional compounds described herein have the formula (I):A G -LR G (I) or a pharma- ceutically acceptable salt thereof, G is a protein moiety that binds to an extracellular target (e.g., an antibody or fragment thereof, a receptor or fragment thereof, an antigenic protein or fragment thereof); L is absent or is a linker; and R G is the moiety that binds to a membrane-bound receptor associated with a degradative pathway.

[0006] In some embodiments, A G A binds to a soluble target (e.g., a plasma protein) or a membrane-associated target (e.g., a membrane-associated protein). In some embodiments, A G is an antibody or fragment thereof, a receptor or fragment thereof, or an antigenic protein or fragment thereof. In some embodiments, A G is an antibody or fragment thereof (e.g., a Fab, Fab', F(ab')2, F(ab)2, a variable fragment (Fv), a domain antibody (dAb), a single domain antibody, or a single chain variable fragment (scFv)). In some embodiments, the antibody or fragment thereof comprises an antigen binding domain that binds to an extracellular target. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, F(ab)2, a variable fragment (Fv), a domain antibody (dAb), a single domain antibody, or a single chain variable fragment (scFv). In some embodiments, the antibody is a monospecific antibody or fragment thereof. In some embodiments, the antibody is a multispecific antibody or fragment thereof. In some embodiments, the antibody is a bispecific antibody or fragment thereof.

[0007] In one aspect, the bispecific antibody or fragment thereof binds to the asialoglycoprotein receptor (ASGPR) and proprotein convertase subtilisin / kexin type 9 (PCSK9). In some embodiments, the bispecific antibody is a full-length antibody. In some embodiments, the bispecific antibody or fragment thereof comprises a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), single domain antibody, or single chain variable fragment (scFv). In some embodiments, the bispecific antibody or fragment thereof comprises a linker. In some embodiments, the linker is (G4S) n linker, where n is an integer from 1 to 20. In some embodiments, n is an integer from 1 to 4 (SEQ ID NO: 42). In some embodiments, n is 4 (SEQ ID NO: 46).

[0008] In some embodiments, the bispecific antibody or fragment thereof comprises one or more of the following: (i) a heavy chain complementarity determining region 1 (HCDR1) amino acid sequence set forth in SEQ ID NO: 9, a heavy chain complementarity determining region 2 (HCDR2) amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain complementarity determining region 3 (HCDR3) amino acid sequence set forth in SEQ ID NO: 11; (ii) the HCDR1 amino acid sequence set forth in SEQ ID NO: 21, the HCDR2 amino acid sequence set forth in SEQ ID NO: 22, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 11; (iii) the HCDR1 amino acid sequence set forth in SEQ ID NO: 31, the HCDR2 amino acid sequence set forth in SEQ ID NO: 32, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 33; (iv) the HCDR1 amino acid sequence set forth in SEQ ID NO: 15, the HCDR2 amino acid sequence set forth in SEQ ID NO: 16, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 17; (v) the HCDR1 amino acid sequence set forth in SEQ ID NO:26, the HCDR2 amino acid sequence set forth in SEQ ID NO:27, and the HCDR3 amino acid sequence set forth in SEQ ID NO:17; (vi) the HCDR1 amino acid sequence set forth in SEQ ID NO: 36, the HCDR2 amino acid sequence set forth in SEQ ID NO: 37, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 38; (vii) a light chain complementarity determining region 1 (LCDR1) amino acid sequence set forth in SEQ ID NO: 12, a light chain complementarity determining region 2 (LCDR2) amino acid sequence set forth in SEQ ID NO: 13, and a light chain complementarity determining region 3 (LCDR3) amino acid sequence set forth in SEQ ID NO: 14; (viii) an LCDR1 amino acid sequence set forth in SEQ ID NO: 23, an LCDR2 amino acid sequence set forth in SEQ ID NO: 24, and an LCDR3 amino acid sequence set forth in SEQ ID NO: 25; (ix) an LCDR1 amino acid sequence set forth in SEQ ID NO: 34, an LCDR2 amino acid sequence set forth in SEQ ID NO: 35, and an LCDR3 amino acid sequence set forth in SEQ ID NO: 14; (x) an LCDR1 amino acid sequence set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence set forth in SEQ ID NO: 19, and an LCDR3 amino acid sequence set forth in SEQ ID NO: 20; (xi) an LCDR1 amino acid sequence set forth in SEQ ID NO: 28, an LCDR2 amino acid sequence set forth in SEQ ID NO: 29, and an LCDR3 amino acid sequence set forth in SEQ ID NO: 30; and / or (xii) an LCDR1 amino acid sequence set forth in SEQ ID NO:39, an LCDR2 amino acid sequence set forth in SEQ ID NO:29, and an LCDR3 amino acid sequence set forth in SEQ ID NO:20.

[0009] In some embodiments, the bispecific antibody or fragment thereof comprises one or more of the following: (i) a heavy chain variable region (VH) amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1; (ii) a VH amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:3; (iii) a light chain variable region (VL) amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2; and / or (iv) a VL amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4.

[0010] In some embodiments, the bispecific antibody or fragment thereof comprises one or more of the following: (i) the VH amino acid sequence set forth in SEQ ID NO:1: (ii) the VH amino acid sequence set forth in SEQ ID NO:3; (iii) the VL amino acid sequence set forth in SEQ ID NO:2; and / or (iv) The VL amino acid sequence set forth in SEQ ID NO:4.

[0011] In some embodiments, a bispecific antibody or fragment thereof comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:5, 6, 7, and / or 8. In some embodiments, a bispecific antibody or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:5, 6, 7, and / or 8.

[0012] In some embodiments, A G is a receptor (e.g., a full-length receptor, a receptor fragment, or a functional variant thereof). In some embodiments, the receptor is a full-length receptor or a functional variant thereof.

[0013] In some embodiments, A G is an antigenic protein or a fragment thereof. In some embodiments, A Gis an antigenic protein or fragment thereof that recognizes pathogenic autoantibodies. In some embodiments, the antigenic protein or fragment thereof is a disintegrin and metalloproteinase with thrombospondin type 1 motile, member 13 (ADAMTS13), steroidogenic cytochrome P450 enzyme 21-hydroxylase, N-methyl-d-aspartate (NMDA) receptor, red blood cells, anti-smooth muscle antibody (ASMA), actin, platelets, signal recognition particle (SRP), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), myosin, sperm, amylase. Alpha 2, type XVII collagen (col17), kallikrein 13, type VII collagen (col7), myeloperoxidase (MPO), type IV collagen, proteinase 3 (PR3), thyrotropin receptor (TSHR), thyroglobulin, thyroid peroxidase (TPO), thyroglobulin, thyroid peroxidase (TPO), platelets, myeloperoxidase (MPO), muscle nicotinic acetylcholine receptor, muscle specific kinase (MuSK), low density lipoprotein receptor protein 4 (LRP4), myo syn, beta 1 adrenergic receptor, adenine nucleotide translocase, aquaporin-4, myelin oligodendrocyte glycoprotein (MOG), heat shock protein 90 (HSP90), heat shock protein A5 (HSPA5), desmoglein-3, parietal cells, mitochondria, phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain containing 7A (THSD7A), cyclic citrullinated protein, RNA binding protein (Ros), La, double-stranded DNA (dsDNA), angiotensin II type 1 receptor (AT1R), endothelin-1 type A receptor (ETAR), insulin, glutamic acid decarboxylase, or protein tyrosine phosphatase.

[0014] In some embodiments, R GR comprises a small molecule, a carbohydrate or carbohydrate derivative (e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, an oligosaccharide, a modified sugar (e.g., a sugar phosphate, e.g., a monosaccharide phosphate (e.g., mannose-6-phosphate), a hexasaccharide diphosphate, a triantennary GalNAc), an antibody molecule or fragment thereof, a peptide, or a pharma- ceutically acceptable salt thereof. In some embodiments, R G binds to a receptor that is associated with a degradation pathway (e.g., receptor-mediated endocytosis or lysosomal degradation). In some embodiments, the degradation pathway comprises receptor-mediated endocytosis or endosomal degradation. In some embodiments, the degradation pathway comprises lysosomal degradation.

[0015] In some embodiments, this degradation pathway is mediated by the mannose-6-phosphate receptor (M6PR), the insulin-like growth factor 2 receptor (IGF2R), or the asialoglycoprotein receptor (ASGPR). G is a binding moiety to the mannose-6-phosphate receptor (M6PR), the insulin-like growth factor 2 receptor (IGF2R), or the asialoglycoprotein receptor (ASGPR). G When R is a binding moiety to the mannose-6-phosphate receptor (M6PR) or the insulin-like growth factor 2 receptor (IGF2R), the binding moiety to the M6PR or IGF2R is a small molecule, a carbohydrate or carbohydrate derivative, or an antibody or fragment thereof. G When R is a binding moiety to the asialoglycoprotein receptor (ASGPR), the binding moiety to the ASGPR is a small molecule, a carbohydrate or carbohydrate derivative, or an antibody or fragment thereof. In some embodiments, the ASGPR binding moiety is ASGPR1 or ASGPR2. In some embodiments, R G comprises an ASGPR ligand. In some embodiments, R G is a binding moiety for the mannose-6-phosphate receptor (M6PR) or the insulin-like growth factor 2 receptor (IGF2R), R Gcomprises a hexasaccharide moiety (e.g., a glucose, galactose, mannose, N-acetylglucose, N-acetylgalactose, N-acetylmannose, mannose-6-phosphate, or mannose-6-phosphate moiety). G comprises multiple hexasaccharide moieties (e.g., multiple glucose, galactose, mannose, N-acetylglucose, N-acetylgalactose, N-acetylmannose, mannose-6-phosphate, or mannose-6-phosphate moieties). G contains one or more N-acetylgalactose (GalNAc) moieties, e.g., contains at least 1, 2, 3, 4, 5, or 6 GalNAc moieties. In some embodiments, R G contains three GalNAc moieties.

[0016] In some embodiments, R G is represented by formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 is -CN, -CH2-CH, -C≡CH, -CH2-N3, -CH2-NH2, -CH2-N(R 4 )-S(O)2-R 5 , -CH2-CO2H, -CO2H, -CH2-OH,-CH2-SH, -CH=CH-R 5 , -CH2-R 5 , -CH2-SR 5 , -CH2-N(R 4 )-R 5 , -CH2-N(R 4 )-C(O)-R 5 , -CH2-N(R 4 )-C(O)-OR 5 , -CH2-N(R 4 )-C(O)-N(R 4 )-R 5 , -CH2-OR 5 , -CH2-OC(O)-R 5, -CH2-OC(O)-N(R 4 )-R 5 , -CH2-OC(O)-OR 5 , CH2-S(O)-R 5 , -CH2-S(O)2-R 5 , -CH2-S(O)2-N(R 4 )-R 5 , -C(O)-NH2, -C(O)-OR 5 , -C(O)-N(R 4 )-R 5 or aryl or heteroaryl, the aryl or heteroaryl being R 5 or optionally replaced by or R 1 is -ZXY, where X is a linker or a drug delivery system, Y is absent or a ligand selected from the group consisting of a small molecule, an amino acid sequence, a nucleic acid sequence, an antibody, an oligomer, a polymer, a gene-derived material, a liposome, a nanoparticle, a dye, and a fluorescent probe, or a combination thereof, and Z is absent or is -C≡C-, -CH═CH-, -CH-, -CH-O-, -C(O)-N(R 4 )-, -CH2-S-, -CH2-S(O)-, -CH2-S(O)2-, -CH2-S(O)2-N(R 4 )-, -C(O)-O-, -CH2-N(R 4 )-, -CH2-N(R 4 )-C(O)-, -CH2-N(R 4 )-S(O)2-, -CH2-N(R 4 )-C(O)-O-, -CH2-N(R 4 )-C(O)-N(R 4 )-, -CH2-OC(O)-, -CH2-OC(O)-N(R 4 )-, -CH2-OC(O)-O-, or aryl or heteroaryl, wherein the aryl or heteroaryl is R 5 is optionally substituted with; R 2 -OH, -N3, -N(R 3 )2, -N(R 3 )-C(O)-R3 , -N(R 3 )-C(O)-N(R 3 )2, -N(R 3 )-C(O)-OR 3 , tetrazole, or triazole, and the tetrazole and triazole are R 3 Optionally substituted with R 1 When is -CH2-OH, R 2 -N3, -N(R 3 )2, -N(R 3 )-C(O)-R 3 , -N(R 3 )-C(O)-N(R 3 )2, -N(R 3 )-C(O)-OR 3 , tetrazole, or triazole, and the tetrazole and triazole are R 3 is optionally substituted with; Each R 3 are independently -H, -(C1-C5)alkyl, halo-substituted (C1-C5)alkyl, or (C3-C6)cycloalkyl, and the -CH2- group of the alkyl or cycloalkyl is selected from -O-, -S-, and -N(R 4 )-, and the -CH3 of the alkyl may be replaced by a heteroatom group selected from -N(R 4 )2, -OR 4 , and -S(R 4 ), which are separated by at least two carbon atoms; Each R 4 are independently -H, -(C1 to C 20 )alkyl, or (C3-C6)cycloalkyl, in which 1 to 6 -CH2- groups of said alkyl or cycloalkyl separated by at least 2 carbon atoms are -O-, -S-, or -N(R 4 )-, and the -CH3 of this alkyl can be replaced by -N(R 4 )2, -OR 4 , and -S(R 4wherein the heteroatom groups are separated by at least two carbon atoms; the alkyl and cycloalkyl may be substituted with 1 to 6 halo atoms; and Each R 5 are independently -H, (C3 to C 20 ) cycloalkyl, or (C1-C 20 ) alkyl, and 1 to 6 -CH2- groups of this alkyl or cycloalkyl separated by at least 2 carbon atoms are -O-, -S-, or -N(R 4 )-, and the -CH3 of this alkyl can be replaced by -N(R 4 )2, -OR 4 , and -S(R 4 ), which heteroatom groups are separated by at least two carbon atoms; and the alkyl and cycloalkyl can be substituted with 1 to 6 halo atoms.

[0017] In some embodiments, R G teeth, [ka] (wherein n is 1, 2, or 3); [ka] (n is 2 or 3) and R G is attached to L at the terminal amine. In some embodiments, R G is selected from any of the following that bind to ASGPR: [ka] [ka] , where R G The * indicates the point of attachment to L.

[0018] In some embodiments, R G is selected from any of the following that bind to M6PR or IGF2R: [ka] [ka] , where R G The * indicates the point of attachment to L.

[0019] In some embodiments, R G comprises an M6PR or IGF2R binding moiety and the extracellular target is TNFR1, IL1R, progranulin, tau, MUC5B, TREM2, or EGFR. In some embodiments, L comprises a heteroalkylene moiety. In some embodiments, the heteroalkylene moiety comprises at least 6, 8, 10, 12, 16, 20, 24, 36, 48, 60, 100, 250, 300, or more carbon atoms. In some embodiments, the heteroalkylene moiety is 4-48 carbon atoms, or 4-36 carbon atoms, or 4-24 carbon atoms, or 4-16 carbon atoms, or 6-12 carbon atoms, or 8-12 carbon atoms. In some embodiments, the heteroalkylene moiety is polyethylene glycol. In some embodiments, L is a chemical moiety formed by the reaction between two reactive groups set forth in Table 2.

[0020] In some embodiments, L is of formula (A): [ka] (In the formula, R 1 is hydrogen or another amino acid side chain; Each R 2 are independently hydrogen or C1-C6 alkyl; R 3is a carbohydrate or carbohydrate derivative; R A is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C 12 Cycloalkyl, C3-C 12 Heterocyclyl, C6-C 12 Aryl, C6-C 12 is heteroaryl; and Each [ka] is independently G , R G , or a connection to another part of L) In some embodiments, R 1 is C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C2-C6 alkenylene, C2-C6 alkynylene, C3-C 12 Cycloalkyl, C3-C 12 Heterocyclyl, C6-C 12 Aryl, C6-C 12 Heteroaryl, C1-C6 alkylene-aryl, C1-C6 alkylene-heteroaryl, C1-C6 alkylene-cycloalkyl, C1-C6 alkylene-heterocyclyl, halogen, or -OR A It is.

[0021] In some embodiments, L is of formula (AI): [ka] (In the formula, R 3 is a carbohydrate or carbohydrate derivative). In some embodiments, -CH3 is in the (L) or (D) configuration. In some embodiments, R 3 is a monosaccharide, disaccharide, oligosaccharide, or polysaccharide, each of which is optionally substituted. In some embodiments, L and / or R Gdoes not include a nitrogen-containing heteroaryl or a nitrogen-containing heterocyclyl. In some embodiments, the nitrogen-containing heteroaryl is triazolyl. In some embodiments, L and / or R G is expressed by the formula (B): [ka] (In the formula, R 10 is a carbohydrate or carbohydrate derivative moiety). 10 is a monosaccharide, disaccharide, oligosaccharide, or polysaccharide, each of which is optionally substituted.

[0022] In some embodiments, extracellular targets include proteins (soluble proteins and membrane-associated proteins, e.g., antibodies or fragments thereof, receptors, growth factors, cytokines, chemokines, enzymes, or hormones), lipoproteins, liposomes, nucleic acids (e.g., oligonucleotides, DNA, RNA), toxins, viral particles, or cells (e.g., prokaryotic cells, eukaryotic cells).

[0023] In certain embodiments, the extracellular target is a soluble protein (e.g., a plasma protein) or a membrane-associated protein. In some embodiments, the extracellular target is a soluble protein (e.g., an antibody, a soluble receptor, a secreted protein, a growth factor, a cytokine, a hormone, a neurotransmitter, or an enzyme). In some embodiments, the A G binds to the soluble protein or a component thereof (e.g., a protein modification (e.g., a sugar)). In some embodiments, the soluble protein is proprotein convertase subtilisin / kexin type 9 (PCSK9), complement factor H-related protein 3 (CFHR3), MICA (MHC class I chain-related gene A), or apolipoprotein-B (Apo-B).

[0024] In some embodiments, the extracellular target is a membrane-associated protein (e.g., a type I, type II, or multipass membrane protein, or a glycosylphosphatidylinositol (GPI)-anchored membrane-associated protein, such as a receptor, a protein channel, or an ion channel). G binds to the membrane-associated protein or a component thereof (e.g., a protein modification (e.g., a sugar)). In some embodiments, the membrane-associated protein is covalently or non-covalently associated with a cell membrane. In some embodiments, the membrane-associated protein is a transmembrane protein or a membrane-anchored protein. In some embodiments, the membrane-associated protein is a type I, type II, or multi-pass membrane protein, or a glycosylphosphatidylinositol (GPI)-anchored membrane-associated protein. In some embodiments, the GPI-anchored membrane-associated protein is a receptor, a protein channel, or an ion channel.

[0025] In some embodiments, the extracellular target is a lipoprotein; and G binds to said lipoprotein or a component thereof. In some embodiments, the component is a lipid or an apolipoprotein. In some embodiments, the lipoprotein is a lipoprotein receptor (LPR) or lipoprotein(a) (Lp(a)).

[0026] In some embodiments, the extracellular target is a pathogenic target, e.g., a protein associated with a harmful or unwanted effect in a sample (e.g., a cell) or a subject. In some embodiments, the pathogenic target is present at an expression level or activity level that causes a harmful or unwanted effect in a sample (e.g., a cell) or a subject. In some embodiments, the pathogenic target is an extracellular secreted protein. In some embodiments, the extracellular secreted protein is a soluble protein. In some embodiments, the pathogenic target is a pathogenic autoantibody or a fragment thereof. In some embodiments, the pathogenic target is a cell surface receptor. In some embodiments, the cell surface receptor is selected from TNF receptor 1 (TNFR1), interleukin-1 receptor (IL1R), PD-L1, epidermal growth factor receptor (EGFR), or transferrin. In some embodiments, the pathogenic target is a neurological target. In some embodiments, the neurological target is a tau protein or aggregate, or an immuno-oncological target. In some embodiments, the immuno-oncological target is progranulin. In some embodiments, the pathogenic autoantibody causes a disease, disorder, condition, or clinical condition, the disease, disorder, condition, or clinical condition being selected from the following: acquired thrombotic thrombocytopenic purpura, Addison's disease, anti-NMDA encephalitis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis, autoimmune idiopathic thrombocytopenia, autoimmune myopathy, autoimmune orchitis, autoimmune pancreatitis, bullous pemphigoid, dry eye, epidermolysis bullosa acquisita, eosinophilic polycythemia vera, and / or eosinophilic purpura. Granulomatosis with vasculitis (EGPA), Goodpasture's disease, Granulomatosis with polyangiitis, Graves' disease, Hashimoto's thyroiditis, Idiopathic interstitial pneumonia, Idiopathic thrombocytopenic purpura (ITP), Microscopic polyangiitis (MPA), Myasthenia gravis, Myocarditis, Neuromyelitis optica (NMO), Ovarian failure, Pemphigus, Pernicious anemia, Primary biliary cholangitis (PBC), Primary membranous nephropathy, Rheumatoid arthritis, Sjogren's syndrome, Systemic lupus erythematosus (SLE), Systemic sclerosis, or Type I diabetes mellitus.

[0027] Exemplary extracellular targets include proprotein convertase subtilisin / kexin type 9 (PCSK9), tumor necrosis factor receptor 1 (TNFR1), interleukin 1 receptor (IL1R), low density lipoprotein, very low density lipoprotein, chylomicrons, apolipoprotein B (ApoB), lipoprotein(a) (Lp(a)), apolipoprotein C3 (ApoCIII), angiopoietin-like 3 (ANGPTL3), angiopoietin-like 4 (ANGPTL4), angiopoietin-like 8 (ANGPTL8), factor 11, and growth and differentiation factor. GDF15, lipoprotein lipase (LPL), interleukin 1-beta (IL1β), interleukin 17 (IL17), complement factor B, complement factor D, myeloperoxidase (MPO), immunoglobulin A (IGA), immunoglobulin E (IgE), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), interleukin 7 (IL7), interleukin 12A (IL12A), interleukin 23 (IL23), tumor necrosis factor A (TNFA), microtubule-associated protein tau (MAPT), complement factor FHR-related protein 3 (FHR3), tissue inhibitor of metalloproteinases 1 (TIMP1), apelin, bone morphogenetic protein 6 (BMP6), bone morphogenetic protein 9 / growth differentiation factor 2 (BMP9 / GDF2), colony-stimulating factor 1 receptor (CSF-1), erythropoietin (EPO), interleukin 5 (IL5), milk fat globule-EGF factor 8 protein (MFGE8), thymic stromal lymphopoietin (TSLP), thrombospondin (TSP), complement component 5 (C5), C-X-C motif chemokine ligand 10 (CXCL10), fibroblast growth factor 23 ( FGF23), insulin-like growth factor 1 (IGF1), interleukin 10 (IL10), interleukin 13 (IL13), interleukin 2 (IL2), interleukin 6 (IL6), vascular endothelial growth factor A (VEGF-A), adenosine deaminase 2 (ADA2), soluble urokinase-type plasminogen activator receptor (suPAR), transforming growth factor beta 1 (TGF-β1), progranulin, alpha-synuclein, toxin, venom, HBV soluble antigen, viral antigen, prion protein, scFv, AAV, and anti-AAV antibody.In some embodiments, the extracellular target is subtilisin / kexin type 9 (PCSK9).

[0028] In another aspect, the disclosure features a combination that includes any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) and at least one additional therapeutic agent. In one embodiment, the combination includes a bispecific antibody or fragment thereof (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof) and at least one additional therapeutic agent. In one embodiment, the combination includes an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof and at least one additional therapeutic agent. In some embodiments, the at least one therapeutic agent is one or more of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof).

[0029] In another aspect, the disclosure features a pharmaceutical composition that includes any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable excipient. In one embodiment, the pharmaceutical composition includes a bispecific antibody or fragment thereof (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof) and a pharma- ceutically acceptable excipient. In one embodiment, the pharmaceutical composition includes an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof.

[0030] In another aspect, the disclosure features a method of targeting an extracellular target (e.g., a soluble protein or a membrane-associated protein) for degradation, the method comprising the use of any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) or related compositions (e.g., in a sample (e.g., a tissue sample, a plasma sample, or a cell) or in a subject). In some embodiments, the extracellular target (e.g., a soluble protein or a membrane-associated protein) is targeted to the liver (e.g., in a subject). In another aspect, the disclosure features a method of modulating (e.g., reducing) the level and / or activity of an extracellular target (e.g., PCSK9), the method comprising the use of any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) or related compositions (e.g., in a sample (e.g., a tissue sample, a plasma sample, or a cell) or in a subject). In some embodiments, the method is an in vitro method, an in vivo method, or an ex vivo method.

[0031] In some embodiments, any of the methods described herein comprises contacting a sample (e.g., a tissue sample, a plasma sample, or a cell) with a bifunctional compound described herein (e.g., a bifunctional compound of formula (I) or a pharma- ceutically acceptable salt thereof) or any of its related compositions. In some embodiments, any of the methods described herein comprises contacting a sample (e.g., a tissue sample, a plasma sample, or a cell) with a bispecific antibody or fragment thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof). In some embodiments, any of the methods described herein comprises contacting a sample (e.g., a tissue sample, a plasma sample, or a cell) with an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof. In some embodiments, the sample (e.g., a tissue sample, a plasma sample, or a cell) is derived from a subject in need. In some embodiments, the sample is a tissue sample, a plasma sample, or a cell. In some embodiments, the sample is a plasma sample.

[0032] In some embodiments, any of the methods described herein include administering to a subject (e.g., a subject in need thereof) any of the bifunctional compounds or related compositions described herein. In some embodiments, the subject is a mammal (e.g., a human). In some embodiments, the level and / or activity of an extracellular target (e.g., PCSK9) is modulated (e.g., reduced) (e.g., in a sample or subject) in response to any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) or related compositions thereof. In some embodiments, the level and / or activity of an extracellular target (e.g., PCSK9) is reduced in response to any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) or related compositions thereof. In one embodiment, the level and / or activity of PCSK9 is reduced in response to any of the bispecific compounds or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or a fragment thereof). In some embodiments, the level and / or activity of an extracellular target (e.g., PCSK9) is reduced by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%) in a sample (e.g., a tissue sample, plasma sample, or cell) or subject.

[0033] In some embodiments, any of the methods described herein include determining the level and / or activity of the extracellular target (e.g., PCSK9) in the sample (e.g., tissue sample, plasma sample, or cells) or subject after the contacting and / or administering. In some embodiments, the level and / or activity of the extracellular target (e.g., PCSK9) is determined by measuring the level and / or activity of the extracellular target in plasma. In some embodiments, the method further includes comparing the level and / or activity of the extracellular target (e.g., PCSK9) in the sample (e.g., tissue sample, plasma sample, or cells) or subject to a baseline value or the level and / or activity of the extracellular target (e.g., PCSK9) in the sample prior to the contacting and / or administering. In some embodiments, the method includes comparing the level and / or activity of PCSK9 in the plasma sample to a baseline value or the level and / or activity of PCSK9 in the plasma sample prior to the contacting and / or administering.

[0034] In another aspect, the disclosure features a method of preventing and / or treating a disease, disorder, condition, or clinical situation in a subject, comprising administering to the subject an effective amount of any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) or related compositions thereof. In yet another aspect, the disclosure features a method of preventing and / or treating a disease, disorder, condition, or clinical situation in a subject, comprising administering to the subject an effective amount of any of the bispecific antibodies or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof) or related compositions thereof. In another aspect, the disclosure features the use of a bifunctional compound (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof) or any of its related compositions thereof to prevent and / or treat a disease, disorder, condition, or clinical situation in a subject. In another aspect, the disclosure features the use of a bispecific antibody or fragment thereof (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof) or any of its related compositions to prevent and / or treat a disease, disorder, condition, or clinical situation in a subject.

[0035] In some embodiments, the disease, disorder, condition, or clinical situation is a proliferative disease (e.g., cancer), a neurological disease (e.g., Alzheimer's disease, neurodegeneration), a cardiovascular disease, a respiratory disease (e.g., asthma), a skin disease, a blood disease, an inflammatory disease, an autoimmune disease, a metabolic disorder, an infectious disease, or a renal disease or disorder. Exemplary diseases, disorders, conditions, and clinical situations include cancer, Alzheimer's disease, atherosclerosis, arteriosclerosis, hyperlipidemia, familial hypercholesterolemia (heterozygous or homozygous), familial chylomicronemia syndrome, inflammation, asthma, allergy, urticaria, IgA nephropathy, membranous nephropathy, transplant rejection, IgE-mediated disorder, or adverse reaction to a therapeutic agent. Additional exemplary diseases, disorders, conditions, and clinical situations include: acute inflammatory demyelinating polyneuropathy (Guillain-Barre syndrome), acute liver failure, anti-glomerular basement membrane disease (Goodpasture's syndrome), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), cutaneous T-cell lymphoma (CTCL); mycosis fungoides; Sezary syndrome, familial hypercholesterolemia, focal segmental glomerular sclerosis (FSGS), hereditary hemochromatosis, hyperviscosity in hypergammaglobulinemia, hyperviscosity in hypergammaglobulinemia, myasthenia gravis, N-methyl-D-aspartate erythropoietin syndrome, and erythropoietin syndrome. The present invention relates to a method for treating a disease, disorder, or condition, including but not limited to, a pulmonary edema ...In some embodiments, the PCSK9-mediated disease or disorder is selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease (e.g., aortic disease and cerebrovascular disease), peripheral arterial disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, and xanthomas. In some embodiments, the disease, disorder, condition, or clinical situation is a disease, disorder, condition, or clinical situation that is treated by therapeutic apheresis.

[0036] In some embodiments, the bifunctional compound described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or any of its pharmaceutical compositions, is delivered by oral, parenteral, topical, mucosal, nasal, buccal, or ophthalmic administration. In some embodiments, the bifunctional compound described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or any of its pharmaceutical compositions, is delivered via parenteral administration (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intraperitoneal, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection). In some embodiments, the bifunctional compound described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or any of its pharmaceutical compositions, is delivered by subcutaneous injection. In some embodiments, any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I), or a pharma- ceutically acceptable salt thereof), or pharmaceutical compositions thereof, are formulated for sustained release (e.g., a depot formulation).

[0037] In some embodiments, the disease, disorder, condition, or clinical situation is a proliferative disease (e.g., cancer), a neurological disease (e.g., Alzheimer's disease, neurodegeneration), a cardiovascular disease, a respiratory disease, a skin disease, a blood disease, an inflammatory disease, an autoimmune disease, a metabolic disorder, an infectious disease, or a renal disease or disorder. Exemplary diseases, disorders, conditions, and clinical situations include cancer, Alzheimer's disease, atherosclerosis, arteriosclerosis, hyperlipidemia, familial hypercholesterolemia, familial chylomicronemia syndrome, inflammation, asthma, allergy, urticaria, IgA nephropathy, membranous nephropathy, transplant rejection, IgE-mediated disorders, or adverse reactions to therapeutic agents.

[0038] In some embodiments, the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or any of its pharmaceutical compositions, can be delivered orally, parenterally, topically, mucosally, nasally, bucally, or ophthalmically. In some embodiments, the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or any of its pharmaceutical compositions, can be delivered via parenteral administration (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-articular, intraperitoneal, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection). In some embodiments, the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or any of its pharmaceutical compositions, can be delivered by subcutaneous injection. In some embodiments, any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or pharmaceutical compositions thereof, are formulated for sustained release (e.g., a depot formulation).

[0039] In one aspect, the disclosure features the use of any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), or related compositions, in the manufacture of a medicament for treating a disease, disorder, condition, or clinical situation modulated by an extracellular target described herein. In another aspect, the disclosure features the use of any of the bispecific antibodies or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof), or related compositions, in the manufacture of a medicament for treating a disease, disorder, condition, or clinical situation modulated by an extracellular target described herein. In one embodiment, the anti-ASGPR-PCSK9 bispecific antibody or fragment thereof, or pharmaceutical composition thereof, is used in the manufacture of a medicament for treating a disease, disorder, condition, or clinical situation modulated by PCSK9.

[0040] In one aspect, the disclosure features a nucleic acid molecule encoding any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof). In another aspect, the disclosure features a nucleic acid molecule encoding any of the bispecific antibodies or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof). In one embodiment, the nucleic acid molecule encodes an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof.

[0041] In one aspect, the disclosure features an expression vector comprising any of the nucleic acid molecules encoding any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof). In another aspect, the disclosure features an expression vector comprising any of the nucleic acid molecules encoding any of the bispecific antibodies or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof). In one embodiment, the expression vector comprises a nucleic acid molecule encoding an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof. In yet another aspect, the disclosure features a host cell comprising any of the expression vectors or nucleic acid molecules described herein.

[0042] In one aspect, the disclosure features a method of making any of the bifunctional compounds described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof). In another aspect, the disclosure features a method of making any of the bispecific antibodies or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof). In one embodiment, the method of making includes culturing any of the host cells described herein under conditions suitable for gene expression, and then purifying and recovering any of the bispecific antibodies or fragments thereof described herein (e.g., an anti-ASGPR-PCSK9 bispecific antibody or fragment thereof) from the cell culture.

[0043] In one aspect, the disclosure features a kit that includes any of the bifunctional compounds (e.g., a bifunctional compound of Formula (I), or a pharma- ceutically acceptable salt thereof), combinations, and / or pharmaceutical compositions described herein. In one embodiment, the kit further includes a means for administering (e.g., orally, parenterally, topically, mucosally, nasally, buccally, or ophthalmically) any of the bifunctional compounds (e.g., a bifunctional compound of Formula (I), or a pharma- ceutically acceptable salt thereof), combinations, and / or pharmaceutical compositions described herein to a subject in need thereof. In one embodiment, the kit includes instructions for administering any of the bifunctional compounds, combinations, and / or pharmaceutical compositions described herein to a subject in need thereof.

[0044] In another aspect, the kit comprises any of the bispecific antibodies or fragments thereof (e.g., anti-ASGPR-PCSK9 bispecific antibodies or fragments thereof), nucleic acids, expression vectors, and / or host cells provided herein. In one embodiment, the kit further comprises means for using or administering (e.g., oral, parenteral, topical, mucosal, nasal, buccal, or ophthalmic) any of the bispecific antibodies or fragments thereof (e.g., anti-ASGPR-PCSK9 bispecific antibodies or fragments thereof), nucleic acids, expression vectors, and / or host cells described herein to a subject in need thereof. In one embodiment, the kit comprises instructions for using or administering (e.g., anti-ASGPR-PCSK9 bispecific antibodies or fragments thereof), nucleic acids, expression vectors, and / or host cells described herein to a subject in need thereof.

[0045] The details of one or more embodiments of the disclosure are described herein. Other features, objects, and advantages of the disclosure will become apparent from the detailed description, examples, and claims. [Brief description of the drawings]

[0046] [Figure 1]1 is a schematic diagram showing an exemplary bifunctional molecule (eg, an anti-ASGPR-PCSK9 bispecific antibody). [Figure 2-1] Figure 2: Graphs showing binding specificity of an exemplary bifunctional compound, anti-ASGPR-PCSK9 bispecific antibody, to ASGPR and PCSK9. Figure 2A shows binding of anti-ASGPR-PCSK9 bispecific antibody to immobilized AGSPR1. Figure 2B shows binding of anti-ASGPR-PCSK9 bispecific antibody to full-length PCSK9. Figure 2C shows binding of anti-ASGPR-PCSK9 bispecific antibody bound to PCSK9 to ASGPR1. Figure 2D shows binding of anti-ASGPR-PCSK9 bispecific antibody bound to ASGPR1 to full-length PCSK9. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 2-4] (As stated above.) [Figure 3-1] Figure 3: Graphs from a mouse model showing the modulation of PCSK9 clearance by administration of an exemplary bifunctional compound, an anti-ASGPR-PCSK9 bispecific antibody. Figure 3A shows that the overall amount of PCSK9 levels in serum decreased with increasing amounts of anti-ASGPR-PCSK9 bispecific antibody administered. Figure 3B shows the rate of PCSK9 clearance when anti-ASGPR-PCSK9 bispecific antibody was administered at various doses (0, 0.3, 1, and 3 mg / kg) over a 4-hour period. Statistics by two-way ANOVA with Dunnett's multiple comparison test. [Figure 3-2] (As stated above.) [Figure 4] 1 is a schematic diagram showing an exemplary bifunctional compound (eg, a GalNAc-labeled PCSK9 antibody conjugate). [Figure 5-1]Figure 5: Graphs showing the binding specificity of an exemplary bifunctional compound, GalNAc trimer conjugated to an anti-PCSK9 antibody, to ASGPR and PCSK9. Figure 5A shows direct binding of GalNAc trimer anti-PCSK9 antibody conjugate to ASGPR1; Figure 5B shows direct binding of GalNAc trimer anti-PCSK9 antibody conjugate to PCSK9; Figure 5C shows the formation of a ternary complex in which the GalNAc trimer anti-PCSK9 antibody conjugate bound to PCSK9 binds to ASGPR1. [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 6-1] Figure 6: Graphs showing PCSK9 clearance in a mouse model modulated by administration of an exemplary bifunctional compound, a GalNAc trimer anti-PCSK9 antibody conjugate. Figure 6A shows the rate of PCSK9 clearance when GalNAc trimer anti-PCSK9 antibody conjugate is administered at various doses (0, 0.3, 1, and 3 mg / kg) over 6 hours. Statistics by two-way ANOVA with Dunnett's multiple comparison test. Figure 6B shows that the total amount of PCSK9 cleared increased with increasing amounts of GalNAc trimer anti-PCSK9 antibody administered. [Figure 6-2] (As stated above.) [Figure 7] Western blot analysis of an exemplary bifunctional compound, GalNAc-labeled anti-apoB antibody, as visualized by apoB100 IgG. [Figure 8] 1 is a schematic diagram showing an exemplary bifunctional compound, a GalNAc trimer anti-PCSK9 Fab fragment conjugate. [Figure 9] 1 is an SDS-PAGE showing purification of an exemplary bifunctional compound, a GalNAc trimer anti-PCSK9 Fab fragment conjugate, and related reaction products. [Figure 10-1]Figure 10: Graphs showing that an exemplary bifunctional compound, a GalNAc trimer anti-PCSK9 Fab fragment conjugate, exhibited high binding affinity to both PCSK9 and ASGPR. Figure 10A shows the binding affinity of a GalNAc trimer anti-PCSK9 Fab fragment conjugate to avidin-bound PCSK9. Figure 10B shows the binding affinity of a GalNAc-labeled anti-PCSK9 Fab fragment to avidin-bound deglycosylated ASGPR. [Figure 10-2] (As stated above.) [Figure 11-1] Figure 11: Graphs showing the clearance of PCSK9 in a mouse model modulated by administration of an exemplary bifunctional compound, a GalNAc trimer anti-PCSK9 Fab fragment conjugate. Figure 11A shows the reduction in the levels of PCSK9 after administration of a GalNAc trimer anti-PCSK9 Fab fragment conjugate. Figure 11B shows the clearance rate of PCSK9 when GalNAc trimer anti-PCSK9 Fab fragment conjugate is administered at various doses (0, 0.3, 1, and 3 mg / kg) over a 4-hour period. [Figure 11-2] (As stated above.) [Figure 12-1] Figure 12: Graphs showing that three exemplary bifunctional compounds, GalNAc trimer anti-MICA Fab fragment conjugates, exhibited high binding affinity to ASGPR. Figure 12A MC-68-LS07 is a Fab conjugate of an isotype control antibody (anti-lysozyme); Figure 12B IC-68-LW07 and Figure 12C WD-78-AD05 are Fab conjugates derived from two different anti-MICA antibodies with affinities for MICA of 69 pM and 2 pM, respectively. [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 13-1] FIG. 13: Graph showing that two exemplary bifunctional compounds, GalNAc trimer anti-MICA Fab fragment conjugates, increased clearance of MICA from serum in a mouse model (FIG. 13A) compared to the control (FIG. 13B). [Figure 13-2] (As stated above.) [Figure 14] 1 is an SDS-PAGE showing purification of an exemplary bifunctional compound, a GalNAc trimer anti-LDLR protein conjugate, and related reaction products. [Figure 15-1] FIG. 15: Graphs showing that an exemplary bifunctional compound, GalNAc trimer anti-LDLR protein, exhibited high binding affinity to both PCSK9 (FIG. 15A) and ASGPR (FIG. 15B). [Figure 15-2] (As stated above.) [Figure 16-1] FIG. 16: Graphs showing that an exemplary bifunctional compound, GalNAc trimer-PCSK9 protein, in two batches (FIG. 16A for batch no. PCSK9-GalNAc 1 and FIG. 16B for batch no. PCSK9-GalNAc 2) showed high binding affinity to PCSK9. [Figure 16-2] (As stated above.) [Figure 17-1] Figure 17: Graphs showing that an exemplary bifunctional compound, a GalNAc trimer-PCSK9 protein conjugate, is cleared more rapidly from serum in a mouse model when compared to unconjugated PCSK9 (Figure 17A), and that anti-PCSK9 antibodies are cleared more rapidly from the circulation compared to PCSK9 protein alone (Figure 17B). Figure 17A: Statistics by two-way ANOVA with Sidak's multiple comparison test. Figure 17B: Statistics by two-way ANOVA with Dunnett's multiple comparison test. [Figure 17-2] (As stated above.) [Figure 18-1] Figure 18: Graphs showing that tri-GalNAc-PCSK9 conjugate 4 functions as a heterobifunctional ligand to promote PCSK9 Ab clearance in vivo. Figure 18A: Synthesis of tri-GalNAc-hPCSK9 4. Figure 18B: Effect of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 90

number

[0047] Described herein are bifunctional compounds for inducing degradation of extracellular targets, as well as compositions and related methods thereof. The bifunctional compounds may provide a means for reducing the levels of otherwise undruggable disease-inducing targets in a sample or subject by inducing degradation, e.g., via cellular degradation pathways. Because no a priori knowledge of the function, structure, or activity of the disease-inducing target is required, the bifunctional compounds and related methods described herein may provide a powerful alternative to traditional treatments (e.g., chemical inhibition) that often rely on compound design based on target structure. For example, when compared to small molecule inhibitors or neutralizing antibodies, the bifunctional compounds described herein remove the entire target molecule. The approaches described herein (e.g., the bifunctional compounds described herein and their uses) are important and provide advantages, especially for certain molecules (e.g., (1) molecules whose presence causes disease, (2) targets with multiple functional sites, or targets with large interaction regions with difficult-to-inhibit partners). In addition, by degrading the target, the activity of the target is restored based on the synthesis rate of the target rather than the pharmacokinetics of the drug, which provides advantages for long-lived molecules. Finally, degradation is irreversible, allowing stoichiometric clearance of the drug from its target (or, if the drug is recycled, superstoichiometric clearance), thus allowing for infrequent dosing.

[0048] Moreover, compared to conventional small molecule inhibitors or neutralizing antibodies, the bifunctional compounds described herein must bind to both the target and the degradation receptor (optionally with a linker connecting these two functional moieties). These individual modules must be appropriately designed to enable the desired functions and properties (e.g., target degradation, recycling, oral availability). However, the approaches described in the prior art cannot show any in vivo activity (particularly any in vivo activity that is attractive for therapeutic applications) due to, for example, (1) simultaneous high affinity binding to both the target and the degradation receptor; (2) appropriate administration (e.g., dose, route of administration, and frequency) for target clearance; and (3) any in vivo activity that is attractive for therapeutic applications, particularly due to the non-obvious combination of the individual modules represented by the bifunctional compounds described herein to achieve rapid and sustained target clearance effect for therapeutic applications. In fact, the approaches described in the prior art require high daily doses and at the same time produce only very delayed and minimal effects on target concentrations, which are not desirable properties for therapeutic use (e.g., WO 2019 / 199621 and WO 2019 / 199634). In contrast, the bifunctional compounds described herein show convincing ASGPR affinity. Compared to the approaches described in the prior art, the bifunctional compounds described herein are carefully designed and appropriately engineered to show excellent binding affinity / specificity to both targets and receptors in various degradation pathways, as well as degradation activity in vivo. For example, the linkers used within the bifunctional compounds significantly improve the affinity, resulting in excellent target clearance in vivo.

[0049] definition Definitions of Certain Chemical Substances Definitions of certain functional groups and chemical terms are explained in more detail below. Chemical elements are defined as follows: thThe elements are identified in accordance with the Periodic Table of the Elements in the CAS format, which is provided on the inside cover of the Ed., and specific functional groups are generally defined as set forth therein. Further, general principles of organic chemistry and specific functional moieties and reactivities are described in detail in the texts set forth in the following publications: Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley&Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0050] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0051] When a range of values ​​is listed, the list is intended to include each value and subrange within the range. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0052] The following terms are intended to have the meanings indicated below and are helpful in understanding the description and intended scope of the present invention.

[0053] As used herein, "alkyl" refers to the radical of a linear or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group has 1 to 12 carbon atoms ("C1-C 12 In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C1-C8 alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C1-C6 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("C1 alkyl"). Examples of C1-C6 alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Further examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Each example of an alkyl group can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents (e.g., 1-5 substituents, 1-3 substituents, or 1 substituent) ("substituted alkyl"). In certain embodiments, an alkyl group is an unsubstituted C 1~ C 10 In certain embodiments, the alkyl group is a substituted C 1~ It is a C6 alkyl.

[0054] As used herein, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C2-C 24 In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C-C10 In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as 2-butenyl) or terminal (such as 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C 2~4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Further examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Each example of an alkenyl group can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents (e.g., 1-5 substituents, 1-3 substituents, or 1 substituent) ("substituted alkenyl"). In certain embodiments, an alkenyl group is an unsubstituted C 1~ C 10 In certain embodiments, the alkenyl group is a substituted C 2~ It is a C6 alkenyl.

[0055] As used herein, the term "alkynyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C 24 In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C-C 10In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C2-C8 alkynyl"). In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C2-C6 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (such as 2-butynyl) or terminal (such as 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents (e.g., 1-5 substituents, 1-3 substituents, or 1 substituent) (a "substituted alkynyl"). In certain embodiments, an alkynyl group is an unsubstituted C 2~10 In certain embodiments, the alkynyl group is a substituted C 2~6 It is alkynyl.

[0056] As used herein, the term "haloalkyl" refers to an acyclic stable straight or branched chain, or combinations thereof, containing at least one carbon atom and at least one halogen selected from the group consisting of F, Cl, Br, and I. The halogens F, Cl, Br, and I may be located at any position of the haloalkyl group. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CCl3, -CH2-CF3, -CH2-CCl 3、 -CH2-CBr 3、 -CH2-CI 3、 -CH2-CH2-CH(CF3)-CH3, -CH2-CH2-CH(Br)-CH3, and -CH2-CH=CH-CH2-CF3. Each example of a haloalkyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted haloalkyl") or substituted with one or more substituents (e.g., 1-5 substituents, 1-3 substituents, or 1 substituent) (a "substituted haloalkyl").

[0057] As used herein, the term "heteroalkyl" refers to an acyclic, stable, linear or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, P, S, and Si may be located at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive (e.g., -CH2-NH-OCH3, and -CH2-O-Si(CH3)3). A "heteroalkyl" is recited, followed by a specific heteroalkyl group (e.g., -CHO, -NR C R D or the like) is described, the term heteroalkyl and -CH2O or -NR C R D It will be understood that the following are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are provided for clarity. Thus, the term "heteroalkyl" is used herein to refer to specific heteroalkyl groups (e.g., -CHO, -NR C R D , or the like). Each example of a heteroalkyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) (a "substituted heteroalkyl").

[0058] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic arrangement) having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic arrangement) ("C6-C 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 10 Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 Aryl; for example, anthracyl. The aryl group is, for example, C6-C 10 The term "membered" refers to the non-hydrogen ring atoms in this moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each example of an aryl group can independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, an aryl group is an unsubstituted C6-C 14 In certain embodiments, the aryl group is a substituted C-C 14 It is aryl.

[0059] As used herein, "heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon atom or at a nitrogen atom, as valence permits. Heteroaryl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups, the point of attachment being either on the aryl ring or on the heteroaryl ring, in such cases the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups in which one ring does not contain heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, and the like), the point of attachment can be on either ring, i.e., on either the ring with heteroatoms (e.g., 2-indolyl) or the ring without heteroatoms (e.g., 5-indolyl). Heteroaryl groups can be described, for example, as 6-10 membered heteroaryl, with the term "member" referring to the non-hydrogen ring atoms in this moiety. Each instance of a heteroaryl group can independently be optionally substituted, i.e., unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents (e.g., 1-5 substituents, 1-3 substituents, or 1 substituent) ("substituted heteroaryl").

[0060] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0061] As used herein, "cycloalkyl" refers to an alkyl group having 3 to 10 ring carbon atoms ("C3-C 10"Cycloalkyl" refers to the radical of a non-aromatic cyclic hydrocarbon group having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C5-C6 cycloalkyl"). 10 Cycloalkyl groups may be described, for example, as C4-C7 membered cycloalkyl, with the term "member" referring to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, but are not limited to, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. ... 10 Cycloalkyl groups include, but are not limited to, the C3-C8 cycloalkyl groups mentioned above, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10), and the like. As the preceding examples illustrate, in certain embodiments, cycloalkyl groups can be monocyclic ("monocyclic cycloalkyl") or can include fused, bridged, or spiro ring systems (e.g., bicyclic systems ("bicyclic cycloalkyl") and can be saturated or partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring, as defined above, is fused to one or more aryl groups (the point of attachment is on the cycloalkyl ring), in such cases the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group can independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, a cycloalkyl group can be an unsubstituted C3-C6 10 In certain embodiments, the cycloalkyl group is a substituted C-C 10 It is cycloalkyl.

[0062] "Heterocyclyl," as used herein, refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and from one to four ring heteroatoms, each of which is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (a "3- to 10-membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment may be at a carbon atom or a nitrogen atom, where valence allows. Heterocyclyl groups can be monocyclic ("monocyclic heterocyclyl") or can be fused, bridged, or spiro ring systems, such as bicyclic systems ("bicyclic heterocyclyl"), and can be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring, as defined above, is fused to one or more cycloalkyl groups (the point of attachment is on either the cycloalkyl ring or on the heterocyclyl ring), or to one or more aryl or heteroaryl groups (the point of attachment is on the heterocyclyl ring). In certain embodiments, the heterocyclyl group includes a 3- to 7-membered heterocyclyl, where the term "member" refers to the non-hydrogen ring atoms in the heterocyclyl ring (i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon). Each instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0063] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclyl ring) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclyl rings) fused to an aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0064] The terms "alkylene," "alkenylene," "alkynylene," "haloalkylene," "heteroalkylene," "cycloalkylene," or "heterocyclylene," by themselves or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, haloalkylene, heteroalkylene, cycloalkyl, or heterocyclyl, respectively. For example, the term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. An alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, or heterocyclylene group may be described, for example, as C1-C6 membered alkylene, C2-C6 membered alkenylene, C2-C6 membered alkynylene, C1-C6 membered haloalkylene, C1-C6 membered heteroalkylene, C3-C8 membered cycloalkylene, or C3-C8 membered heterocyclylene (the term "membered" refers to the non-hydrogen atoms within the moiety). In the case of heteroalkylene and heterocyclylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Additionally, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- may represent both -C(O)2R'- and -R'C(O)2-.

[0065] As used herein, the term "cyano" or "-CN" refers to a substituent having a carbon atom attached to a nitrogen atom by a triple bond (eg, C≡N).

[0066] As used herein, the term "halogen" or "halo" refers to fluorine, chlorine, bromine, or iodine.

[0067] As used herein, the term "hydroxy" refers to --OH.

[0068] As used herein, the term "nitro" refers to a substituent having two oxygen atoms attached to a nitrogen atom (eg, --NO.sub.2).

[0069] As used herein, "oxo" refers to carbonyl (i.e., --C(O)--).

[0070] symbol [ka] as used herein with respect to a compound of formula (I), refers to a point of attachment to another moiety or functional group within said compound.

[0071] The alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups defined herein are optionally substituted. In general, the term "substituted", whether preceded by the term "optionally", means that at least one hydrogen present on the group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent (e.g., a substituent that upon replacement results in a stable compound (e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction)). Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when multiple positions in any given structure are substituted, the substituents are the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds (e.g., any of the substituents described herein) that result in the formation of a stable compound. The present disclosure contemplates all such combinations to yield stable compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and form a stable moiety.

[0072] Two or more substituents may optionally be bonded to form an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, found to be bonded to a cyclic base structure. In one embodiment, the ring-forming substituents are bonded to adjacent members of the base structure. For example, two ring-forming substituents bonded to adjacent members of the cyclic base structure form a fused ring structure. In another embodiment, the ring-forming substituents are bonded to a single member of the base structure. For example, two ring-forming substituents bonded to a single member of the cyclic base structure form a spirocyclic structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0073] The bifunctional compounds described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms (e.g., enantiomers and / or diastereomers). For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers (e.g., racemates and mixtures enriched in one or more stereoisomers). In certain embodiments, the stereochemistry depicted in the compounds is relative rather than absolute. Isomers may be isolated from mixtures by methods known to those skilled in the art (e.g., chiral high pressure liquid chromatography (HPLC), and formation and crystallization of chiral salts); or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure further includes the compounds described herein as individual isomers substantially free of other isomers or as mixtures of various isomers.

[0074] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). Upon reduction, the "S" form of a compound is substantially free of the "R" form of the compound and is therefore in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" indicates that a compound contains more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, or more than 99.9% by weight of an enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0075] In the compositions provided herein, the enantiomerically pure compounds may be present together with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R-compound may comprise, for example, about 90% of excipients and about 10% of the enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such a composition may comprise, for example, at least about 95% by weight of the R-compound and up to about 5% by weight of the S-compound, based on the total weight of the compound. For example, a pharmaceutical composition comprising an enantiomerically pure S-compound may comprise, for example, about 90% of excipients and about 10% of the enantiomerically pure S-compound. In certain embodiments, the enantiomerically pure S-compound in such a composition may comprise, for example, at least about 95% by weight of the S-compound and up to about 5% by weight of the R-compound, based on the total weight of the compound. In certain embodiments, the active ingredient may be formulated with little or no excipients or carriers.

[0076] The bifunctional compounds described herein may also include one or more isotopic substitutions. For example, H may be in any isotopic form, such as 1 H, 2 H (D or deuterium), and 3H (T or tritium); C can be in any isotopic form (e.g., 12 C. 13 C, and 14 C); O can be in any isotopic form (e.g., 16 O, and 18 O); and the like.

[0077] The term "pharmaceutical acceptable salt" is meant to include salts of bifunctional compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. In cases where the bifunctional compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutical acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. In cases where the bifunctional compounds described herein contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and the like, and those derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic acid or galacturonic acid and the like (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific bifunctional compounds of the present disclosure contain both basic and acidic functional groups that allow the compounds to be converted into either base or acid addition salts. These salts can be prepared by methods known to those skilled in the art. Other pharma- ceutically acceptable carriers known to those skilled in the art are suitable for the present invention.

[0078] The term "polyethylene glycol" or "PEG" as used herein refers to a linear, branched, or star-shaped configuration composed of (OCHCH) groups. In certain embodiments, the polyethylene or PEG group is -(OCHCH) t *-, where t is 4 to 40, "-" indicates the end directed towards the self-immolative spacer, and "*-" indicates the point of attachment to the terminal group R', where R' is OH, OCH, or OCHCHC(=O)OH. In another embodiment, the polyethylene or PEG group is -(CHCHO). t *-, where t is 4 to 40, "-" refers to the end toward the self-immolative spacer, and "*-" indicates the point of attachment to the terminal group R'', where R'' is H, CH, or CHCHC(=O)OH.

[0079] Other definitions The following definitions are of more general terms used throughout this disclosure.

[0080] The articles "a" and "an" refer to one or to more than one (e.g., at least one) of the grammatical referent of the article. By way of example, "an element" means one element or more than one element. The term "and / or" means either "and" or "or", unless otherwise indicated.

[0081] The term "about" is used herein to mean within typical tolerances in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the numbers in the series or range.

[0082] "Obtain" or "obtaining", as used herein, refers to taking ownership of a value (e.g., a numerical value), or an image, or a physical entity (e.g., a sample) by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" refers to performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. "Indirectly obtaining" refers to receiving a value or physical entity from another party or source (e.g., a third party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process that involves a physical change of a physical substance, or the use of a machine or device. An example of directly obtaining a value is obtaining a sample from a human subject. Directly obtaining a value includes performing a process that uses a machine or device (e.g., a mass spectrometer) to obtain mass spectrometry data.

[0083] The terms "administer", "administering" or "administration" as used herein refer to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing a bifunctional compound of the present invention or a pharmaceutical composition thereof.

[0084] The term "amino acid" refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified (e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0085] The term "antibody" as used herein refers to a protein, polypeptide, or fragment thereof having an amino acid sequence derived from an immunoglobulin molecule that specifically binds to an antigen. The amino acid sequence of an antibody or fragment thereof usually includes at least one immunoglobulin variable domain sequence. Typically, antibodies, as well as antigen-binding fragments and engineered variants thereof, are produced in a subject (e.g., a human subject) in response to the presence of an antigen and are capable of specifically binding to the antigen non-covalently and reversibly (i.e., via the antigen-binding moiety). A naturally occurring "antibody" is a glycoprotein that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and each VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the host tissue or immune system (e.g., effector cells) and factors including the first component (Clq) of the classical complement system.

[0086] Both light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that both the variable domain of the light chain portion (VL) and the variable domain of the heavy chain portion (VH) determine antigen recognition and specificity. Conversely, the constant domain of the light chain (CL) and the constant domain of the heavy chain (CH1, CH2, or CH3) confer important biological properties (e.g., secretion, transplacental transfer, Fc receptor binding, complement binding, and the like). By convention, the numbering of the constant regions increases as one moves away from the antigen-binding site or amino-terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal domains of the heavy and light chains, respectively.

[0087] The amino acid assignment for each variable region domain follows the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia, Al-Lazikani et al., J. Mol. Biol. 273, 927-948 (1997). The numbering of the heavy chain constant region is according to the EU index as described in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0088] The term "antibody" as used herein includes, but is not limited to, whole antibodies, human antibodies, camelid antibodies, intact monoclonal antibodies (e.g., antibodies produced using hybridoma technology), anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies to an antibody of the present disclosure), and antigen-binding antibody fragments (e.g., F(ab')2, Fv fragments, diabodies, single chain antibodies, scFv fragments, or scFv-Fc). Also included are genetically engineered intact antibodies and fragments thereof (e.g., chimeric antibodies, humanized antibodies, minibodies, linear antibodies, multivalent or multispecific (e.g., bispecific) hybrid antibodies, and the like). Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Thus, the term "antibody" is used broadly to include any protein that contains an antibody antigen-binding site and is capable of specifically binding to its antigen.

[0089] In some embodiments, the antibody is a multispecific antibody, e.g., it comprises a plurality of immunoglobulin variable domain sequences, a first immunoglobulin variable domain sequence of the plurality having binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality having binding specificity for a second epitope. In some embodiments, the multispecific antibody is a bispecific antibody. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In some embodiments, the bispecific antibody binds to asialoglycoprotein receptor (ASGPR) and proprotein convertase subtilisin / kexin type 9 (PCSK9). In some embodiments, the multispecific antibody is a trispecific antibody (e.g., an antibody having specificity for three antigens).

[0090] The phrase "antibody fragment" as used herein refers to one or more portions of an antibody. In some embodiments, these portions are portions of the constant domains of an antibody (e.g., fragment crystallizable (Fc) domain, constant (C) domain, etc.). In other embodiments, these portions are antigen-binding fragments that retain the ability to non-covalently, reversibly, and specifically bind antigen, and may be referred to herein as antigen-binding domains. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), single domain antibody, or single chain variable fragment (scFv).

[0091] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, which usually determines the class to which the antibody belongs.

[0092] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0093] The term "antigen" refers to a molecule that elicits an immune response, and also refers to a molecule that is recognized by components of the immune response, such as antibodies and immunocompetent cells. The immune response may include either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can be an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA that includes a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens may be synthetic or derived from biological samples, or may be macromolecules other than polypeptides, including, but not limited to, tissue samples, tumor samples, cells, or fluids with other biological components.

[0094] Unless otherwise specified, the terms "bifunctional compound", "bifunctional compound(s)", "bifunctional compound of the disclosure", or "bifunctional compounds of the disclosure" refer to one or more bifunctional compounds of formula (I), and pharma- ceutically acceptable salts thereof, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers, and isotopically labeled compounds (including deuterium substitutions).

[0095] As used herein, the term "biological neutralization" refers to a molecule that binds to a target in such a way as to reduce (e.g., impede) the ability of the target to perform its normal biological function. For example, a neutralizing anti-PCSK9 antibody may bind to PCSK9 and impede the function of PCSK9 (e.g., to bind to LDLR and cause its degradation).

[0096] The term "combination" as used herein refers to either a fixed combination in one unit dosage form or combined administration, where the compound of the present invention and the combination partner (e.g., another drug described below, also referred to as a "therapeutic agent" or "adjunct") can be administered independently at the same time or separately within a time interval, particularly when these time intervals allow the combination partners to exhibit a cooperative effect (e.g., synergistic effect). The single components can be packaged in a kit or packaged separately. One or both of the components (e.g., powder or liquid) can be reconstituted or diluted to the desired dose before administration.

[0097] The terms "complementarity determining region" and "CDR" are used interchangeably herein to refer to sequences of amino acids in an antibody or fragment thereof (e.g., within the antibody variable region (VL and / or VH) that confer antigen specificity and binding affinity). Generally, there are three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3, numbered consecutively from the N-terminus) and three CDRs in each heavy chain variable region (HCDR1, HCDR2, and HCDR3, numbered consecutively from the N-terminus).

[0098] The locations of CDRs and framework regions may be determined using a variety of definitions known in the art, such as Kabat, Chothia, IMGT, AbM, and combined definitions (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); Al-Lazikani et al., Nucleic Acids Res., 29:207-209 (2001); al., J. Mol. Biol., 273:927-748 (1997); also see the World Wide Web at bioinf.org.uk / abs / info.html). The definition of an antigen-binding site is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0099] For example, Table 1 lists CDR definitions according to Chothia, Kabat (Gawinowicz et al. 1991), combined, and IMGT. In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align. The IMGT tool is available on the World Wide Web at imgt.org.

[0100] [Table 1]

[0101] As used herein, the terms "condition," "disease," "disorder," and "clinical condition" are used interchangeably.

[0102] An "effective amount" of a bifunctional compound described herein refers to an amount sufficient to induce a desired biological response (i.e., to treat a condition). As will be appreciated by those skilled in the art, an effective amount of a bifunctional compound may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatments. For example, in the treatment of cancer, an effective amount of a compound of the present invention may reduce tumor burden or halt tumor growth or spread. In some embodiments, an effective amount of a bifunctional compound described herein is equimolar to the concentration of the target or in slight excess of concentration relative to the target. In some embodiments, an effective amount of a bifunctional compound described herein is the concentration required to obtain a binary complex between the bifunctional compound and the target. In some embodiments, an effective amount of a bifunctional compound described herein does not include a high excess concentration over the target concentration (e.g., 100-fold, 250-fold, 500-fold, or higher fold of a bifunctional compound described herein relative to the target).

[0103] A "therapeutically effective amount" of a bifunctional compound refers to an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with a condition. In some embodiments, a therapeutically effective amount refers to an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with a condition. A therapeutically effective amount of a bifunctional compound refers to an amount of a therapeutic agent that, alone or in combination with other therapeutic agents, provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids the symptoms or causes of a condition, or enhances the therapeutic effectiveness of another therapeutic agent.

[0104] The term "extracellular", as used herein, refers to the space outside the plasma membrane of one or more cells.

[0105] The term "extracellular target", as used herein, refers to an entity (e.g., one or more molecules) that is present in the space outside the plasma membrane of one or more cells. An extracellular target may be a protein or non-protein entity. Exemplary extracellular targets include proteins (soluble proteins and membrane-associated proteins, such as antibodies, receptors, growth factors, cytokines, chemokines, enzymes, hormones, neurotransmitters, or combinations thereof), lipoproteins, nucleic acids (e.g., oligonucleotides, DNA, RNA), toxins, liposomes, viral particles, and cells (e.g., prokaryotic cells, eukaryotic cells).

[0106] The terms "reducing extracellular levels" or "inhibiting extracellular levels," as used herein, refer to reducing or decreasing the concentration and / or activity of a target located in the space outside the plasma membrane of one or more cells, as compared to the concentration and / or activity of the target in the absence of a bifunctional compound described herein. In certain embodiments, the reduction in the concentration and / or activity of a target located in the space outside the plasma membrane of one or more cells is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, as compared to the concentration and / or activity of the target in the absence of a bifunctional compound described herein. In certain embodiments, the reduction in the concentration and / or activity of a target located outside the plasma membrane of one or more cells is greater than about 30% (e.g., greater than about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater) compared to the concentration and / or activity of the target in the absence of a bifunctional compound described herein.

[0107] The term "pathogenic autoantibody" refers to an antibody against one or more of the subject's own antigens. Pathogenic autoantibodies may cause injury and / or inflammation in the subject. For example, pathogenic autoantibodies are involved in several diseases, such as lupus erythematosus, rheumatoid arthritis, primary biliary cirrhosis, membranous nephropathy, IgA nephropathy, IgE-mediated allergic reactions, type 1 diabetes, and myasthenia gravis.

[0108] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds made up of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids they may contain. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which there are many varieties, commonly referred to in the art as proteins.

[0109] "Prevention," "prevent," and "preventing," as used herein, refer to treatments that include administering a therapeutic agent, e.g., a bifunctional compound described herein (e.g., a bifunctional compound of Formula (I)) prior to the onset of a disease, disorder, or condition to prevent the physical manifestations of said disease, disorder, or condition. In some embodiments, "prevention," "prevent," and "preventing" require that a sign or symptom of the disease, disorder, or condition has not yet developed or been observed. In some embodiments, treatment includes prevention, while in other embodiments, treatment does not include prevention.

[0110] "Proliferative disease" refers to a disease resulting from the abnormal expansion of cells by proliferation (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). Proliferative diseases may be associated with: 1) pathological proliferation of normally quiescent cells; 2) pathological migration of cells from normal locations (e.g., metastasis of tumor cells); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase); 4) pathological angiogenesis such as proliferative retinopathy and tumor metastasis; or 5) evasion of host immune surveillance and elimination of tumor cells. Exemplary proliferative diseases include cancer (i.e., "malignant neoplasms"), benign neoplasms, and angiogenesis.

[0111] "Reduce", "reduce" or "lower" refers to a negative change of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, in the context of a particular disease (e.g., a PCSK9-mediated disease or disorder), "reduce", "reduce" or "lower" refers to a reduction in the level, expression, or activity of an extracellular target (e.g., PCSC9) to a level that is considered in the literature to be below the normal range in subjects without such disorder, or to a level at which the symptoms of the disease (e.g., a PCSK9-mediated disease or disorder) are alleviated or ameliorated. This reduction can be, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (e.g., 100%).

[0112] "Reference" means a standard or control condition. "Reference level known in the art" refers to an experimentally determined control or threshold level identified in the prior art or known, or to a level identified in a sample from a control patient or a control patient population. In one embodiment, the reference is an untreated subject or a subject administered a placebo or normal saline, medium, buffer, and / or control. In one embodiment, the reference is a healthy subject.

[0113] "Sequence identity" refers to the similarity between amino acid sequences or between nucleic acid sequences, expressed as the similarity between the sequences. Sequence identity is often measured as a percentage of identity (or similarity or homology), and the higher the percentage, the more similar the sequences are. Homologs or variants of a given gene or protein will have a relatively high degree of sequence identity when aligned using standard methods. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications (e.g., gaps or overhangs).

[0114] An exemplary approach for determining the degree of identity may use the BLAST program, e.g. -3 and e -100A probability score between indicates closely related sequences. In addition, other programs and alignment algorithms are described, for example, in Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp, 1988, Gene 73:237-244; Higgins and Sharp, 1989, CABIOS 5:151-153; Corpet et al., 1988, Nucleic Acids Research 16:10881-10890; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; and Altschul et al., 1994, Nature Genet. 6:119-129. The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al. 1990, J. Mol. Biol. 215:403-410) is readily available from a variety of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0115] The term "single-chain Fv" or "scFv" as used herein typically refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises an internal polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. scFVs may also have engineered internal disulfide bridges that enhance stability. For a discussion of scFvs, see Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (1994) Springer-Verlag, New York, pp. 269-315.

[0116] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent), or an adult subject (e.g., young adult, middle-aged adult, or elderly adult)), and / or other non-human animals, e.g., mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals (e.g., cows, pigs, horses, sheep, goats, cats, and / or dogs), and birds (e.g., commercially relevant birds, e.g., chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be male or female and may be at any stage of development. The non-human animal may be a transgenic animal.

[0117] A "subject in need" is a subject or patient who would benefit from administration of a bifunctional compound of the present disclosure, for example, for use in treating a disease, disorder, or condition. In some embodiments, a "subject in need" is a subject or patient who has been diagnosed with, is at risk of having, has been predetermined to have, or is suspected of having a disease or disorder (e.g., a PCSK9-mediated disease or disorder). In a non-limiting example, a "subject in need" is a subject or patient who has, is at risk of developing, or is susceptible to a PCSK9-mediated disease or disorder.

[0118] As used herein, the terms "treatment", "treat", and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of one or more of the symptoms, signs, or underlying causes of a disease, disorder, or condition (e.g., as described herein), for example, by administering a therapeutic agent (e.g., by administering a bifunctional compound described herein (e.g., a bifunctional compound of Formula (I))). In certain embodiments, treating includes alleviating, reversing, alleviating, delaying the onset, or inhibiting the progression of a symptom of a disease, disorder, or condition. In certain embodiments, treating includes alleviating, reversing, alleviating, delaying the onset, or inhibiting the progression of a symptom of a disease, disorder, or condition. In certain embodiments, treating includes alleviating, reversing, alleviating, reducing, or delaying the onset of an underlying cause of a disease, disorder, or condition. In some embodiments, "treatment", "treat", and "treating" require that a sign or symptom of a disease, disorder, or condition has developed or is observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease or condition (e.g., prophylactic treatment). For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. In some embodiments, treatment includes prevention, and in other embodiments, treatment does not include prevention.

[0119] As used herein, a "vector" refers to a nucleic acid (polynucleotide) molecule capable of transporting another polynucleotide to which it has been linked. In some embodiments, a vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. In other embodiments, the vector is a viral vector (e.g., AAV), and additional DNA segments can be ligated to the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes operably linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Expression vectors can contain additional nucleic acid sequences to promote and / or facilitate expression of introduced sequences (e.g., initiation, termination, enhancer, promoter, and secretion sequences). Non-limiting examples of vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, with plasmids being the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0120] The terms "PCSK9", "hPCSK9", or "proprotein convertase subtilisin / kexin type 9" refer synonymously to the naturally occurring human proprotein convertase belonging to the proteinase K subfamily of the secreted subtilase family. PCSK9 is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. PCSK9 plays a role in cholesterol homeostasis and may have a role in cortical neuronal differentiation. Mutations in the PCSK9 gene are the cause of autosomal dominant familial hypercholesterolemia (Burnett and Hooper, Clin. Biochem. Rev. (2008) 29(1):11-26). "PCSK9" refers to the proprotein convertase subtilisin kexin 9 gene or protein. PCSK9 is also known as FH3, PC9, FHCL3, LDLCQ1, HCHOLA3, NARC-1, or NARCl. The term PCSK9 includes human PCSK9 (the amino acid and nucleotide sequences of which can be found, by way of example, at Gene ID: 255738; mouse PCSK9 (the amino acid and nucleotide sequences of which can be found, by way of example, at Gene ID: 100102); rat PCSK9 (the amino acid and nucleotide sequences of which can be found, by way of example, at Gene ID: 298296).

[0121] The terms "PCSK9-mediated disease or disorder" or "disease or disorder associated with PCSK9", as used herein, refer to a disease or disorder associated with the activity of PCSK9, including hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic disease and cerebrovascular disease), peripheral arterial disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, and xanthomas.

[0122] The term "hypercholesterolemia" or "dyslipidemia" includes, for example, familial and non-familial hypercholesterolemia. Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevated serum cholesterol bound to low-density lipoproteins (LDL). Familial hypercholesterolemia includes both heterozygous FH and homozygous FH. Hypercholesterolemia (or dyslipidemia) is the presence of high levels of cholesterol in the blood. This is in the form of hyperlipidemia (elevated lipid levels in the blood) and hyperlipoproteinemia (elevated lipoprotein levels in the blood). Hyperlipidemia is the elevation of lipids in the bloodstream. The lipids include cholesterol, cholesterol esters, phospholipids, and triglycerides. Hyperlipidemia includes, for example, types I, IIa, IIb, III, IV, and V. Hypertriglyceridemia refers to high blood levels of triglycerides. Elevated triglyceride levels, even in the absence of hypercholesterolemia, are associated with atherosclerosis and predispose to cardiovascular disease.

[0123] The term "sitosterolemia" or "phytosterolemia" refers to a rare autosomal recessive disorder of lipid metabolism characterized by excessive absorption of sitosterol from the gastrointestinal tract and reduced biliary excretion of dietary sterols (i.e., leading to hypercholesterolemia, tendon and tuberous xanthomas, and premature development of atherosclerosis) and altered cholesterol synthesis.

[0124] The term "atherosclerosis" includes hardening of arteries associated with the accumulation of fatty substances, cholesterol, cellular waste products, calcium, and fibrin in the inner lining of arteries. The resulting buildup is called plaque.

[0125] The term "atherosclerosis" or "arteriosclerotic vascular disease (ASVD)" refers to a specific form of arteriosclerosis involving thickening, hardening, and loss of elasticity of the arterial wall as a result of invasion and accumulation of white blood cells, containing both live, activated white blood cells (inflammatory products) and remnants of dead cells, including cholesterol and triglycerides. Atherosclerosis is a syndrome that affects arterial blood vessels due to a chronic inflammatory response of white blood cells in the arterial wall.

[0126] The term "coronary heart disease", also known as atherosclerotic disease, atherosclerotic cardiovascular disease, coronary heart disease, or ischemic heart disease, is the most common type of heart disease that leads to heart attacks. The disease is caused by plaque that builds up along the inner walls of the arteries of the heart, narrowing the lumen of the arteries and reducing blood flow to the heart.

[0127] The term "xanthomas" refers to the skin manifestation of lipidosis in which lipids accumulate in large foam cells in the skin. Xanthomas are associated with hyperlipidemia.

[0128] The term "elevated Lp(a) concentration" as used herein refers to a serum Lp(a) concentration above 30 mg / dL (75 nmol / L). "Elevated serum Lp(a)" means a serum Lp(a) level above about 14 mg / dL. In certain embodiments, a patient is considered to have elevated serum Lp(a) if the serum Lp(a) level measured in the patient is greater than about 15 mg / dL, about 20 mg / dL, about 25 mg / dL, about 30 mg / dL, about 35 mg / dL, about 40 mg / dL, about 45 mg / dL, about 50 mg / dL, about 60 mg / dL, about 70 mg / dL, about 80 mg / dL, about 90 mg / dL, about 100 mg / dL, about 20 mg / dL, about 140 mg / dL, about 150 mg / dL, about 180 mg / dL, or about 200 mg / dL. Serum Lp(a) levels can be measured in a patient after a meal. In some embodiments, Lp(a) levels are measured after a period of fasting (e.g., after fasting for 8 hours, 8 hours, 10 hours, 12 hours, or more). Although any clinically acceptable diagnostic method may be used in connection with the present disclosure, representative methods for measuring serum Lp(a) in patients include, but are not limited to, immunoturbidimetry, ELISA, nephelometry, immunoturbidimetry, and dissociation-enhanced lanthanide fluorescent immunoassay.

[0129] "Elevated triglyceride levels" or "ETL" refers to any degree of triglyceride levels that are determined to be undesirable or targeted for regulation.

[0130] The term "sepsis" is a systemic response characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea, and organ dysfunction. Sepsis can result from septicemia (i.e., organisms, their metabolic end products, or toxins in the bloodstream), including bacteremia (i.e., bacteria in the blood), and toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxins in the blood). The term "sepsis" also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitemia (i.e., parasitic or protozoan parasites in the blood). Thus, sepsis and septic shock (acute circulatory failure due to sepsis, often associated with multiple organ failure and high mortality) can be caused by many organisms.

[0131] The terms "CFHR3" or "complement factor H-related protein 3 gene" refer synonymously to the gene encoding the human protein complement factor H-related protein 3 (FHR3).

[0132] The terms "FHR3" or "complement factor H-related protein 3" refer synonymously to the naturally occurring human complement factor H-related protein 3, a secreted protein that belongs to the complement factor H-related protein family.

[0133] The term "CFHR3-mediated disease or disorder" or "CFHR3-associated disease or disorder," as used herein, refers to a disease or disorder associated with abnormal activity of CFHR3, including nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0134] The term "FHR3-mediated disease or disorder" or "disease or disorder associated with FHR3", as used herein, refers to a disease or disorder associated with the activity of FHR3, and includes nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0135] The term "nephropathy," as used herein, refers to a disease or disorder of the kidney.

[0136] The term "age-related macular degeneration," as used herein, refers to an eye disease that affects the macula of the eye, causing blindness over time.

[0137] The term "atypical hemolytic uremic syndrome" as used herein refers to a disease affecting kidney function due to abnormal blood clotting in the kidney. Atypical hemolytic uremic syndrome is characterized by three major features related to abnormal clotting: hemolytic anemia, thrombocytopenia, and renal failure.

[0138] The term "hemolytic anemia," as used herein, refers to the premature breakdown of red blood cells.

[0139] The term "thrombocytopenia," as used herein, refers to a decrease in the level of circulating platelets, which are used in aiding in clotting.

[0140] The term "hepatocellular carcinoma (HCC)" as used herein refers to cancer of the liver.

[0141] The term "asialoglycoprotein receptor" or "ASGPR" refers synonymously to the protein or gene encoding the ASGPR protein, and includes any naturally occurring form, homolog, mutant, functional fragment, or variant of ASGPR that maintains the activity of ASGPR (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homolog, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. ASGPR may also be referred to herein as CLEC4H1, C-type lectin domain family 4 member H1, hepatic lectin H1, or HL-1. In some embodiments, the ASGPR protein is encoded by a nucleic acid identified by NCBI sequence references NM_00167, NM_080914, homologs or functional fragments thereof. In some embodiments, the ASGPR protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P07306 or P07307. In some embodiments, the ASGPR protein comprises a major subunit (48 kDa) and a minor subunit (40 kDa). The ASGPR protein, or a polypeptide encoded by the ASGPR gene, is involved in serum glycoprotein homeostasis (e.g., binding, internalization, and clearance of glycoproteins containing terminal galactose (Gal) or N-acetylgalactosamine (GalNAc) residues).

[0142] The term "mannose-6-phosphate receptor" or "M6PR" refers synonymously to this protein or gene encoding the M6PR protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of M6PR that maintains the activity of M6PR (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. M6PR may also be referred to herein as CD-MPR, cation-dependent mannose-6-phosphate receptor, CD Man-6-P receptor, CD-M6PR, MPR 46, MPR46, M6PR, MPRD, or SMPR. In some embodiments, the M6PR protein is encoded by a nucleic acid identified by NCBI sequence references NM_002355, NM_000876, homologs or functional fragments thereof. In some embodiments, the M6PR protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P20645 or P11717. As used herein, M6PR includes the cation-independent mannose-6-phosphate receptor (CI-M6PR) and the cation-dependent mannose-6-phosphate receptor (CD-M6PR), which may require divalent cations to recognize targets. The M6PR protein, or a polypeptide encoded by the M6PR gene, is involved in the transport of mannose-6-phosphate (eg, binding to glycoproteins having terminal mannose-6-phosphate residues).

[0143] The term "insulin-like growth factor 2 receptor" or "IGF2R" refers synonymously to this protein or gene encoding IGF2R, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of IGF2R that maintains the activity of IGF2R (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the mutant, variant, or homologue has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IGF2R may also be referred to herein as MPRI, CI Man-6-P receptor, CI-M6PR, M6P / IGF2R, IGF-II receptor, M6P / IGF2 receptor, CI Man-6-P receptor, MPR 300, CI-MPR, CD222, CIMPR, M6P-R, MPR1, or cation-independent mannose-6 phosphate receptor. In some embodiments, the IGF2R protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000876, a homologue or functional fragment thereof. In some embodiments, the IGF2R protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P11717. The IGF2R protein, or the protein encoded by the IGF2R gene, is a protein receptor involved in trans-Golgi network trafficking.

[0144] The terms "tumor necrosis factor A" and "TNFA" refer interchangeably to the protein or gene encoding the TNFA protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of TNFA that maintains the activity of TNFA (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. TNFA may also be referred to herein as tumor necrosis factor, TNF-alpha, TNFSF2, tumor necrosis factor ligand superfamily member 2, cachectin, TNF-A, DIF, tumor necrosis factor ligand 1F, TNLG1F, or APC1 protein. In some embodiments, the TNFA protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000594, a homolog or functional fragment thereof. In some embodiments, the TNFA protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P01375. Tumor necrosis factor A protein, or a polypeptide encoded by a TNFA gene, is a cytokine involved in, for example, the initiation of systemic inflammation.

[0145] The terms "tumor necrosis factor receptor 1" and "TNFR1" refer interchangeably to the protein or gene encoding the TNFR1 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of TNFR1 that maintains the activity of TNFR1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring form. TNFR1 may also be referred to herein as TNFRSF1A, CD120a, FPF, MS5, TBP1, TNF-R, TNF-RI, TNF-R55, TNFAR, TNFR1, TNFR1-d2, TNFR55, TNFR60, p55, p55-R, p60, tumor necrosis factor receptor superfamily member 1A, TNF receptor superfamily member 1A. In some embodiments, the TNFR1 protein is encoded by a nucleic acid identified by NCBI sequence references NM_001065, NM_001346091, NM_001346092, homologs or functional fragments thereof. In some embodiments, the TNFR1 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P19438. The TNFR1 protein, or the polypeptide encoded by the TNFR1 gene, is a membrane receptor that specifically binds tumor necrosis factor-alpha (TNF-alpha).

[0146] The terms "Interleukin-1 receptor" or "IL1R" refer synonymously to this protein or gene encoding the IL1R protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL1R that maintains the activity of IL1R (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL1R may also be referred to herein as CD121 antigen-like family member A, interleukin-1 receptor type 1, interleukin-1 receptor alpha, IL-1R-alpha, IL-1RT-1, D2S1473, IL-1R-1, IL-1RT1, CD121A, P80, EC 3.2.2.6, D2S1473, interleukin-1 receptor type 2, interleukin-1 receptor beta, IL-1R-beta, IL-1RT-2, CDw121b, IL-1R-2, CD121b, IL1RB, IL1R2c, or IL1R2. In some embodiments, the IL1R protein is encoded by a nucleic acid identified by NCBI sequence references NM_000877, NM_173343, homologs or functional fragments thereof. In some embodiments, an IL1R protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P14778 or P27930. As used herein, IL1R includes interleukin 1 receptor, type I, and interleukin 1 receptor, type II. The IL1R protein, or the polypeptide encoded by the IL1R gene, is a cytokine receptor that specifically binds interleukin-1.

[0147] The terms "interleukin-1 alpha" or "IL1-alpha" refer synonymously to this protein or gene encoding the IL1-alpha protein and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL1-alpha that maintains the activity of IL1-alpha (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring form. IL1-alpha may also be referred to herein as hematopoietin 1, IL1A, IL-1A, IL1, IL1-alpha, IL1F1, interleukin 1 alpha, IL-1 alpha, pro-interleukin-1-alpha, or pre-interleukin 1 alpha. In some embodiments, the IL1-alpha protein is encoded by a nucleic acid identified by NCBI sequence references NM_000575, NM_001371554, homologs or functional fragments thereof. In some embodiments, the IL1-alpha protein refers to a polypeptide, homologs, variants, mutants, or functional fragments identified by UniProt ID P01583. The IL1-alpha protein, or a polypeptide encoded by the IL1-alpha gene, is a cytokine mediator of the inflammatory response.

[0148] The terms "interleukin-1 beta" or "IL1-beta" refer synonymously to this protein or gene encoding the IL1-beta protein and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL1-beta that maintains the activity of IL1-beta (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL1-beta may also be referred to herein as interleukin 1 beta, IL1F2, catabolin, pro-interleukin-1-beta, leukocyte pyrogen, leukocyte intrinsic mediator, mononuclear cell factor, or lymphocyte activating factor. In some embodiments, the IL1-beta protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000576, a homolog or functional fragment thereof. In some embodiments, the IL1-beta protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P01584. The IL1-beta protein, or a polypeptide encoded by the IL1-beta gene, is a cytokine mediator of the inflammatory response.

[0149] The terms "Interleukin-17" or "IL17" refer synonymously to this protein or the gene encoding the IL17 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL17 that maintains the activity of IL17 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL17 may also be referred to herein as IL17A, CTLA8, IL-17, IL-17A, IL17, CTLA-8, interleukin 17A, cytotoxic T lymphocyte-associated antigen 8, or cytotoxic T lymphocyte-associated serine esterase 8. In some embodiments, the IL17 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_002190, a homologue or functional fragment thereof. In some embodiments, the IL17 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q16552. The IL17 protein, or the polypeptide encoded by the IL17 gene, is a cytokine produced in response to stimulation with IL-23.

[0150] The terms "Interleukin-12A" or "IL12A" refer synonymously to this protein or the gene encoding the IL12A protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL12A that maintains the activity of IL12A (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL12A may also be referred to herein as IL-12 p35, CLMF, IL-12A, NFSK, NKSF1, P35, interleukin 12A, cytotoxic lymphocyte maturation factor 1, cytotoxic lymphocyte maturation factor 35KDa subunit, or NF cell stimulatory factor chain 1. In some embodiments, the IL12A protein is encoded by a nucleic acid identified by NCBI sequence references NM_000882, NM_001354582, NM_001354583, homologs or functional fragments thereof. In some embodiments, the IL12A protein refers to a polypeptide, homologs, variants, mutants, or functional fragments identified by UniProt ID P29459. The IL12A protein, or the polypeptide encoded by the IL12A gene, is a subunit of the cytokine IL12, which is required for T cell-dependent induction of interferon gamma.

[0151] The terms "Interleukin-12B" or "IL12B" refer synonymously to this protein or the gene encoding the IL12B protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL12A that maintains the activity of IL12B (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring form. IL12B may also be referred to herein as CLMF, CLMF P40, CLMF2, IL-12B, IMD28, NKSF, NKSF2, IMD29 natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor p40, or interleukin-12 subunit p40. In some embodiments, the IL12B protein is encoded by a nucleic acid identified by NCBI sequence reference NM_002187, a homologue or functional fragment thereof. In some embodiments, the IL12B protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P29460. The IL12B protein, or the polypeptide encoded by the IL12B gene, is a subunit of the cytokine IL12, involved in the development of Th1 cells.

[0152] The term "Interleukin 23" or "IL23" refers synonymously to a heterodimeric protein comprising an IL23A subunit and an IL12B subunit (shared with IL-12), or to genes encoding the subunits of the IL23 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of IL23 that maintains the activity of IL23 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. In some embodiments, IL-23 refers to IL-23A. IL23 may also be referred to herein as SGRF, IL23P19, IL-23A, P19, interleukin-23 subunit P19, JKA3 induced upon T cell activation, or interleukin 23 P19 subunit. In some embodiments, the IL23 protein is encoded by a nucleic acid identified by NCBI sequence NM_016584, a homolog or functional fragment thereof. In some embodiments, the IL23 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q9NPF7. The IL23 protein is a cytokine involved in, among other things, the maintenance and expansion of Th17.

[0153] The terms "interleukin 2" or "IL2" refer synonymously to the protein or gene encoding the IL2 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL2 that maintains the activity of IL2 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL2 may also be referred to herein as TCGF, ​​lymphokine, T cell growth factor, cell growth factor, aldesleukin, or involved in the control of T cell clonal expansion. In some embodiments, the IL2 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000586, a homologue or functional fragment thereof. In some embodiments, the IL2 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P60568. The IL2 protein, or the polypeptide encoded by the IL2 gene, is a cytokine signaling molecule.

[0154] The term "Interleukin 5" or "IL5" refers synonymously to the protein or gene encoding the IL5 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of IL5 that maintains the activity of IL5 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL5 may also be referred to herein as TRF, EDF, eosinophil differentiation factor, colony stimulating factor, eosinophil, or B cell differentiation factor I. In some embodiments, the IL5 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000879, a homologue or functional fragment thereof. In some embodiments, the IL5 protein refers to a polypeptide, homologue, variant, mutant or functional fragment identified by UniProt ID P05113. The IL5 protein, or a polypeptide encoded by the IL5 gene, is a cytokine associated with stimulation of the immune system, for example in an allergic response.

[0155] The terms "Interleukin 6" or "IL6" refer synonymously to this protein or the gene encoding the IL6 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL6 that maintains the activity of IL6 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL6 may also be referred to herein as BSF2, HGF, HSF, IFNB2, IL-6, BSF-2, CDF, IFN-beta-2, B cell stimulatory factor 2, CTL differentiation factor, hybridoma growth factor, B cell differentiation factor, interleukin, beta 2, or interleukin BSF-2. In some embodiments, the IL6 protein is encoded by a nucleic acid identified by NCBI sequence references NM_000600, NM_001318095, NM_001371096, homologs or functional fragments thereof. In some embodiments, the IL6 protein refers to a polypeptide, homologs, variants, mutants, or functional fragments identified by UniProt ID P05231. The IL6 protein, or the polypeptide encoded by the IL6 gene, is a pro-inflammatory cytokine and an anti-inflammatory myokine.

[0156] The terms "Interleukin-10" or "IL10" refer synonymously to this protein or gene encoding the IL10 protein and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL10 that maintains the activity of IL10 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL10 may also be referred to herein as CSIF, GVHDS, IL-10, IL10A, TGIF, T cell proliferation inhibitory factor, or cytokine synthesis inhibitory factor. In some embodiments, the IL10 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000572, a homologue or functional fragment thereof. In some embodiments, the IL10 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P22301. The IL10 protein, or a polypeptide encoded by the IL10 gene, is a cytokine involved in various pathways in the immune system, such as enhancing B cell survival and antibody production.

[0157] The terms "Interleukin-13" or "IL13" refer synonymously to this protein or the gene encoding the IL13 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IL13 that maintains the activity of IL13 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IL13 may also be referred to herein as P600, bronchial hyperresponsiveness-1, allergic rhinitis, MGC116786, MGC116788, MGC116789, ALRH, BHR1, NC30, or interleukin-13. In some embodiments, the IL13 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_002188, a homologue or functional fragment thereof. In some embodiments, the IL13 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P35225. The IL13 protein, or a polypeptide encoded by the IL13 gene, is a cytokine associated with various pathways in the immune system, such as mediating physiological changes induced by allergic inflammation.

[0158] The terms "immunoglobulin E" or "IgE" refer synonymously to this protein or gene encoding the IgE protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IgE that maintains the activity of IgE (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring form. IgE may also be referred to herein as IGHE or immunoglobulin epsilon. In some embodiments, the IgE protein is encoded by a nucleic acid identified by NCBI sequence reference AH005278.2, a homologue or functional fragment thereof. In some embodiments, the IgE protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P01854. In some embodiments, IgE comprises two heavy chains (ε chains) and two light chains, the ε chains comprising four Ig-like constant domains (Cε1-Cε4). The IgE protein is a type of antibody found in mammals, for example, on the surface of mast cells and basophils.

[0159] The terms "insulin-like growth factor 1" or "IGF1" refer synonymously to this protein or gene encoding the IGF1 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of IGF1 that maintains the activity of IGF1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. IGF1 may also be referred to herein as IGF-I, IGF1A, IGFI, MGF, IBP1, insulin-like growth factor 1, IGF, somatomedin-C, mechano growth factor, or insulin-like growth factor IB. In some embodiments, the IGF1 protein is encoded by a nucleic acid identified by NCBI sequence references NM_000618, NM_001111283, NM_001111284, NM_001111285, homologs or functional fragments thereof. In some embodiments, the IGF1 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P05019. The IGF1 protein, or the polypeptide encoded by the IGF1 gene, is an endocrine hormone.

[0160] The terms "erythropoietin" or "EPO" refer synonymously to the protein or gene encoding the EPO protein and include any naturally occurring form, homologue, mutant, functional fragment, or variant of EPO that maintains the activity of EPO (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. EPO may also be referred to herein as EP, MVCD2, erythropoietin, hematopoietin, haemopoietin, ECYT5, DBAL, or epoetin. In some embodiments, the EPO protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000799, a homologue or functional fragment thereof. In some embodiments, the EPO protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P01588. The EPO protein, or a polypeptide encoded by the EPO gene, is, for example, a hormone involved in red blood cell production.

[0161] The terms "programmed death-ligand 1" or "PD-L1" refer synonymously to this protein or the gene encoding the PD-L1 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of PD-L1 that maintains an activity of PD-L1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. PD-L1 may also be referred to herein as CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, CD274 molecule, programmed cell death ligand 1, hPD-L1, cluster of differentiation 274, B7 homolog 1. In some embodiments, the PD-L1 protein is encoded by a nucleic acid identified by NCBI sequence references NM_001314029, NM_001267706, NM_014143, homologs or functional fragments thereof. In some embodiments, the PD-L1 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q9NZQ7. The PD-L1 protein, or a polypeptide encoded by the PD-L1 gene, is a transmembrane protein involved in suppressing the adaptive immune system, for example as a checkpoint protein.

[0162] The terms "programmed cell death 1" or "PD-1" refer synonymously to this protein or the gene encoding the PD-1 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of PD-1 that maintains an activity of PD-1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. PD-1 may also be referred to herein as PDCD1, CD279, PD-1, PD1, SLEB2, hPD-1, hPD-l, hSLE1, HPD-L, systemic lupus erythematosus susceptibility 2, programmed cell death protein 1, or programmed cell death 1. In some embodiments, the PD-1 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_005018, a homologue or functional fragment thereof. In some embodiments, the PD-1 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q15116. The PD-1 protein, or a polypeptide encoded by the PD-1 gene, is a cell surface protein that, for example, behaves as an immune checkpoint protein.

[0163] The term "microtubule-associated protein tau" or "MAPT" refers synonymously to this protein or the gene encoding the MAPT protein, and includes any of the naturally occurring forms, isoforms, homologues, mutants, functional fragments, or variants of MAPT that maintain the activity of MAPT (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the naturally occurring form. MAPT may also be referred to herein as DDPAC, FTDP-17, MAPTL, MSTD, MTBT1, MTBT2, PPND, PPP1R103, TAU, PHF-tau, MGC138549, FLJ31424, DDPAC, microtubule-associated protein tau, G protein beta 1 / gamma 2 subunit-interacting factor 1, protein phosphatase 1 regulatory subunit 103, neurofibrillary tangle protein, paired helical filaments-tau, or tau protein. In some embodiments, the MAPT protein is encoded by a nucleic acid identified by NCBI sequence references NM_001123066, NM_001123067, NM_001203251, NM_001203252, NM_005910, homologs or functional fragments thereof. In some embodiments, MAPT protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P10636. MAPT protein, or a polypeptide encoded by the MAPT gene, is a protein involved in, for example, regulating cellular stability and recruiting cell signaling proteins.

[0164] The terms "milk fat globule-EGF factor 8 protein," "MFGE8," or "lactadherin" refer synonymously to this protein or gene encoding the MFGE8 protein, and include any naturally occurring form, isoform, homolog, mutant, functional fragment, or variant of MFGE8 that maintains the activity of MFGE8 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homolog, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. MFGE8 may also be referred to herein as milk fat globule-EGF factor 8 protein, BA46, EDIL1, HMFG, HsT19888, MFG-E8, MFGM, OAcGD3S, SED1, SPAG10, hP47, milk fat globule EGF, mammary epithelial antigen BA46, sperm associated antigen 10, sperm surface protein HP47, medin, or O-acetyl disialoganglioside synthase. In some embodiments, the MFGE8 protein is encoded by a nucleic acid identified by NCBI sequence references NM_001114614, NM_001310319, NM_001310320, NM_001310321, NM_005928, homologs or functional fragments thereof. In some embodiments, the MFGE8 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q08431. The MFGE8 protein, or the polypeptide encoded by the MFGE8 gene, is a cell adhesion protein.

[0165] The term "thymic stromal lymphopoietin" or "TSLP" refers synonymously to the protein or gene encoding the TSLP protein, and includes any naturally occurring form, homolog, mutant, functional fragment, or variant of TSLP that maintains the activity of TSLP (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homolog, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. TSLP may also be referred to herein as thymic stromal lymphopoietin. In some embodiments, the TSLP protein is encoded by a nucleic acid identified by NCBI sequence references NM_033035, NM_138551, homologs or functional fragments thereof. In some embodiments, the TSLP protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q969D9. The TSLP protein, or a polypeptide encoded by the TSLP gene, is a cytokine involved in the maturation of T cells through activation of antigen presenting cells.

[0166] The terms "thrombospondin" or "TSP" refer synonymously to the protein or gene encoding the TSP protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of TSP that maintains an activity of TSP (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. TSP may also be referred to herein as thymic stromal lymphopoietin, THBS1, THBS, THBS-1, TSP, TSP-1, TSP1, thrombospondin 1, thrombospondin 2, thrombospondin 3, thrombospondin 4, thrombospondin 5, THBS-2, THBS-3, THBS-4, THBS-5, THBS2, THBS3, THBS4, THBS5, TSP-2, TSP2, TSP-3, TSP3, TSP-4, TSP4, TSP-5, TSP5, or glycoprotein G. In some embodiments, the TSP protein is encoded by a nucleic acid identified by NCBI sequence references NM_003246, XM_011521971.2, NM_001252607.2, NM_001252608.2, NM_007112.5, NM_003248.6, NM_001306212.2, NM_001306213.2, NM_001306214.2, homologs or functional fragments thereof. In some embodiments, the TSP protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P07996, P35442, P49746, or P35443. The TSP proteins, or polypeptides encoded by the TSP genes, are a family of proteins involved in the formation and maintenance of the extracellular matrix.

[0167] The term "fibroblast growth factor 23" or "FGF23" refers synonymously to the protein or gene encoding the FGF23 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of FGF23 that maintains the activity of FGF23 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the naturally occurring form. FGF23 may also be referred to herein as ADHR, FGFN, HPDR2, HYPF, PHPTC, fibroblast growth factor 23, tumor-derived hypophosphatemia-inducing factor, or HFTC2. In some embodiments, the FGF23 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_020638, a homologue or functional fragment thereof. In some embodiments, the FGF23 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q9GZV9. The FGF23 protein, or the polypeptide encoded by the FGF23 gene, is a protein involved in phosphate and vitamin D metabolism and regulation.

[0168] The term "tissue inhibitor of metalloproteinases 1" or "TIMP1" refers synonymously to this protein or the gene encoding the TIMP1 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of TIMP1 that maintains the activity of TIMP1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. TIMP1 may also be referred to herein as CLGI, EPA, EPO, HCI, TIMP, TIMP-1, tissue inhibitor of metalloproteinase 1, fibroblast collagenase inhibitor, collagenase inhibitor, epididymal secretory sperm binding protein, erythrocyte-potentiating activity, erythrocyte-potentiating activity, or TIMP metallopeptidase inhibitor 1. In some embodiments, the TIMP1 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_003254, a homologue or a functional fragment thereof. In some embodiments, the TIMP1 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P01033 or Q6FGX5. The TIMP1 protein, or a polypeptide encoded by the TIMP1 gene, is, for example, a protein involved in the inhibition of matrix metalloproteinases.

[0169] The term "adenosine deaminase 2" or "ADA2" refers synonymously to this protein or the gene encoding the ADA2 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of ADA2 that maintains an activity of ADA2 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring form. ADA2 may also be referred to herein as ADGF, feline eye syndrome chromosomal region, candidate 1, feline eye syndrome critical region protein 1, CECR1, IDGFL, adenosine deaminase CECR1, EC 3.5.4.4, SNEDS, VAIHS, or PAN. In some embodiments, the ADA2 protein is encoded by a nucleic acid identified by NCBI sequence references NM_001282225.2, NM_177405.3, NM_001282226.2, NM_001282227.2, NM_001282228.2, NM_001282229.2, homologs or functional fragments thereof. In some embodiments, the ADA2 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q9NZK5. The ADA2 protein, or the polypeptide encoded by the ADA2 gene, is involved in the metabolism of adenosine and related purine molecules.

[0170] The term "apelin" or "APLN" refers synonymously to the protein or gene encoding the APLN protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of APLN that maintains the activity of APLN (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. APLN may also be referred to herein as APEL, XNPEP2, APJ endogenous ligand, or AGTRL1 ligand. In some embodiments, the APLN protein is encoded by a nucleic acid identified by NCBI sequence reference NM_017413, a homologue or functional fragment thereof. In some embodiments, the APLN protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q9ULZ1. The APLN protein, or a polypeptide encoded by the APLN gene, is a protein involved in numerous pathways, such as the regulation of blood pressure.

[0171] The terms "apolipoprotein B" or "ApoB" refer synonymously to this protein or the gene encoding the ApoB protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of ApoB that maintains an activity of ApoB (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. ApoB may also be referred to herein as FLDB, LDLCQ4, apoB-100, apoB-48, apolipoprotein B, FCHL, apolipoprotein B-100, apolipoprotein B48, or FCHL2. In some embodiments, the ApoB protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000384, a homologue or functional fragment thereof. In some embodiments, the ApoB protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P04114. The APLN protein, or a polypeptide encoded by the ApoB gene, is the major apolipoprotein of various lipoprotein particles, such as low density lipoprotein (LDL) particles.

[0172] The terms "lipoprotein lipase" or "LPL" refer synonymously to this protein or gene encoding the LPL protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of LPL that maintains the activity of LPL (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. LPL may also be referred to herein as HDLCQ11, LIPD, EC 3.1.1.34, phospholipase A1, EC 3.1.1.32, or EC 3.1.1. In some embodiments, the LPL protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000237, a homologue or functional fragment thereof. In some embodiments, the LPL protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P06858. The LPL protein, or the polypeptide encoded by the LPL gene, is an enzyme that hydrolyzes triglycerides in various lipoproteins.

[0173] The terms "lipoprotein(a)" or "Lp(a)" refer synonymously to the protein or gene encoding the Lp(a) protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of Lp(a) that maintains the activity of Lp(a) (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. Lp(a) may also be referred to herein as AK38, APOA, LP, apolipoprotein (A), lipoprotein, Lp(A), Apo(A), EC 3.4.21.7, EC 3.4.21, EC 3.4.21, LPA, or antiangiogenic AK38 protein. In some embodiments, the Lp(a) protein is encoded by a nucleic acid identified by NCBI sequence reference NM_005577, a homolog or functional fragment thereof. In some embodiments, the Lp(a) protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P08519. The Lp(a) protein, or the polypeptide encoded by the Lp(a) gene, is a low density lipoprotein variant, including apolipoprotein(a). Lipoprotein(a) has been identified as a risk factor for certain cardiovascular diseases and conditions, such as atherosclerosis, coronary heart disease, and stroke.

[0174] The terms "apolipoprotein C3" or "ApoCIII" refer synonymously to this protein or gene encoding the ApoCIII protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of ApoCIII that maintains the activity of ApoCIII (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. ApoCIII may also be referred to herein as APOC3, HALP2, apolipoprotein-C3, or apolipoprotein C-III. In some embodiments, the ApoCIII protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000040, a homologue or functional fragment thereof. In some embodiments, the ApoCIII protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P02656. The ApoCIII protein, or the polypeptide encoded by the ApoCIII gene, is a protein secreted from the small intestine and liver and found in triglyceride-rich lipoproteins.

[0175] The term "angiopoietin-like 3" or "ANGPTL3" refers synonymously to this protein or gene encoding the ANGPTL3 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of ANGPTL3 that maintains the activity of ANGPTL3 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. ANGPTL3 may also be referred to herein as ANG-5, ANGPT5, ANL3, FHBL2, angiopoietin 5, or angiopoietin-like protein 3. In some embodiments, the ANGPTL3 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_014495, a homologue or functional fragment thereof. In some embodiments, the ANGPTL3 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q9Y5C1. The ANGPTL3 protein, or the polypeptide encoded by the ANGPTL3 gene, is a secreted factor that behaves as a dual inhibitor of lipoprotein lipase (LPL) and endothelial lipase (EL).

[0176] The term "angiopoietin-like 4" or "ANGPTL4" refers synonymously to this protein or the gene encoding the ANGPTL4 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of ANGPTL4 that maintains the activity of ANGPTL4 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. ANGPTL4 may also be referred to herein as ANGPTL2, ARP4, FIAF, HARP, HFARP, NL2, PGAR, TGQTL, UNQ171, pp1158, hepatic fibrinogen / angiopoietin-related protein, angiopoietin-related protein 4, peroxisome proliferator-activated receptor (PPAR) gamma-induced angiopoietin-related protein, hepatic angiopoietin-related protein, PPARG angiopoietin-related protein, fasting-induced adipose factor, or angiopoietin-like protein 4. In some embodiments, the ANGPTL4 protein is encoded by a nucleic acid identified by NCBI sequence references NM_001039667, NM_016109, NM_139314, homologs or functional fragments thereof. In some embodiments, the ANGPTL4 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q9BY76. The ANGPTL4 protein, or the polypeptide encoded by the ANGPTL4 gene, is the secretory target of certain peroxisome proliferator-activated receptors.

[0177] The terms "angiopoietin-like 8," "ANGPTL8," or "ribasin" refer synonymously to this protein or gene encoding the ANGPTL8 protein, and include any naturally occurring form, homolog, mutant, functional fragment, or variant of ANGPTL8 that maintains the activity of ANGPTL8 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homolog, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. ANGPTL8 may also be referred to herein as PRO1185, PVPA599, RIFL, TD26, C19orf80, betatrophin, refeeding-induced fat and liver protein, betatrophin, hepatocellular carcinoma-associated protein TD26, betatrophin variant 1, betatrophin variant 2, or angiopoietin-like protein 8. In some embodiments, the ANGPTL8 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_018687, a homologue or functional fragment thereof. In some embodiments, the ANGPTL8 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q6UXH0. The ANGPTL8 protein, or the polypeptide encoded by the ANGPTL8 gene, is an inhibitor of lipoprotein lipase (LPL).

[0178] The term "bone morphogenetic protein 6" or "BMP6" refers synonymously to this protein or the gene encoding the BMP6 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of BMP6 that maintains the activity of BMP6 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. BMP6 may also be referred to herein as VGR, VGR1, VGR-1, VG-1-R, VG-1-related protein, or vegetable-related growth factor (TGFB-related). In some embodiments, the BMP6 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_001718, a homologue or functional fragment thereof. In some embodiments, the BMP6 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P22004. The BMP6 protein, or a polypeptide encoded by the BMP6 gene, is involved in the induction of osteogenic markers in mesenchymal stem cells.

[0179] The term "bone morphogenetic protein 9 / growth differentiation factor 2" or "BMP9 / GDF2" refers synonymously to the protein or gene encoding the BMP9 / GDF2 protein and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of BMP9 / GDF2 that maintains the activity of BMP9 / GDF2 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. BMP9 / GDF2 may also be referred to herein as BMP-9, BMP9, HHT5, or growth differentiation factor 2. In some embodiments, the BMP9 / GDF2 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_016204, a homologue or functional fragment thereof. In some embodiments, the BMP9 / GDF2 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID Q9UK05. The BMP9 / GDF2 protein, or a polypeptide encoded by the BMP9 / GDF2 gene, plays a role in the maintenance of nerve cells and their response to acetylcholine.

[0180] The term "colony stimulating factor 1 receptor" or "CSF-1" refers synonymously to this protein or gene encoding the CSF-1 protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of CSF-1 that maintains the activity of CSF-1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. CSF-1 may also be referred to herein as MCSF, CSF1, MGC31930, colony stimulating factor 1, macrophage colony stimulating factor 1, or macrophage colony stimulating factor. In some embodiments, the CSF-1 protein is encoded by a nucleic acid identified by NCBI sequence references NM_172212, NM_000757, NM_172210, NM_172211, homologs or functional fragments thereof. In some embodiments, the CSF-1 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P09603. The CSF-1 protein, or the polypeptide encoded by the CSF-1 gene, is a hematopoietic growth factor involved in the proliferation, differentiation, and survival of immune cells.

[0181] The terms "growth differentiation factor 15" or "GDF15" refer synonymously to this protein or the gene encoding the GDF15 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of GDF15 that maintains the activity of GDF15 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. GDF15 may also be referred to herein as GDF-15, MIC-1, MIC1, NRG-1, NAG-1, PDF, PLAB, PTGFB, growth differentiation factor 15, TGF-PL, macrophage inhibitory cytokine 1, NSAID-activated gene 1 protein, NSAID (nonsteroidal anti-inflammatory drug)-activated protein 1, placental TGF-beta, placental bone morphogenetic protein, or nonsteroidal anti-inflammatory drug-activated gene-1. In some embodiments, the GDF15 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_004864, a homolog or functional fragment thereof. In some embodiments, the GDF15 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q99988. The GDF15 protein, or a polypeptide encoded by the GDF15 gene, plays a role in regulating inflammatory pathways and apoptosis.

[0182] The terms "complement factor B" or "CFB" refer synonymously to the protein or gene encoding the CFB protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of CFB that maintains the activity of CFB (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. CFB may also be referred to herein as Cfb, AI195813, AI255840, Bf, C2, Fb, H2-Bf, AHUS4, ARMD14, BFD, CFAB, CFBD, FBI12, GBG, PBF2, properdin factor B, C3 / C5 convertase, EC 3.4.21.47, BF, glycine-rich beta-glycoprotein, C3 proaccelerator, C3 precursor activator, or complement factor B. In some embodiments, the CFB protein is encoded by a nucleic acid identified by NCBI sequence reference NM_001710, homologs or functional fragments thereof. In some embodiments, the CFB protein refers to a polypeptide, homologs, variants, mutants, or functional fragments identified by UniProt ID P00751. The CFB protein, or a polypeptide encoded by the CFB gene, is a component of the alternative pathway of complement activation.

[0183] The terms "complement factor D" or "CFD" refer synonymously to this protein or gene encoding the CFD protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of CFD that maintains the activity of the CFD (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. CFD may also be referred to herein as Cfd, ADN, PFD, DF, EC 3.4.21.46, EC 3.4.21, C3 proactivator convertase, C3 convertase proactivator, properdin factor D esterase, properdin factor D, factor D (complement), complement factor D, CFD, adipsin, or complement-factor D. In some embodiments, the CFD protein is encoded by a nucleic acid identified by NCBI sequence reference NM_001928, a homolog or functional fragment thereof. In some embodiments, the CFD protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P00746. The CFD protein, or a polypeptide encoded by the CFD gene, is a component of the alternative pathway of complement activation.

[0184] The terms "complement component 5" or "C5" refer synonymously to this protein or gene encoding the C5 protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of C5 that maintains the activity of C5 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. C5 may also be referred to herein as C5D, C5a, C5b, CPAMD4, ECLZB, complement component 5, complement C5, C5a anaphylatoxin, prepro-C5, or C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4. In some embodiments, the C5 protein is encoded by a nucleic acid identified by NCBI sequence references NM_001735, NM_001317163, NM_001317164, homologs or functional fragments thereof. In some embodiments, the C5 protein refers to a polypeptide, homologs, variants, mutants, or functional fragments identified by UniProt ID P01031. In some embodiments, the C5 protein includes C5a and C5b. The C5 protein, or the polypeptide encoded by the C5 gene, is a component of the complement system and is cleaved into C5a and C5b.

[0185] The terms "CXC motif chemokine ligand 10" or "CXCL10" refer synonymously to this protein or gene encoding the CXCL10 protein and include any naturally occurring form, homologue, mutant, functional fragment, or variant of CXCL10 that maintains the activity of CXCL10 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. CXCL10 may also be referred to herein as IFI10, INP10, IP-10, SCYB10, crg-2, gIP-10, GIP-10, Mob-1, C7, mob-1, gamma IP10, gamma-IP10, small inducible cytokine G10, interferon inducible cytokine IP-10, 10KDa interferon gamma inducible protein, CXC motif chemokine ligand 10, CXC motif chemokine 10, small inducible chemokine subfamily B (Cys-X-Cys), member 10, or interferon gamma inducible protein 10. In some embodiments, the CXCL10 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_001565, a homologue or functional fragment thereof. In some embodiments, a CXCL10 protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P02778. The CXCL10 protein, or the polypeptide encoded by the CXCL10 gene, is a cytokine secreted in response to interferon-gamma and binds to the cell surface chemokine receptor CXCR3.

[0186] The terms "myeloperoxidase" or "MPO" refer synonymously to the protein or gene encoding the MPO protein, and include any naturally occurring form, homologue, mutant, functional fragment, or variant of MPO that maintains the activity of MPO (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. MPO may also be referred to herein as EC 1.11.1.7, EC 1.11.2.2, or 1.11.1. In some embodiments, the MPO protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000250, a homologue or functional fragment thereof. In some embodiments, the MPO protein refers to a polypeptide, homologue, variant, mutant, or functional fragment identified by UniProt ID P05164. The MPO protein, or the polypeptide encoded by the MPO gene, is a peroxidase of the XPO subfamily of peroxidases. Elevated levels of MPO are associated with the severity of coronary artery disease.

[0187] The term "Progranulin" refers synonymously to the protein or gene encoding the Progranulin protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of Progranulin that maintains the activity of Progranulin (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the naturally occurring form. Progranulin may also be referred to herein as GRN, CLN11, GEP, GP88, PCDGF, PEPI, PGRN, PGranulin, granulin precursor, proepithelin, acrogranin, epithelin precursor, granulin-epithelin, granulin, or PC cell-derived growth factor. In some embodiments, the Progranulin protein is encoded by a nucleic acid identified by NCBI sequence references NM_001012479, NM_002087, homologs or functional fragments thereof. In some embodiments, the Progranulin protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P28799. The Progranulin protein, or the polypeptide encoded by the Progranulin gene, is a protein involved in the regulation of cell growth, survival, repair, and inflammation, for example in the central nervous system.

[0188] The term "transforming growth factor beta 1" or "TGF-beta 1" refers synonymously to the protein or gene encoding the TGF-beta 1 protein and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of TGF-beta 1 that maintains the activity of TGF-beta 1 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. TGF-beta 1 may also be referred to herein as CED, DPD1, LAP, IBDIMDE, TGFB, TGF beta, transforming growth factor beta 1, IBDIMDE, TGFB1, TGFB-1, TGF-beta 1, latency-associated peptide, diaphyseal dysplasia 1, progressive, or prepro transforming growth factor beta-1. In some embodiments, the TGF-beta 1 protein is encoded by a nucleic acid identified by NCBI sequence reference NM_000660, a homolog or functional fragment thereof. In some embodiments, the TGF-beta 1 protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID P01137. The TGF-beta 1 protein, or the polypeptide encoded by the TGF-beta 1 gene, is a cytokine involved in many regulatory pathways.

[0189] The term "vascular endothelial growth factor A" or "VEGF-A" refers synonymously to the protein or gene encoding the VEGF-A protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of VEGF-A that maintains the activity of VEGF-A (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. VEGF-A may also be referred to herein as MVCD1, VEGFA, VEGF, VPF, vascular endothelial growth factor A, vascular endothelial growth factor A121, vascular endothelial growth factor A165, or vascular permeability factor. In some embodiments, the VEGF-A protein is encoded by a nucleic acid identified by NCBI sequence references NM_003376, NM_001025366, NM_001025367, NM_001025368, NM_001025369, homologs or functional fragments thereof. In some embodiments, the VEGF-A protein refers to a polypeptide, homologs, variants, mutants, or functional fragments identified by UniProt ID P015692. The VEGF-A protein, or the polypeptide encoded by the VEGF-A gene, is a member of the platelet-derived growth factor / vascular endothelial growth factor family.

[0190] The term "urokinase-type plasminogen activator receptor" or "suPAR" refers synonymously to this protein or gene encoding the suPAR protein, and includes any naturally occurring form, homologue, mutant, functional fragment, or variant of suPAR that maintains the activity of suPAR (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, a homologue, mutant, or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. suPAR may also be referred to herein as PLAUR, CD87, CD87 antigen, MO3, U-PAR, UPAR, URKR, plasminogen activator, urokinase receptor, monocyte activation antigen Mo3, U-plasminogen activator receptor type 2, or urokinase plasminogen activator surface receptor. In some embodiments, the suPAR protein is encoded by a nucleic acid identified by NCBI sequence references NM_001005376, NM_001005377, NM_001301037, NM_002659, homologs or functional fragments thereof. In some embodiments, the suPAR protein refers to a polypeptide, homolog, variant, mutant, or functional fragment identified by UniProt ID Q03405. The suPAR protein, or the polypeptide encoded by the uPAR gene, is a soluble form of the urokinase plasminogen activator receptor (uPAR) and is a biomarker of inflammation and immune system activation.

[0191] As used herein, the term "therapeutic apheresis" refers to an extracorporeal procedure to selectively remove abnormal cells or substances in the blood that are associated with or cause certain disease states.

[0192] Bifunctional compounds The present disclosure provides a first moiety capable of binding to an extracellular target (i.e., AG ) and a second moiety capable of binding to a membrane-bound receptor associated with a degradative pathway (i.e., A G In some embodiments, the bifunctional compounds described herein have the formula (I): A G -LR G (I), or a pharma- ceutically acceptable salt thereof, wherein: A G is a protein moiety that binds to an extracellular target (e.g., an antibody or fragment thereof, a receptor or fragment thereof, or an antigenic protein or fragment thereof); L is absent or is a linker; and R G is the moiety that binds to a membrane-bound receptor associated with a degradative pathway.

[0193] Each of these components is described in more detail herein.

[0194] Targeted binding moiety (A G ) The targeting binding moiety (i.e., A G ) can be a protein moiety that binds to an extracellular target molecule. In some embodiments, A G is the portion of the bifunctional moiety that confers target specificity to the bifunctional compound; G may be an antibody molecule or fragment thereof, a receptor molecule, or an antigen or fragment thereof.

[0195] In some embodiments, A G is an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises an antigen-binding domain that binds to an extracellular target. G is a full-length antibody. In one embodiment, A Gis an antibody fragment. Antibody fragments may include Fab, Fab', F(ab')2, F(ab)2, variable fragments (Fv), domain antibodies (dAbs), single domain antibodies, or single chain variable fragments (scFv). Antibodies may further be monospecific or multispecific (e.g., bispecific or trispecific) antibody molecules. In certain embodiments, A is an antibody fragment. G comprises an anti-ApoB antibody, an anti-PCSK9 antibody, an anti-MICA antibody, an anti-EGFR antibody, or an anti-LDL antibody. G In one embodiment, the antibody comprises an anti-ApoB antibody. G In one embodiment, A comprises an anti-PCSK9 antibody. G In one embodiment, A comprises an anti-MICA antibody. G In one embodiment, A comprises an anti-EGFR antibody. G comprises an anti-LDL antibody. G comprises an antibody fragment, including an anti-ApoB Fab fragment, an anti-PCSK9 Fab fragment, an anti-MICA Fab antibody, an anti-EGFR Fab antibody, or an anti-LDL Fab fragment. G comprises an anti-ApoB Fab fragment. G comprises an anti-PCSK9 Fab fragment. G comprises an anti-MICA Fab fragment. G comprises an anti-EGFR Fab fragment. G contains an anti-LDL Fab fragment.

[0196] In some embodiments, A Gcomprises an antigenic protein or a fragment thereof (e.g., a peptide). The antigenic protein may comprise a fragment of a cell surface antigen (e.g., a human Cluster of Differentiation (CD) protein). For example, the antigenic protein may comprise one or more of the following sequences: CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD2, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD 37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD77, CD79, CD80, or other CD proteins. In some embodiments, the antigen protein or fragment thereof recognizes a pathogenic autoantibody.Exemplary antigenic proteins include: a disintegrin and metalloproteinase with thrombospondin type 1 moiety, member 13 (ADAMTS13), steroidogenic cytochrome P450 enzyme 21-hydroxylase, N-methyl-d-aspartate (NMDA) receptor, red blood cells, anti-smooth muscle antibody (ASMA), actin, platelets, signal recognition particle (SRP), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), myosin, sperm, amylase. Alpha 2, type XVII collagen (col17), kallikrein 13, type VII collagen (col7), myeloperoxidase (MPO), type IV collagen, proteinase 3 (PR3), thyrotropin receptor (TSHR), thyroglobulin, thyroid peroxidase (TPO), thyroglobulin, thyroid peroxidase (TPO), platelets, myeloperoxidase (MPO), muscle nicotinic acetylcholine receptor, muscle specific kinase (MuSK), low density lipoprotein receptor protein 4 (LRP4), myo Syn, beta 1 adrenergic receptor, adenine nucleotide translocase, aquaporin-4, myelin oligodendrocyte glycoprotein (MOG), heat shock protein 90 (HSP90), heat shock protein A5 (HSPA5), desmoglein-3, parietal cells, mitochondria, phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain containing 7A (THSD7A), cyclic citrullinated protein, RNA binding protein (Ros), La, double-stranded DNA (dsDNA), angiotensin II type 1 receptor (AT1R), endothelin-1 type A receptor (ETAR), insulin, glutamic acid decarboxylase, protein tyrosine phosphatase, and proprotein convertase subtilisin kexin type 9 (PCSK9).

[0197] In some embodiments, A G is a receptor or a fragment thereof. G A may be a full-length receptor, a receptor fragment, or a functional variant thereof. In some embodiments, A GA is a cell surface receptor, or a fragment thereof. Exemplary receptors include epidermal growth factor receptor, low density lipoprotein receptor (LDLR), integrins, immune receptors (e.g., Toll-like receptors, T cell receptors), fibroblast growth factor receptors, insulin receptors, receptor tyrosine kinases, olfactory receptors, adrenergic receptors, or ephrin receptors. In one embodiment, A G comprises LDLR or a fragment thereof.

[0198] A G binds to an extracellular target, which may be a soluble (e.g., plasma) protein, a membrane-associated protein, or a lipoprotein. In some embodiments, the extracellular target molecule is an antibody, a lipoprotein, a plasma membrane protein, a capsid, a virus, a soluble receptor, a secreted protein, a growth factor, a cytokine, a chemokine, a hormone, a neurotransmitter, an exosome, a cell, or a combination thereof. In some embodiments, the extracellular target is a soluble protein, or a component thereof (e.g., a protein modification, e.g., a sugar). The soluble protein may be an antibody, a soluble receptor, a secreted protein, a growth factor, a cytokine, a hormone, a neurotransmitter, or an enzyme. The soluble protein may be a monomer, or an oligomer (e.g., a dimer, a trimer, a tetramer, or a higher order species). In some embodiments, the soluble protein may include a post-translational modification, such as a sugar (e.g., a monosaccharide, an oligosaccharide, a phosphate group, a lipid, or an additional amino acid). In some embodiments, the soluble protein can be found in blood, lymph, or adipose tissue. Exemplary soluble proteins include proprotein convertase subtilisin / kexin type 9 (PPCSK9), complement factor H-related protein 3 (CFHR3), MICA (MHC class I chain-related gene A), and apolipoprotein-B (Apo-B).

[0199] In some embodiments, the extracellular target is a membrane-associated protein, or a component thereof (e.g., a protein modification, e.g., a sugar). A membrane-associated protein may be covalently or non-covalently associated with a cell membrane, e.g., a transmembrane protein or a membrane-anchored protein. In some embodiments, a membrane-anchored protein comprises a transmembrane domain (e.g., an alpha helix, or a loop). In some embodiments, a membrane-anchored protein comprises a channel or a pore. In some embodiments, the membrane-associated protein is selected from a type I, type II, or multi-pass membrane protein, or a glycosylphosphatidylinositol (GPI)-anchored membrane-associated protein, e.g., a receptor, a protein channel, or an ion channel. Exemplary membrane-associated proteins include: TNFR1 (tumor necrosis factor receptor 1), IL1R (interleukin 1 receptor), EGFR (epidermal growth factor receptor), CXCR4 (CXC chemokine receptor type 4), TSP1 (thrombospondin 1), NKG2D (encoded by KLRK1), uPAR (urokinase plasminogen activator receptor), GFRAL, IL4R, thymic stromal lymphopoietin receptor, IL7R, TGFβ receptor, PD-L1, or sToll receptor.

[0200] In some embodiments, the extracellular target is a lipoprotein. Lipoproteins can include lipids and proteins and can include various plasma lipoprotein particles. Exemplary lipoproteins include lipoprotein(a), low density lipoprotein (LDL), chylomicron, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and high density lipoprotein (HDL).

[0201] In some embodiments, the extracellular target is a pathogenic target (i.e., a target associated with (e.g., causing) a harmful or undesirable effect in a cell or subject). The pathogenic target may be associated with a particular disease, disorder, condition, or clinical situation, or a symptom thereof. In some embodiments, the pathogenic target is an extracellular secreted protein, e.g., a soluble protein (PCSK9), an extracellular lipoprotein (e.g., LDLR), an infectious agent (virus, bacteria, or parasite). In some embodiments, the pathogenic target is a pathogenic autoantibody. In some embodiments, the pathogenic target is an anti-drug antibody. In some embodiments, the pathogenic target is a membrane-associated protein (e.g., TSP1, uPAR). In some embodiments, the pathogenic target is a cell surface receptor, such as TNF receptor 1 (TNFR1), interleukin-1 receptor (IL1R), PD-L1, epidermal growth factor receptor (EGFR), CXCR4, NKG2D, GFRAL, IL4R, thymic stromal lymphopoietin receptor, IL-7R, TGFβ receptor, or sToll receptor. In some embodiments, the pathogenic target is a neurological target, such as tau protein or aggregates; or an immuno-oncological target, such as progranulin. In some embodiments, the pathogenic target is a toxin or drug (e.g., an antagonist, e.g., any agent used to reverse the deleterious effects of a previously administered drug or treatment, e.g., a sedative or narcotic).

[0202] Receptor binding moiety (R G ) Receptor binding moiety (R G ) is a moiety that binds to a membrane-bound receptor associated with a degradative pathway.

[0203] In some embodiments, R G comprises a small molecule, a carbohydrate or carbohydrate derivative, an antibody molecule, a peptide, or a pharma- ceutically acceptable salt thereof. G In some embodiments, R G comprises a carbohydrate or carbohydrate derivative. In some embodiments, R Gcomprises an antibody molecule. In some embodiments, R G contains peptides. In some implementations, R G comprises a hexasaccharide moiety (e.g., a glucose, galactose, mannose, N-acetylglucose, N-acetylgalactose, N-acetylmannose, mannose-6-phosphate, or mannose-6-phosphonate moiety). G comprises multiple hexasaccharide moieties (e.g., multiple glucose, galactose, mannose, N-acetylglucose, N-acetylgalactose, N-acetylmannose, mannose-6-phosphate, or mannose-6-phosphonate moieties). G contains one or more N-acetylgalactose (GalNAc) moieties, e.g., contains at least 1, 2, 3, 4, 5, or 6 GalNAc moieties. In some embodiments, R G contains three GalNAc moieties.

[0204] In some embodiments, R G R comprises a binding moiety for the asialoglycoprotein receptor (ASGPR). ASGPR is a C-type lectin expressed on the surface of hepatocytes that regulates the levels of plasma glycoproteins that terminate with galactose (Gal) or N-acetylgalactosamine (GalNAc) sugars. ASGPR binds to glycoproteins that terminate with galactose (Gal) or N-acetylgalactosamine (GalNAc) sugars and is internalized primarily via receptor-mediated endocytosis in coated pits on the basolateral membrane of hepatocytes. Upon internalization, the ligand-receptor complex is transported to an intralysosomal compartment. Calcium sequestration and subsequent acidification of the endosomal compartment promotes dissociation of the ligand-receptor complex, and the receptor is recycled back to the plasma membrane while the cargo (ligand) is sorted to the lysosome for degradation. In some embodiments, the binding moiety for the ASGPR comprises a carbohydrate or carbohydrate derivative, a small molecule, an antibody molecule or fragment thereof, a peptide, or a pharma- ceutical acceptable salt thereof (e.g., R Gcomprises Gal, GalNAc, a small molecule, an antibody molecule or fragment thereof, a peptide, or a pharma- ceutically acceptable salt thereof). In some embodiments, the binding moiety to the ASGPR comprises a galactose (Gal) or an N-acetylgalactosamine (GalNAc) sugar. In some embodiments, the binding moiety to the ASGPR is a GalNAc trimer, or a cross-linked GalNAc trimer and monomer (e.g., R G comprises a GalNAc trimer, or a cross-linked GalNAc trimer and monomer). In some embodiments, R G comprises a GalNAc trimer or a cross-linked GalNAc trimer.

[0205] The asialoglycoprotein receptor (ASGPR) has the ability to efficiently support the delivery of glycoproteins terminated with galactose (Gal) or N-acetylgalactosamine (GalNAc) sugars to lysosomes for degradation of extracellular target molecules (e.g., growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells), which are utilized by the A-glycosylation of the bifunctional compounds described herein. G The receptor binding portion of the bifunctional compound (R G ) contains one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups. G ) binds to the asialoglycoprotein receptor (ASGPR), thereby delivering extracellular target molecules to lysosomes for degradation via lysosomal degradation.

[0206] In some embodiments, R G teeth, [ka] [ka] (In the formula, R GThe * indicates the attachment point to the linker (L). The compound includes a moiety selected from

[0207] In some embodiments, R G comprises a galactose (Gal) group or an N-acetylgalactose (GalNAc) group, where the Gal or GalNAc group comprises a bridged ketal moiety. G teeth, [ka] (In the formula, R G The * indicates the attachment point to the linker (L). The compound includes a moiety selected from

[0208] In some embodiments, R Gcontains binding moieties to the mannose-6-phosphate receptor (M6PR) or the insulin-like growth factor 2 receptor (IGF2R). Mannose-6-phosphate (M6P) groups are added to the N-linked oligosaccharides of lysosomal hydrolases as they translocate through the cis-Golgi network. The M6P groups can then be recognized by two independent transmembrane M6P receptors (MPRs) present in the trans-Golgi network: the cation-independent M6P receptor (CI-MPR, also known as insulin-like growth factor 2 receptor (IGF2R)) and / or the cation-dependent M6P receptor (CD-MPR, also known as M6PR). In the trans-Golgi network, the M6P receptors can bind to the M6P groups on tagged lysosomal hydrolases at pH 6.5-6.7 and then help package the hydrolases into transport vesicles for delivery to their late endosomes. The cation-independent M6P receptor (CI-MPR, also known as insulin-like growth factor 2 receptor (IGF2R)) is also present on the cell surface, where it can bind lysosomal enzymes that have escaped the cell and deliver them to late endosomes. Once inside the endosome, which is normally at pH 6, lysosomal hydrolases dissociate from the MPR, and during maturation of the endosome into a lysosome, the pH drops to pH 5, and the hydrolases begin to digest plasma membrane-invaginated material delivered from the early endosome. The MPR can then be recycled from the endosome to the cell surface and then back to the Golgi complex.

[0209] In some embodiments, R G comprises a binding moiety to M6PR, the binding moiety comprising a small molecule, a carbohydrate or carbohydrate derivative, an antibody molecule or fragment thereof, a peptide, or a pharma- ceutically acceptable salt thereof. Gcomprises a binding moiety to M6PR, the binding moiety comprising a saccharide (e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, or hexasaccharide (e.g., galactose (Gal), N-acetylgalactosamine (GalNAc), cross-linked GalNAc, mannose-6-phosphate (M6P), mannose-6-phosphate, phosphonate dimer or polymer), an antibody molecule or fragment thereof, a peptide, or a pharma- ceutically acceptable salt thereof (e.g., R G includes a small molecule, a sugar (eg, a hexasaccharide), a phosphonic acid dimer or polymer, an antibody molecule or fragment thereof, a peptide, or a pharma- ceutically acceptable salt thereof.

[0210] With the ability to efficiently aid in the delivery of M6P-tagged proteins to lysosomes, M6PR and / or IGF2R can be utilized for the degradation of extracellular target molecules (e.g., growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells), which can be mediated by the A of the bifunctional compounds described herein. G The bifunctional compound binds to the receptor-binding portion (R G ) comprises a high affinity ligand for M6PR and / or IGF2R. Such receptor binding moieties (R G ) group binds to M6PR or IGF2R, thereby delivering the extracellular target molecule to the lysosome and degrading it via lysosomal degradation.

[0211] In some embodiments, R G teeth, [ka] [ka] (In the formula, R G The * indicates the attachment point to the linker (L). The compound includes a moiety selected from

[0212] In some embodiments, R G is the formula A(A): [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 are -CN, -CH2-CN, -C≡CH, -CH2-N3, -CH2-NH2, -CH2-N(R 4 )-S(O)2-R 5 , -CH2-CO2H, -CO2H, -CH2-OH, -CH2-SH, -CH=CH-R 5 , -CH2-R 5 , -CH2-SR 5 , -CH2-N(R 4 )-R 5 , -CH2-N(R 4 )-C(O)-R 5 , -CH2-N(R 4 )-C(O)-OR 5 , -CH2-N(R 4 )-C(O)-N(R 4 )-R 5 , -CH2-OR 5 , -CH2-OC(O)-R 5 , -CH2-OC(O)-N(R 4 )-R 5 , -CH2-OC(O)-OR 5 , CH2-S(O)-R 5 , -CH2-S(O)2-R 5 , -CH2-S(O)2-N(R 4 )-R 5 , -C(O)-NH2, -C(O)-OR 5 , -C(O)-N(R 4 )-R 5 or aryl or heteroaryl, the aryl or heteroaryl being R 5 or optionally replaced by or R 1is -ZXY, where X is a linker or a drug delivery system, Y is absent or a ligand selected from the group consisting of a small molecule, an amino acid sequence, a nucleic acid sequence, an antibody, an oligomer, a polymer, a gene-derived material, a liposome, a nanoparticle, a dye, a fluorescent probe, or a combination thereof, and Z is absent or is -C≡C-, -CH═CH-, -CH-, -CH-O-, -C(O)-N(R 4 )-, -CH2-S-, -CH2-S(O)-, -CH2-S(O)2-, -CH2-S(O)2-N(R 4 )-, -C(O)-O-, -CH2-N(R 4 )-, -CH2-N(R 4 )-C(O)-, -CH2-N(R 4 )-S(O)2-, -CH2-N(R 4 )-C(O)-O-, -CH2-N(R 4 )-C(O)-N(R 4 )-, -CH2-OC(O)-, -CH2-OC(O)-N(R 4 )-, -CH2-OC(O)-O-, or aryl or heteroaryl, wherein the aryl or heteroaryl is R 5 is optionally substituted with; R 2 -OH, -N3, -N(R 3 )2, -N(R 3 )-C(O)-R 3 , -N(R 3 )-C(O)-N(R 3 )2, -N(R 3 )-C(O)-OR 3 , tetrazole, or triazole, and the tetrazole and triazole are R 3 Optionally substituted with R 1 is -CH2-OH, R 2 -N3, -N(R 3 )2, -N(R 3 )-C(O)-R 3 , -N(R 3 )-C(O)-N(R 3 )2, -N(R 3 )-C(O)-OR 3, tetrazole, or triazole, and the tetrazole and triazole are R 3 is optionally substituted with; Each R 3 are independently -H, -(C1-C5)alkyl, halo-substituted (C1-C5)alkyl, or (C3-C6)cycloalkyl, and the -C2- group of the alkyl or cycloalkyl is selected from -O-, -S-, and -N(R 4 )-, and the -CH3 of the alkyl may be replaced by a heteroatom group selected from -N(R 4 )2, -OR 4 , and -S(R 4 ), which are separated by at least two carbon atoms; Each R 4 are independently -H, -(C1 to C 20 )alkyl, or (C3-C6)cycloalkyl, in which 1 to 6 -CH2- groups of said alkyl or cycloalkyl separated by at least 2 carbon atoms are -O-, -S-, or -N(R 4 )-, and the -CH3 of this alkyl can be replaced by -N(R 4 )2, -OR 4 , and -S(R 4 wherein the heteroatom groups are separated by at least two carbon atoms; the alkyl and cycloalkyl may be substituted with 1 to 6 halo atoms; and Each R 5 are independently -H, (C3 to C 20 ) cycloalkyl, or (C1-C 20 ) alkyl, and 1 to 6 -CH2- groups of this alkyl or cycloalkyl separated by at least 2 carbon atoms are -O-, -S-, or -N(R 4 )-, and the -CH3 of this alkyl can be replaced by -N(R 4 )2, -OR 4 , and -S(R 4), which heteroatom groups are separated by at least two carbon atoms; and the alkyl and cycloalkyl can be substituted with 1 to 6 halo atoms.

[0213] In some embodiments, R G teeth, [ka] (wherein n is 1, 2, or 3); [ka] (n is 2 or 3) Selected from; R G is attached to L at the terminal amine.

[0214] In some embodiments, R G is any compound or moiety described in U.S. Pat. Nos. 9,617,293 and 10,039,778, each of which is incorporated herein by reference in its entirety.

[0215] Linker (L) The linker moiety (L) is G ) and a receptor binding moiety (R G ) is an optional moiety within the bifunctional compounds described herein that links the linker moiety to the aryl group. In some embodiments, the linker moiety is absent. In some embodiments, the linker moiety is present.

[0216] In some embodiments, the linker is (G4S) nlinker, where n is an integer between 1 and 20 (see, e.g., Hust, M., et al. Single chain Fab (scFab) fragment. BMC Biotechnol 7, 14 (2007); Koerber JT, et al. An improved single-chain Fab platform for efficient display and recombinant expression. J Mol Biol. 427(2):576-586 (2015)). In some embodiments, n is an integer between 1 and 4 (SEQ ID NO: 42). In some embodiments, the linker is a G4S linker (SEQ ID NO: 43). In some embodiments, the linker is a (G4S)2 linker (SEQ ID NO: 44). In some embodiments, the linker is a (G4S)3 linker (SEQ ID NO: 45). In some embodiments, the linker is a (G4S)4 linker (SEQ ID NO: 46).

[0217] In one embodiment, the linker moiety has the formula (LI): [ka] or a pharma- ceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof; During the ceremony, L 1 is a bond, O, NR', C(O), C 1~9 Alkylene, C 1~9 Heteroalkylene, *C(O)-C 1~6 Alkylene, *C(O)-C 1~6 Heteroalkylene, *C 1~6 Alkylene-C(O), and *C 1~6 heteroalkylene-C(O), * is selected from the group consisting of L in formula (I). 1 means a point of attachment to a targeting ligand; X 1 and X 2 are each independently selected from the group consisting of a bond, carbocyclyl, heterocyclyl, and heteroaryl; L 2 is a bond, O, NR', C(O), C 1~6 Alkylene, C 1~6 Heteroalkylene, and *C(O)NR'-C 1~6 alkylene; * is selected from the group consisting of L 2 X 2 means a point of attachment to; or X 1 -L 2 -X 2 forms a spiroheterocyclyl; L 3 is a bond, C 1~6 Alkylene, C 2~6 Alkenylene, C 2~6 Alkynylene, C 1~6 Heteroalkylene, C(O), S(O)2, O, NR', *C(O)-C 1~9 Alkylene, *C(O)-C 1~6 Alkylene-O, and *C(O)-C 1~9 heteroalkylene; * is selected from L 3 X in (LI) 2 means the attachment point to; L 1 , X 1 , X 2 , L 2 , and L 3 may be simultaneously bonded; and R' is hydrogen or C 1~6 It is an alkyl.

[0218] In one embodiment, L 3 is a bond, -O-, -C(O)-, -S(O)2-, C 1~6 Alkylene, C 2~6 Alkynylene, and C 1~6 In one embodiment, X is selected from the group consisting of heteroalkylene. 1 and X 2 In one embodiment, one of X is not a bond. 1 and X 2 One of is a bond and the other is carbocyclyl or heterocyclyl.

[0219] In one embodiment, X 1 and X 2 One of X is a bond and the other is heterocyclyl. 1 and X 2 are each independently selected from piperidinyl and piperazinyl. 1 and X 2 are both piperidinyl. 1 -L 2 -X 2 -teeth, [ka] In certain embodiments, the linker has the formula: [ka] or a pharma- ceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0220] In one embodiment, -X 1 -L 2 -X 2 -R a Structures substituted with 0 to 4 occurrences of [ka] and forming a spiroheterocyclyl having each R a is independently 1~6 Alkyl, C 1~6 Alkoxyl and C 1~6 hydroxyalkyl.

[0221] In one embodiment, -X 1 -L 2 -X 2 -R b Structures substituted with 0 to 4 occurrences of [ka] wherein Y is selected from CH, oxygen, and nitrogen; and each R b is independently 1~6 Alkyl, C 1~6 Alkoxyl and C 1~6 hydroxyalkyl.

[0222] In one embodiment, X 1 and X 2 Each is a bond. 3 are independently -C(O)-, C 2~6 Alkynylene or C 1~6 heteroalkylene; and L 1 is -C(O)-, C 1~8 Alkylene, C 1~8 Heteroalkylene, and *C 1~6 In one embodiment, L is alkylene-C(O). 3 -C(O)-, -OC 1~6 Alkylene, C 2~6 Alkynylene, and C 1~6 heteroalkylene; and L 1 is C 1~8 Alkylene or C 1~8 In one embodiment, L is heteroalkylene. 3 is -C(O)- or C 1~6 heteroalkylene; and L 1 is C 1~8 Alkylene or C 1~8 In one embodiment, L is heteroalkylene. 3 is a bond or -O-; and L 1 is -C(O)- or C 1~8 In one embodiment, L is heteroalkylene. 3 is -O-, -C(O)-, -S(O)2-, and C 1~6 heteroalkylene; and L 1 is C 1~8 Alkylene or C 1~8 In one embodiment, L is heteroalkylene.2 is -C(O)-, -NR'-, or C 1~6 In one embodiment, L is an alkylene. 2 is -C(O)-, -O-, or C 1~6 In one embodiment, L is an alkylene. 2 is C 1~6 In one embodiment, L is an alkylene. 2 is -C(O)-, C 1~6 Alkylene, C 1~6 Heteroalkylene, and *C(O)NR'-C 1~6 In one embodiment, Y is selected from the group consisting of CH, CH(C 1~3 alkyl), C(C 1~3 alkyl), oxygen, NH, or N(C 1~3 alkyl).

[0223] In some embodiments, the linker comprises a peptide. In some embodiments, the peptide sequence is composed of naturally occurring amino acids. In some embodiments, the peptide sequence comprises at least one synthetically derived amino acid, e.g., at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 15, at least 20, or more synthetically derived amino acids. In some embodiments, the peptide has a linear structure. In some embodiments, the peptide has a branched structure. In some embodiments, the peptide has a branched structure, e.g., with at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 branch points. In some embodiments, the peptide has a cyclic structure.

[0224] In some embodiments, the linker comprises a peptide, and the peptide sequence comprises at least 2 amino acid residues. In some embodiments, the peptide sequence comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid residues. In some embodiments, the peptide sequence is about 1 to about 10 amino acid residues. In some embodiments, the peptide sequence is about 1 to about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acid residues. In some embodiments, the peptide sequence is about 10 to about 100 amino acid residues. In some embodiments, the peptide sequence is about 25 to about 100 amino acid residues. In some embodiments, the peptide sequence is about 50 to about 100 amino acid residues.

[0225] In some embodiments, the linker comprises an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkylene, carbonyl, ester, carbonate, amine, amide, carbamate, urea, hydroxyl, aryl ring, heteroaryl ring, cycloalkyl ring, or heterocyclyl ring. In some embodiments, the linker comprises a heteroalkylene moiety. Exemplary heteroalkylene moieties include polyethylene oxide (PEO), polypropylene glycol (PPG), polyglycerin (PG), polyoxamine (POX), polybutylene oxide (PBO), polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-glycolic acid) (PLGA), polycaprolactone (PCL), polydioxanone (PDO), polyanhydrides, polyacrylides, polyvinyls, polyorthoesters, dextran, cyclodextran, chitosan, or other carbohydrate-based polymers. In some embodiments, the heteroalkylene moiety contains at least 6, 8, 10, 12, 16, 20, 24, 36, 48, 60, 100, 250, 300, or more carbon atoms. In some embodiments, the heteroalkylene moiety is 4 to 48 carbon atoms, or 4 to 36 carbon atoms, or 4 to 24 carbon atoms, or 4 to 16 carbon atoms, or 6 to 12 carbon atoms, or 8 to 12 carbon atoms.

[0226] In some embodiments, the linker comprises a polyethylene glycol moiety (i.e., "PEG"). Polyethylene glycol includes ethylene glycol polymers containing various numbers of linked monomers, such as PEG1, PEG2, PEG3, PEG4, PEG6, PEG8, PEG10, PEG12, PEG14, PEG16, PEG18, PEG20, PEG30, PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000, or PEG2000000, and any mixtures thereof. In some embodiments, the linker comprises a polyethylene glycol moiety having 4 to 48 carbon atoms, or 4 to 36 carbon atoms, or 4 to 24 carbon atoms, or 4 to 16 carbon atoms, or 6 to 12 carbon atoms, or 8 to 12 carbon atoms.

[0227] The linkers of the bifunctional compounds described herein, or components thereof, can be readily formed by reaction between two reactive groups. Non-limiting examples of such chemical moieties are set forth in Table 2. In some embodiments, the linkers are prepared by reaction of two or more reactive groups described in Table 2. In some embodiments, the linkers comprise chemical moieties described in Table 2.

[0228] [Table 2]

[0229] [Table 3]

[0230] [Table 4]

[0231] [Table 5]

[0232] [Table 6]

[0233] In the formula, R in Table 2 32 , H, C 1~4 R in Table 2 is alkyl, phenyl, pyrimidine, or pyridine. 35 , H, C 1~6 Alkyl, phenyl, or C substituted with 1 to 3 -OH groups 1~4 each R7 in Table 2 is H, C 1~6 Alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1~4 R in Table 2 is independently selected from alkoxy, and C1-4 alkyl substituted with -C(=O)OH; 37 is independently selected from H, phenyl, and pyridine; q in Table 2 is 0, 1, 2, or 3.

[0234] In some embodiments, the linker comprises a moiety described in Table 3.

[0235] [Table 7]

[0236] [Table 8]

[0237] In some embodiments, the linker is selected from the following: *-(CH2)nC(=O)NHNHC(=O)(CH2)nON=CH2X1C(=O)-**; *-(CH2)nX3C(=O)-**; *-(CH2)nC(=O)-**; *-(CH2)nC(=O)NHNHC(=O)(CH2)nON=CH2X1C(=O)NH(CH2)nCH(C=(O)NH2)-**; *-(CH2)nX3C(=O)NH(CH2)nCH(C=(O)NH2)-**; *-(CH2)nC(=O)NH(CH2)nCH(C=(O)NH2)-**, *-((CH2)nO)t(CH2)mC(=O)-**; *-((CH2)nO)t(CH2)m-**; *-(CH2)nC(=O)NH((CH2)nO)t(CH2)m-**; *-(CH2)n-;*-(CH2)nNHC(=O)(CH2)m-**; *-(CH2)nNHC(=O)(CH2)nC(=O)NH(CH2)m-**; *-((CH2)nO)t(CH2)nNHC(=O)(CH2)m-**; *-((CH2)nO)tCH2)mC(=O)NH(CH2)m-**; *((CH2)nO)t(CH2)nNHC(=O)(CH2)m-**; *-(CH2)nO(CH2)m-**; *-(CH2)nNH(CH2)n-**; *-(CH2)nNH(CH2)mC(=O)-**; *-(CH2)nX3(CH2)m-**; *-((CH2)nO)t(CH2)nX3(CH2)m-**; *-(CH2)nNHC(=O)(CH2)nX3(CH2)m-**; *-((CH2)nO)t(CH2)nNHC(=O)(CH2)nX3(CH2)m-**; *-((CH2)nO)t(CH2)nC(=O)NH(CH2)m-**; *-(CH2)mNHC(=O)((CH2)nO)t(CH2)m-**; *-(CH2)nC(=O)NH(CH2)m-**; *-(CH2)nNHC(=O)((CH2)nO)t(CH2)m-**; *-(CH2)nNHC(=O)(CH2)nO(CH2)m-**; *-(CH2)nNH(CH2)m-**; *-((CH2)nO)tCH2)nC(=O)NH(CH2)m-**; *-(CH2)nNHC(=O(CH2)nX3(CH2)m-**; *-C(=O)-; *-C(=O)(CH2)nC(=O)-**; *-C(=O)((CH2)nO)t(CH2)mC(=O)-**; *-C(=O)((CH2)nO)t(CH2)m-**; *-((CH2)nO)t(CH2)mX3(CH2)nO(CH2)nNHC(=O)((CH2)nO)t(CH2)mC(=O)-**, *-C(=O)(CH2)nC(=O)NH((CH2)nO)t(CH2)m-**; *-C(=O)NHNHC(=O)(CH2)nON=CH2X1C(=O)NH(CH2)nCH(C=(O)NH2)-**; *-X3C(=O)NH(CH2)nCH(C=(O)NH2)-**; *-C(=O)(CH2)nC(=O)NHNHC(=O)(CH2)nON=CH2X1C(=O)-**; *-C(=O)(CH2)nX3C(=O)-**; *-C(=O)(CH2)nNHC(=O)(CH2)nC(=O)NH(CH2)m-**; *-C(=O)((CH2)nO)t(CH2)nNHC(=O)(CH2)m-**; *-C(=O)((CH2)nO)tCH2)mC(=O)NH(CH2)m-**; *-C(=O)(CH2)nO(CH2)m-**; *-C(=O)(CH2)n-**; *-C(=O)NH((CH2)nO)t(CH2)m-**; *-C(=O)(CH2)nNH(CH2)n-**; *-C(=O)(CH2)nNH(CH2)mC(=O)-**; *-C(=O)(CH2)nX3(CH2)m-**; *-C(=O)((CH2)nO)t(CH2)nX3(CH2)m-**; *-C(=O)(CH2)nNHC(=O)(CH2)m-**; *-C(=O)(CH2)nNHC(=O)((CH2)nO)t(CH2)m-**; *-C(=O)(CH2)nNHC(=O)(CH2)nO(CH2)m-**; *-C(=O)(CH2)nNH(CH2)m-**; *-C(=O)((CH2)nO)tCH2)nC(=O)NH(CH2)m-**; *-C(=O)(CH2)nNHC(=O(CH2)nX3(CH2)m-**; *-C(=O)NH(CH2)nX3(CH2)m-**; *-C(=O)NH(CH2)nNHC(=O)(CH2)m-**; *-C(=O)NH(CH2)nNHC(=O)(CH2)nO(CH2)m-**; *-C(=O)NH(CH2)nNHC(=O)(CH2)nX3(CH2)m-**; *-C(=O)NH(CH2)nNHC(=O)-**; *-C(=O)NH((CH2)nO)t(CH2)nX3(CH2)m-**; *-C(=O)(CH2)nNHC(=O)(CH2)nX3(CH2)m-**; *-C(=O)((CH2)nO)t(CH2)nNHC(=O)(CH2)nX3(CH2)m-**; *-C(=O)((CH2)nO)t(CH2)nC(=O)NH(CH2)m-**; *-C(=O)(CH2)mNHC(=O)((CH2)nO)t(CH2)m-**, or *-C(=O)(CH2)nC(=O)NH(CH2)m-** (wherein, X1 is, [ka] and X3 is [ka] For X3, * is R G indicates the point of attachment to A G For all other species, * indicates the point of attachment to R G indicates the attachment point to A G (showing the attachment points for

[0238] In some embodiments, the linker is selected from the following: *-(CH2)nC(=O)NHNHC(=O)(CH2)nON=CH2X1C(=O)-**; *-(CH2)nX3C(=O)-**; *-((CH2)nO)t(CH2)mC(=O)-**; *-(CH2)nC(=O)-**; *-(CH2)nC(=O)NHNHC(=O)(CH2)nON=CH2X1C(=O)NH(CH2)nCH(C=(O)NH2)-**;*-(CH2)nX3C(=O)NH(CH2)nCH(C=(O)NH2)-**; *-C(=O)-; *-C(=O)(CH2)nC(=O)-**; *-C(=O)((CH2)nO)t(CH2)mC(=O)-**; *-((CH2)nO)t(CH2)mX3(CH2)nO(CH2)nNHC(=O)((CH2)nO)t(CH2)mC(=O)-**; *-C(=O)((CH2)nO)t(CH2)m-**; and *-C(=O)(CH2)nC(=O)NH((CH2)nO)t(CH2)m-** (In the formula, X1 is, [ka] and X3 is [ka] For X3, * is R G indicates the point of attachment to A G For all other species, * indicates the point of attachment to R G indicates the attachment point to A G (showing the attachment points for

[0239] In some embodiments, the linker is not a peptide. In some embodiments, the linker is a peptide, but does not include an amino acid residue selected from alanine, glycine, serine, or threonine. In some embodiments, the linker is an unmodified peptide (e.g., a peptide that does not include modifications, such as chemical modifications). In some embodiments, the linker is not a glycopeptide.

[0240] In some embodiments, the linker has formula (B): [ka] (In the formula, R 3 does not include a structure that is a carbohydrate or carbohydrate derivative (e.g., a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide, each of which is optionally substituted). In some embodiments, -CH3 is in the (L) or (D) configuration.

[0241] In some embodiments, the linker does not include an aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some embodiments, the linker does not include a heteroaryl or heterocyclyl (e.g., a nitrogen-containing heteroaryl or a nitrogen-containing heterocyclyl). In some embodiments, the linker does not include a nitrogen-containing heteroaryl (e.g., a triazolyl) or a nitrogen-containing heterocyclyl. In some embodiments, the linker has the formula (C): [ka] (In the formula, R 10 does not contain a structure that is a carbohydrate or carbohydrate derivative moiety (e.g., a monosaccharide, disaccharide, oligosaccharide, or polysaccharide, each of which is optionally substituted). 10 contains a glucose, galactose, mannose, glucosamine, galactosamine, or mannosamine moiety.

[0242] In some embodiments, the linker does not include a compound or moiety disclosed in WO 2019 / 199621, which is incorporated by reference in its entirety.

[0243] In some embodiments, the bifunctional compound of formula (I) is a bispecific antibody. In some embodiments, the bifunctional compound of formula (I) is a bispecific antibody, wherein R G comprises an anti-ASGPR antibody or a fragment thereof. In some embodiments, the bifunctional compound of formula (I) is an anti-ASGPPR-PCSK9 bispecific antibody. In certain embodiments, G comprises an antibody or fragment thereof (anti-PCSK9 antibody or fragment thereof), L is absent, and R G is an antibody or fragment thereof (e.g., an anti-ASGPR antibody or fragment thereof). In certain embodiments, the bifunctional compound comprises an antibody as described in Example 2 and, for example, in Figures 1-3.

[0244] In some embodiments, the bifunctional compound of formula (I) comprises an anti-PCSK9 antibody (e.g., a modified anti-PCSK9 antibody). G comprises an anti-PCSK9 antibody, L comprises a polyethylene glycol moiety (e.g., a PEG, PEG, or PEG moiety), and R G comprises a GalNAc moiety (e.g., multiple GalNAc moieties, e.g., 2, 3, or 4 GalNAc moieties). In some embodiments, a bifunctional compound of formula (I) comprises a GalNAc-tagged anti-PCK9 antibody (e.g., depicted as tri-GalNAc-functionalized PCK9 antibody 10 in FIG. 20B), e.g., as described in Examples 3 and 9-11 and FIGS. 5-6. In some embodiments, a bifunctional compound of formula (I) comprises a GalNAc-tagged anti-PCSK9 antibody, e.g., as depicted in FIG. 20C.

[0245] In some embodiments, the bifunctional compound of formula (I) is a bifunctional compound set forth in Table 4.

[0246] [Table 9]

[0247] [Table 10]

[0248] [Table 11]

[0249] [Table 12]

[0250] [Table 13]

[0251] [Table 14]

[0252] [Table 15]

[0253] [Table 16]

[0254] [Table 17]

[0255] Pharmaceutical Compositions, Kits, and Administration The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising a bifunctional compound described herein (e.g., a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof), and, optionally, a pharma- ceutically acceptable excipient. In certain embodiments, the pharmaceutical compositions described herein comprise a bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof, and, optionally, a pharma- ceutically acceptable excipient. In certain embodiments, in the pharmaceutical composition, the bifunctional compound of Formula (I) or a pharma- ceutically acceptable salt thereof is provided in an effective amount. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

[0256] The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology. In general, such methods of preparation include the step of bringing into association the bifunctional compound of formula (I) ("active ingredient") with the carrier and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single or multi-dose unit.

[0257] Pharmaceutical compositions can be prepared, packaged, and / or sold in a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition that contains a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage (e.g., one-half or one-third of such a dosage, etc.).

[0258] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

[0259] The term "pharmaceutical acceptable excipient" refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the bifunctional compound with which it is formulated. Pharmaceutically acceptable excipients useful in preparing pharmaceutical compositions of the invention are any of those known in the art of pharmaceutical formulation, including inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Pharmaceutically acceptable excipients useful in the preparation of pharmaceutical compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0260] Compositions of the present disclosure may be administered orally, parenterally (e.g., subcutaneously, intramuscularly, intravenously, and intradermally), by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, the bifunctional compounds or compositions thereof can be administered intravenously and / or orally.

[0261] The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intraperitoneal, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, subcutaneously, intraperitoneally, or intravenously. In some embodiments, the compositions described herein are administered by subcutaneous injection (e.g., depot subcutaneous injection). Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents (e.g., solutions in 1,3-butanediol). Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0262] The pharma- ceutically acceptable composition of the present disclosure may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also usually added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If necessary, certain sweeteners, flavorings or colorings may also be added. In some embodiments, the oral formulations provided are formulated for immediate release or sustained / delayed release.

[0263] In some embodiments, the compositions are suitable for buccal or subcutaneous administration (e.g., tablets, lozenges, and troches). Provided compounds may also be in microencapsulated form. Alternatively, the pharmaceutically acceptable compositions of the present disclosure may be administered in the form of suppositories for rectal administration. The pharmaceutically acceptable compositions of the present disclosure may also be administered topically, particularly when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0264] For ophthalmic use, the pharma- ceutically acceptable compositions provided may be formulated as a micronized suspension or may be formulated in an ointment such as petrolatum.

[0265] In order to prolong the effect of a drug, it is often desirable to delay the absorption of the drug by subcutaneous or intramuscular injection. This can be accomplished by using a suspension of crystalline or amorphous material with low water solubility. The rate of absorption of the drug then depends on its rate of dissolution, which may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle or hydrogel vehicle. In some embodiments, the compositions described herein are formulated for sustained release (e.g., depot formulations (e.g., subcutaneous depot injections)). Exemplary depot formulations may include excipients such as hydrogels, peptides, phospholipids, or polyethylene glycols.

[0266] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, one of skill in the art will appreciate that such compositions are generally suitable for administration to any animal. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to a variety of animals are well understood, and an ordinarily skilled veterinary pharmacologist may design and / or perform such modifications with routine experimentation.

[0267] The bifunctional compounds provided herein may be formulated in dosage unit form (e.g., single unit dosage form) for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on various factors, such as the severity of the disease and disorder being treated; the activity of the specific active ingredient being used; the specific composition being used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the specific active ingredient being used; the duration of treatment; the drugs used in combination or simultaneously with the specific active ingredient being used; and similar factors known in the medical field.

[0268] The exact amount of bifunctional compound required to achieve an effective dose will vary between subjects, depending on, for example, the species, age, and general health of the subject, the severity of side effects or disorders, the identity of the specific bifunctional compound, the mode of administration, and the like. The desired dosage can be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations).

[0269] In certain embodiments, an effective amount of a bifunctional compound for single or multiple daily administration to a 70 kg adult may include from about 0.0001 mg to about 3000 mg, from about 0.0001 mg to about 2000 mg, from about 0.0001 mg to about 1000 mg, from about 0.001 mg to about 1000 mg, from about 0.01 mg to about 1000 mg, from about 0.1 mg to about 1000 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 100 mg, from about 10 mg to about 1000 mg, or from about 100 mg to about 1000 mg of the compound per unit dosage form.

[0270] In certain embodiments, the bifunctional compound of Formula (I) may be at a dosage level sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, more preferably from about 1 mg / kg to about 25 mg / kg of the subject's body weight per day, one or more times per day, to obtain the desired therapeutic effect.

[0271] It will be understood that the dosage ranges described herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent may be determined by a physician or person skilled in the art and may be less than or equal to the amount administered to an adult.

[0272] It will also be understood that the bifunctional compounds or compositions thereof described herein may be administered in combination with one or more additional therapeutic agents. The bifunctional compounds or compositions thereof may be administered in combination with additional therapeutic agents that improve their bioavailability, reduce and / or alter their metabolism, inhibit their excretion, and / or alter their distribution in the body. It will also be understood that the treatments utilized may achieve the desired effect on the same disorder and / or may achieve different effects.

[0273] The bifunctional compound or composition thereof may be administered simultaneously with, prior to, or subsequent to one or more additional therapeutic agents that may be useful, for example, as a combination therapy. Therapeutic agents include therapeutically effective agents. Therapeutic agents also include prophylactically effective agents. Each additional therapeutic agent may be administered at a dose and / or time schedule determined for that therapeutic agent. These additional therapeutic agents may also be administered in a single dose, together with each other and / or with the bifunctional compound or composition thereof described herein, or may be administered separately in various doses. The particular combination employed in the regimen will take into consideration the compatibility of the compound of the present invention with the additional therapeutic agents and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, additional therapeutic agents utilized in combination are expected to be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower compared to the levels utilized individually.

[0274] Exemplary additional therapeutic agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-diabetic agents, anti-inflammatory agents, immunosuppressants, and analgesics. Therapeutic agents include small organic molecules, such as drug compounds (e.g., compounds approved by the U.S. Food and Drug Administration as defined in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules bound to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.

[0275] Also encompassed by the present disclosure are kits (e.g., pharmaceutical packs). The kits of the present invention may be useful, for example, for the prevention and / or treatment of diseases, disorders, conditions, or clinical situations described herein. The kits provided may include the pharmaceutical composition or bifunctional compound of the present invention and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the kits provided may optionally further include a second container containing a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound of the present invention. In some embodiments, the pharmaceutical composition or compound provided in this container and the second container are combined to form a unit dosage form.

[0276] Thus, in one aspect, provided is a kit comprising a first container containing a bifunctional compound described herein, or a pharma- ceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof. In certain embodiments, the kit of the present disclosure comprises a first container containing a bifunctional compound described herein, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the kit is useful in preventing and / or treating a disease, disorder, condition, or clinical situation described herein (e.g., a proliferative disease or a non-proliferative disease) in a subject. In certain embodiments, the kit further comprises instructions for administering the bifunctional compound, or a pharma- ceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof to a subject to prevent and / or treat a disease, disorder, condition, or clinical situation described herein.

[0277] How to use Described herein are bifunctional compounds useful for the degradation of extracellular targets. In some embodiments, the bifunctional compounds (e.g., bifunctional compounds of formula (I)) can be used to reduce or suppress the levels of extracellular targets by activating degradation pathways (e.g., receptor-mediated endocytosis, endosomal degradation, or lysosomal degradation). The bifunctional compounds can be useful, for example, as research tools and / or treatments for diseases, disorders, conditions, or clinical conditions described herein.

[0278] Conventional therapeutic agents (e.g., therapeutic agents directed against proteins) often produce the desired effect by interfering with the function of the protein (e.g., enzyme inhibition) or by recruiting immune effectors, as in the case of many monoclonal antibody drugs. Typically, due to the reversible nature of conventional drug / target interactions, the efficacy of such conventional therapies requires superstoichiometric drug concentrations to maintain inhibition that may be lost over time as drug concentration decreases. However, the methods described herein, including the use of bifunctional compounds for targeted protein degradation, may show improved efficacy at substoichiometric, stoichiometric, or superstoichiometric concentrations, where efficacy is limited by resynthesis of the target molecule (e.g., protein) rather than drug concentration. In certain embodiments, the methods described herein, including the use of bifunctional compounds, show improved efficacy at stoichiometric concentrations.

[0279] In one aspect, the disclosure features a method of targeting an extracellular target molecule (e.g., a soluble or membrane-bound protein in a cell) for degradation, the method including administering a bifunctional compound described herein (e.g., a bifunctional compound of Formula (I)). The method can be an in vitro method, an in vivo method, or an ex vivo method. In some embodiments, the method includes contacting a sample (e.g., a cell or tissue sample) with a bifunctional compound described herein. In some embodiments, the method includes administering to a subject a bifunctional compound described herein.

[0280] In some embodiments, the extracellular target molecule is associated with a disease, disorder, condition, or clinical condition in the subject. In some embodiments, the method includes modulating the level and / or activity of the extracellular target molecule in the subject in response to the bifunctional compound. In some embodiments, the method includes reducing the level and / or activity of the extracellular target molecule in the subject in response to administration of a bifunctional compound described herein compared to the level and / or activity of the extracellular target molecule in a subject not administered a bifunctional compound described herein. In some embodiments, the method includes reducing the level and / or activity of an extracellular target molecule in a subject by, for example, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., greater than about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) in response to administration of a bifunctional compound, e.g., compared to a baseline or the level and / or activity of the extracellular target molecule in a subject to which a bifunctional compound described herein has not been administered. In some embodiments, the method includes reducing a symptom associated with a disease, disorder, condition, or clinical condition in a subject in response to administration of a bifunctional compound described herein, compared to a baseline or the symptom associated with a disease, disorder, condition, or clinical condition in a subject to which a bifunctional compound described herein has not been administered. In some embodiments, the methods include reducing a symptom associated with a disease, disorder, condition, or clinical condition in a subject by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more in response to administration of a bifunctional compound, e.g., compared to a baseline or a symptom associated with a disease, disorder, condition, or clinical condition in a subject to which a bifunctional compound described herein has not been administered.

[0281] In some embodiments, the disease, disorder, condition, or clinical condition is associated with an increased level and / or activity of an extracellular target molecule (e.g., a soluble or membrane-bound protein, such as an antibody, lipoprotein, receptor, growth factor, cytokine, chemokine, enzyme, hormone, neurotransmitter, or a combination thereof). The level and / or activity of the extracellular target molecule may be increased, for example, by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, e.g., compared to a baseline. In some embodiments, the disease, disorder, condition, or clinical condition is associated with an increased level and / or activity of an extracellular target molecule, and administration of a bifunctional compound described herein returns the level of the extracellular target molecule to normal (e.g., healthy levels).

[0282] In some embodiments, the disease, disorder, condition, or clinical situation is a proliferative disease (e.g., cancer), a neurological disease (e.g., Alzheimer's disease, neurodegeneration), a cardiovascular disease, a respiratory disease, a skin disease, a blood disease, an inflammatory disease, a metabolic disorder, an infectious disease, or a renal disease or disorder. Exemplary diseases, disorders, conditions, and clinical situations include: acute inflammatory demyelinating polyneuropathy (Guillain-Barre syndrome), acute liver failure, anti-glomerular basement membrane disease (Goodpasture's syndrome), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), cutaneous T-cell lymphoma (CTCL), mycosis fungoides, Sézary syndrome, familial hypercholesterolemia, focal segmental glomerulosclerosis (FSGS), hereditary hemochromatosis, hyperviscosity in hypergammaglobulinemia, hyperviscosity in hypergammaglobulinemia, myasthenia gravis, N-methyl-D-aspartate receptor antagonist (NARA), and / or vasculitis. Laginate receptor antibody encephalitis, paraproteinaceous demyelinating neuropathy; chronic acquired demyelinating polyneuropathy, polycythemia vera; polycythemia; thrombotic microangiopathy, thrombotic thrombocytopenic purpura (TTP), vasculitis, fulminant Wilson's disease, dilated cardiomyopathy, graft-versus-host disease (GVHD), lipoprotein(a) hyperlipoproteinemia, multiple sclerosis, neuromyelitis optica spectrum disorder (NMOSD), childhood autoimmune neuropsychiatric disorders with streptococcal infections (PANDAS), Sydenham's chorea, peripheral vascular disease, sickle cell disease, voltage-gated potassium channel (VGKC) antibody-associated disease.

[0283] In some embodiments, the disease, disorder, condition, or clinical situation includes: cancer, Alzheimer's disease, atherosclerosis, arteriosclerosis, hyperlipidemia, familial hypercholesterolemia, familial chylomicronemia syndrome, inflammation, asthma, urticaria, IgA nephropathy, and membranous nephropathy.

[0284] In some embodiments, the disease, disorder, condition, or clinical situation involves the removal of pathogenic antibodies that can cause autoimmune diseases (e.g., membranous nephropathy, IgF nephropathy, myasthenia gravis, and allergies).

[0285] In some embodiments, the disease, disorder, condition, or clinical situation involves removal of anti-drug antibodies, e.g., clearance of the anti-drug antibodies allows for effective treatment.

[0286] In some embodiments, the disease, disorder, condition, or clinical situation comprises atherosclerosis and the target is low density lipoprotein, very low density lipoprotein, chylomicron, PCSK9, ANGPTL3, ANGPTL4, ANGPTL8. In some embodiments, the disease, disorder, condition, or clinical situation comprises a central nervous system disease (e.g., Alzheimer's disease, Parkinson's disease, muscular dystrophy) and the target is tau or TNFR1. In some embodiments, the disease, disorder, condition, or clinical situation comprises a respiratory disease (e.g., cancer) and the target is EGFR or MICA.

[0287] In some embodiments, the disease, disorder, condition, or clinical situation is caused by or associated with a pathogenic autoantibody (e.g., the presence of a pathogenic autoantibody). Exemplary diseases, disorders, conditions, or clinical situations associated with said pathogenic autoantibodies include: acquired thrombotic thrombocytopenic purpura, Addison's disease, anti-NMDA encephalitis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis, autoimmune idiopathic thrombocytopenia, autoimmune myopathy, autoimmune orchitis, autoimmune pancreatitis, bullous pemphigoid, dry eye, epidermolysis bullosa acquisita, eosinophilic granulomatosis with polyangiitis (EGPA). , Goodpasture's disease, granulomatosis with polyangiitis, Graves' disease, Hashimoto's thyroiditis, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura (ITP), microscopic polyangiitis (MPA), myasthenia gravis, myocarditis, neuromyelitis optica (NMO), ovarian failure, pemphigus, pernicious anemia, primary biliary cholangitis (PBC), primary membranous nephropathy, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus (SLE), systemic sclerosis, and type I diabetes.

[0288] In some embodiments, the methods described herein include reducing proprotein convertase subtilisin / kexin type 9 (PCSK9) levels in a subject by, for example, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, for example, compared to baseline or to PCSK9 levels in a subject not administered a bifunctional compound described herein. PCSK9 significantly affects plasma low-density lipoprotein cholesterol (LDL-C) levels through regulation of the hepatic low-density lipoprotein receptor (LDLR), the primary pathway by which cholesterol is removed from circulation. PCSK9 binds to LDLR and targets it for lysosomal degradation, thereby increasing plasma LDL-C levels and subsequently increasing coronary heart disease risk. (Maxwell KN, Proc. Natl. Acad. Sci., 101, 2004, 7100-7105; Park, SW, J. Biol. Chem. 279, 2004, 50630-50638; Lagace TA, et al. J. Clin. Invest. 2006, 116(11):2995-3005). Overexpression of mouse or human PCSK9 in mice has been shown to increase overall and LDL-C levels and dramatically reduce hepatic LDLR protein, with no observed effects on levels of mRNA, SREBP, or the nuclear-cytoplasmic ratio of SREBP protein. (Maxwell KN, Proc. Natl. Acad. Sci. 101, 2004, 7100-7105). Furthermore, mutations in PCSK9 that cause loss of PCSK9 function in mouse models have been shown to reduce total and LDL-C levels (Cohen, JC, et al., N. Engl. J. Med., 354, 2006, 1264-1272), thus indicating that modulation of PCSK9 leads to reduced LDLR protein levels.Exemplary diseases, disorders, conditions, and clinical situations associated with PCSK9 include hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral arterial disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or chylomicrons), elevated triglycerides, sepsis, and xanthomas.

[0289] In some embodiments, the methods described herein include reducing complement factor H-related protein 3 (FHR3) levels in a subject by, for example, about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, for example, compared to a baseline or FHR3 levels in a subject to which a bifunctional compound described herein has not been administered. Complement-mediated immune responses are tightly regulated by several endogenously produced proteins that regulate activity and distinguish between healthy self (non-activated) and damaged or non-self pathogenic activated cells. These complement regulatory proteins range from those that bind to cell surfaces (i.e., CR1, MCP, DAF) to circulating proteins (i.e., Factor H and C4BP) that are recruited to host self-surfaces by binding to polysaccharides such as glycosaminoglycans on self-surfaces and inactivate complement (Mol Immuno 47(13):2187-2197). Complement regulation is tightly controlled to maintain homeostasis, and its dysregulation and deficiency have been implicated in many diseases, as they cause it to target host cells.

[0290] Factor H (FH), the major negative regulator of alternative complement pathway activation, belongs to a family that also includes five other related family members that are believed to have arisen from non-allelic homologous recombination and interlocus gene conversion: complement factor H-related protein 1 (FHR1), complement factor H-related protein 2 (FHR2), complement factor H-related protein 3 (FHR3), complement factor H-related protein 4 with isoforms 4A and 4B (FHR4A and FHR4B), and complement factor H-related protein 5 (FHR5). FHR3, unlike factor H, lacks a complement regulatory domain essential for complement inactivation and also competes with factor H, resulting in excessive activation of complement. Thus, the present invention provides bifunctional compounds for use in regulating the concentration of complement factor H protein (specifically FHR3) that remove competitors for factor H, thereby restoring factor H-mediated regulation to treat disorders caused by excessive complement activation. Exemplary diseases, disorders, conditions, and clinical situations associated with FHR3 include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC). EXAMPLES

[0291] In order that the disclosure described herein may be more fully understood, the following examples are provided. The examples described in this application are provided to illustrate the bifunctional compounds, pharmaceutical compositions, and methods provided herein, and should not be construed in any way as limiting the scope thereof.

[0292] The bifunctional compounds provided herein can be prepared from readily available starting materials using modifications to the specific synthetic protocols shown below that would be known to one of ordinary skill in the art. Where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, it will be understood that other process conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the reactants or solvents used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures.

[0293] In addition, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The selection of a suitable protecting group for a particular functional group, as well as suitable conditions for protection and deprotection, are well known in the art. For example, numerous protecting groups and their introduction and removal are described in Greene et al., Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[0294] The reactants may be purified or analyzed according to any suitable method known in the art. For example, the formation of products may be confirmed by spectroscopic means, such as nuclear magnetic resonance (NMR) spectroscopy (e.g., 1 H or 13 C), infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry (MS)), or by chromatographic methods (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography (TLC)).

[0295] Abbreviations used in the examples below and elsewhere in the specification are set forth in Table 5.

[0296] [Table 18]

[0297] Example 1: Synthesis of exemplary ASGPR receptor ligands Synthesis of intermediates Type A: Synthesis of benzyl 5-hydroxypentanoate (int-A1) [ka] Step 1: To a solution of dihydro-2H-pyran-2,6(3H)-dione (20 g, 175.44 mmol) and BnOH (20.8 g, 192.98 mmol) in DCM (150 mL) at 0 °C, DMAP (0.32 g, 2.62 mmol) and Et3N (29 mL, 210.5 mmol) were added. The reaction mixture was allowed to warm to room temperature and then stirred for 2 days. The reaction mixture was evaporated to dryness and the resulting residue was dissolved in DCM (200 mL) and washed with 3M HCl (100 mL x 2). The organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (eluent: PE:EA = 20:1 to 10:1) to give 5-(benzyloxy)-5-oxopentanoic acid as a colorless oil. 1 H NMR(400MHz,CDCl3)δ ppm 7.38-7.28(m,5H),5.12(s,2H),2.46-2.40(m,4H),2.00-1.93(m,2H).

[0298] Step 2: To a solution of 5-(benzyloxy)-5-oxopentanoic acid (30 g, 135.1 mmol) in THF (200 mL) was added BH3-SMe2 (20.2 ml, 202.7 mmol) dropwise at 0 °C under N2 protection. The mixture was allowed to warm to room temperature and stirred for 16 h. TLC showed that the starting material was completely consumed. The reaction was carefully quenched with H2O (8 mL). The resulting mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography (eluent: PE:EA = 20:1 to 2:1) to give benzyl 5-hydroxypentanoate (int-A1). 1 H NMR(400MHz,CDCl3)δ ppm 7.41-7.33(m,5H),5.14(s,2H),3.66(t,2H,J=6Hz),2.43(t,2H,J=7.2Hz),1.80-1.73(m,2H),1.65-1.58(m,2H).

[0299] Type B: Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidin-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (int-B1) [ka] Step 1: To a solution of (2R,3R,4R,5R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride (100.0 g, 0.132 mol) in pyridine (1 L) was added acetic anhydride (473 g, 4.64 mol) at 0° C., and the reaction mixture was stirred at room temperature for 72 hours. The resulting precipitate was collected, washed with HO (200 mL×2), and dried in vacuum to obtain (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate. 1 H NMR(400MHz,CDCl3)δ ppm 5.68(d,1H,J=8.8Hz),5.46(d,1H,J=9.2Hz),5.35(d,1H,J=3.2Hz),5.07(dd,1H,J1=11.2Hz,J2=3.2Hz),4.47-4. 39(m,1H),4.18-4.07(m,2H),4.02-3.98(m,1H),2.16(s,3H),2.11(s,3H),2.03(s,3H),2.00(s,3H),1.93(s,3H).

[0300] Step 2: To a solution of (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (100 g, 0.257 mol) in 1,2-dichloroethane (500 mL) cooled to 0° C., TMSOTf (85.5 g, 0.385 mol) was added, the mixture was stirred for 10 min, then heated to 50° C. and stirred for 3 h. TLC showed that the starting material was completely consumed. After cooling, the resulting mixture was treated with saturated aqueous NaHCO3 solution (1000 mL) at 0° C. and extracted with DCM (500 mL×2). The combined organic layers were dried over Na2SO4 and concentrated. The residue was dried under high vacuum overnight to give (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetate. 1 H NMR(400MHz,CDCl3)δ ppm 6.00(d,1H,J=2.8Hz),5.47-5.46(m,1H),4.93-4.90(m,1H),4.27-4.18(m,2H) ,4.13-4.09(m,1H),4.02-3.98(m,1H),2.13(s,3H),2.07(s,6H),2.06(s,3H).

[0301] Step 3: (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetate (65 g, 197.4 mmol) and benzyl 5-hydroxypentanoate (int-A1) (41 g, 197.4 mmol) were dissolved in DCM (600 mL). Molecular sieves (50 g) were added and the reaction was stirred for 30 min, followed by TMSOTf (6.5 g, 29.6 mmol). The reaction mixture was then stirred overnight at room temperature. TLC showed that the starting material was completely consumed. The reaction mixture was filtered to remove the molecular sieves. The filtrate was treated with saturated aqueous NaHCO3 (500 ml) and extracted with DCM (500 mL x 2). The combined organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (eluent: PE:EA = 2:1 to 1:2) to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-(benzyloxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate. 1 H NMR(400MHz,CDCl3)δ ppm 7.37-7.32(m,5H),5.60(d,1H,J=8.4Hz),5.35(d,1H,J=2.4Hz),5.25(dd,1H,J1=11.6Hz,J2=3.6Hz),5.11(s,2H),4.63(d,1H,J1=8.4Hz),4.15 -4.11(m,2H),3.98-3.86(m,3H),3.55-3.45(m,1H),2.41-2.36(m,2H), 2.14(s,3H),2.03(s,3H),2.00(s,3H),1.91(s,3H),1.72-1.55(m,4H).

[0302] Step 4: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-(benzyloxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (90 g, 167.4 mmol) was dissolved in a mixture of EtOAc (250 mL) and MeOH (250 mL) and then wet Pd / C (4.5 g, 10%) was added. The reaction mixture was degassed and hydrogenated under hydrogen gas and then stirred overnight. TLC showed that the starting material was completely consumed. The reaction mixture was filtered and the filtrate was concentrated to dryness to give 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid. 1 H NMR(400MHz,CDCl3)δ ppm 5.95(d,1H,J=8.4Hz),5.35(d,1H,J =2.4Hz),5.25(dd,1H,J1=11.6Hz,J2=3.6Hz),4.66(d,1H,J1=8Hz),4.16-4.11(m,2H),4.01-3.90(m,3H),3 .56-3.49(m,1H),2.40-2.34(m,2H),2.16(s,3H),2.06(s,3H),2.01(s,3H),1.98(s,3H),1.72-1.55(m,4H).

[0303] Step 5: To a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (69 g, 154.2 mmol) and NHS-OH (19.5 g, 169.62 mmol) in DCM (600 mL) was added DIC (19.4 g, 154.2 mmol) and DMAP (36 mg, 0.29 mmol). The reaction mixture was stirred at room temperature for 3 h. TLC showed that the starting material was completely consumed. The resulting mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography (eluent: PE:EA = 2:1 to 1:4) to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidin-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (int-B1). 1 H NMR(400MHz,CDCl3)δ ppm 5.83(d,1H,J=8.4Hz),5.33(d,1H,J=2.4Hz),5.25(dd,1H,J1=11.6Hz,J2=3.6Hz),4.67(d,1H,J1=8Hz),4.13-4.07(m,2H),4. 00-3.87(m,3H),2.86-2.82(m,4H),2.73-2.55(m,2H),2.14(s,3H),2.02(s,3H),1.97(s,3H),1.91(s,3H),1.72-1.55(m,4H).

[0304] Type C: Synthesis of tetraacetic acid (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((16-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-16-(((benzyloxy)carbonyl)amino)-5,11,21,27-tetraoxo-14,18-dioxa-6,10,22,26-tetraazahentriacontane-1,31-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) (int-C1) [ka] Step 1: Aqueous NaOH (3.2 mL, 5 M) was added to a mixture of 2-amino-2-(hydroxymethyl)propane-1,3-diol (20 g, 165.28 mmol) in DMSO (32 mL) at 20° C., followed by dropwise addition of tert-butyl acrylate (74 g, 578.48 mmol). The mixture was stirred at 20° C. for 24 h. The resulting mixture was diluted with EtOAc (1 L) and washed with H2O (800 mL). The organic layer was dried over Na2SO4 and concentrated to give crude di-tert-butyl 3,3′-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate, which was used directly in the next step. t r =1.308 minutes, [M+H]+506.3

[0305] Step 2: Aqueous NaHCO3 (1 L) was added to a solution of di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (160 g, 316.20 mmol, crude) in EtOAc (1 L), followed by dropwise addition of benzyl carbonochloridate (53.9 g, 316.20 mmol). The reaction mixture was stirred at room temperature for 3 h. TLC showed the reaction was complete. The organic layer was separated, washed with H2O (500 ml), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (eluent: PE:EA = 20:1 to 5:1) to obtain di-tert-butyl 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate. 1 H NMR(400MHz,CDCl3)δ ppm 7.36-7.26(m,5H),5.29(brs,1H),5.02(s,2H),3.65-3.61(m,12H),2.43(t,6H,J=6.4Hz),1.43(s,27H).

[0306] Step 3: A solution of di-tert-butyl 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (37 g, 57.83 mmol) in formic acid (100 mL) was stirred at room temperature for 8 hours. The resulting mixture was concentrated to dryness to give 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid. 1 H NMR(400MHz,CDCl3)δ ppm 7.39-7.30(m,5H),5.35(brs,1H),5.06(s,2H),3.71-3.65(m,12H),2.59(t,6H,J=6.8Hz).

[0307] Step 4: HOBT (38.6 g, 286.34 mmol), EDCI (54.9 g, 286.34 mmol), and EtN (28.9 g, 286.34 mmol) were added to a mixture of 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (27 g, 57.27 mmol) and tert-butyl (3-aminopropyl)carbamate (49.8 g, 286.34 mmol) in DMF (600 mL), and the reaction mixture was stirred at room temperature for 6 h. TLC showed the reaction was complete. The resulting mixture was diluted with HO (1 L) and extracted with DCM (800 mL x 3). The combined organic layers were washed with aqueous NH4Cl (1 L), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (eluent: DCM:MeOH = 50:1 to 20:1) to give benzyl di-tert-butyl (10-(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,10,19-triyl)tricarbamate. 1 H NMR(400MHz,CDCl3)δ ppm 7.37-7.29(m,5H),6.85(brs,3H),5.55(brs,1H),5.14(brs,3H),5.02(s,2H),3.69-3.64(m,1 2H),3.31-3.23(m,6H),3.15-3.08(m,6H),2.43-2.36(m,6H),1.65-1.56(m,6H),1.42(s,27H).

[0308] Step 5: To a solution of benzyl di-tert-butyl (10-(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,10,19-triyl)tricarbamate (35 g, 37.23 mmol) in MeOH (500 mL) was added a solution of HCl in 1,4-dioxane (46 ml, 186.17 mmol, 4 M). The reaction mixture was stirred at room temperature for 3 h. The resulting mixture was concentrated to dryness to give the HCl salt of benzyl (1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)carbamate. 1 H NMR(400MHz,MeOD)δ ppm 7.41-7.32(m,5H),5.06(s,2H),3.70-3.64(m,12H),3.33-3.29(m,6H),2.97-2.93(m,6H),2.48-2.45(m,6H),1.88-1.82(m,6H).

[0309] Step 6: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidin-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (int-B1) (24 g, 44.05 mmol) was added to a mixture of (1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)benzylcarbamate (10 g, 13.35 mmol) and DIPEA (17.2 g, 133.5 mmol) in DMF (250 mL) and the reaction mixture was stirred at room temperature for 12 h. TLC showed the reaction was complete. The resulting mixture was diluted with HO (500 mL) and extracted with DCM (300 mL × 3). The combined organic layers were washed with aqueous HCl (300 mL, 4 M), dried over NaSO, and concentrated. The residue was purified by silica gel column chromatography (eluent: DCM:MeOH = 30:1 to 15:1) to give tetraacetic acid (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((16-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanediol. The compound (int-C1) was obtained as a result of the synthesis of the compound (2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) (int-C2). 1H NMR(400MHz,DMSO-d6)δ ppm 7.85-7.80(m,6H),7.72(brs,3H),7.34-7.30(m,5H),6.52(s,1H),5.19(d,3H,J=3.2Hz),4.95-4.92(m,5H),4.46(d,3H,J= 8.4Hz),4.00(brs,9H),3.86-3.84(m,3H),3.69-3.67(m,3H),3.54-3.41(m,15H),3.01(brs,12H),2.27-2.24(m,6H),2.08.

[0310] Type D: Synthesis of N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-aminooxy)-3,6,9,12-tetraoxapentadecane-15-amino)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide) (int-D1) [ka] Step 1: Synthesis of tetraacetic acid (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((16-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-16-amino-5,11,21,27-tetraoxo-14,18-dioxa-6,10,22,26-tetraazahentriacontane-1,31-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl). Tetraacetic acid (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((16-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxypropoxy)methyl)-16-(((benzyloxy)carbonyl) in MeOH (5 mL). To a solution of (5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)bis(oxy)amino)-5,11,21,27-tetraoxo-14,18-dioxa-6,10,22,26-tetraazahentriacontane-1,31-diyl)bis(oxy)bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) (int-C1) (142.8 mg, 0.074 mmol) was added Pd / C (10 wt%, 30 mg) and acetic acid (100 μL). The reaction mixture was hydrogenated under hydrogen gas at 50 PSI for 18 hours. Upon completion, the reaction was filtered, the filter cake was washed with MeOH, and the combined organics were concentrated to give the title product (135 mg). LCMS m / z (M+1) + =1794.9.

[0311] Step 2: Synthesis of N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-amino-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide) Tetraacetic acid (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((16-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-16- A solution of amino-5,11,21,27-tetraoxo-14,18-dioxa-6,10,22,26-tetraazahentriacontane-1,31-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) (343 mg, 0.191 mmol) was treated with a solution of methylamine (2.0 M in MeOH, 2 mL). The reaction was maintained at room temperature for 2 hours and then concentrated to give the title product (310 mg). LCMS m / z [(M+2H) / 2] + =708.9.

[0312] Step 3: Synthesis of N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-((1,3-dioxoisoindolin-2-yl)oxy)-3,6,9,12-tetraoxapentadecane-15-amido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide). To a solution of N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-amino-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide) (70.8 mg, 0.05 mmol) in DMF (1 mL) was added 2,5-dioxopyrrolidin-1-yl 1-((1,3-dioxoisoindolin-2-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-oate (38.1 mg, 0.075 mmol) and N,N-diisopropylethylamine were added. The mixture was stirred at room temperature for 2 h and then purified by reverse phase silica gel chromatography to give the title product (57 mg) after lyophilization. LCMS m / z[(M+2H) / 2] + =905.5.

[0313] Step 4: Synthesis of N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-(aminooxy)-3,6,9,12-tetraoxapentadecane-15-amido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide (int-D1) N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-((1,3-dioxoisoindolin-2-yl)oxy)-3,6,9,12-tetraoxapentadecane-1 in MeOH (1 mL). To a solution of 5-amido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide) (57 mg, 0.032 mmol) at room temperature was added hydrazine hydrate (63.1 mg, 1.26 mmol). The mixture was stirred at room temperature for 2 hours and then purified by reverse phase silica gel chromatography to give N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-(aminooxy )-3,6,9,12-tetraoxapentadecane-15-amido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide (int-D1) (18.1 mg) was obtained. LCMS m / z[M+H] + =1679.8.

[0314] Example 2: Preparation and analysis of anti-ASGPR-PCSK9 bispecific antibodies A bispecific antibody was prepared that contains both an anti-ASGPR domain and an anti-PCSK9 domain (see, e.g., FIG. 1). Each domain was designed based on published antibodies targeting ASGPR and PCSK9 (see, e.g., Bon et al. (2017) MABS 9(8):1360-1369 and Chaparro-Riggers et al. (2012) J Biol Chem 287(14):11090-11097). An exemplary ASGPR-PCSK9 bispecific antibody sequence is shown below:

[0315] PCSK9 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPFGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS (SEQ ID NO: 1)

[0316] PCSK9 VL DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 2)

[0317] ASGPR VH DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 3)

[0318] ASGPR VL SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSLERIGYLSYVFGGGTKLTVL (SEQ ID NO: 4)

[0319] ASGPR scFv (VH-VL with (G4S)4 linker (SEQ ID NO: 46))* [ka]

[0320] ASGPR_4F3_scFv-PCSK9_Fab_DANAPA_hIgG1_KiH_0 [ka]

[0321] ASGPR_4F3_scFv-PCSK9_Fab_DANAPA_hIgG1_KiH_1* [ka]

[0322] ASGPR_4F3_scFv-PCSK9_Fab_DANAPA_hIgG1_KiH_2 [ka]

[0323] *(G4S) n The linker is shown in underlined font (G4S(SEQ ID NO:43); (G4S)4(SEQ ID NO:46)).

[0324] The CDR sequences of exemplary ASGPR-PCSK9 bispecific antibodies are shown in Table 6 below.

[0325] [Table 19]

[0326] The anti-PSCK9 heavy chain was synthesized as a fusion of the variable domain with a constant silent hIgG1 domain (the hIgG1 domain could be modified to promote heterodimerization). A PCSK9 light chain plasmid was also synthesized. For the anti-ASGPR arm, it was produced as a single-chain fragment variable (scFv) in a VH-VL orientation with a 4xG4S (SEQ ID NO: 46) linker between the variable domain fused to the constant IgG1 domain (the hIgG1 domain could be modified to promote heterodimerization). The bispecific antibodies were co-expressed transiently in Expi293F (Thermo-Fisher Scientific) cells. Briefly, transfections were performed using PEI Max as the transfection reagent. Cells were grown in shake flasks on an orbital shaker (115 rpm) in a humidified incubator (85%) at 5% CO2. Anti-PSCK9 light and heavy chain plasmids were combined with ASGPR plasmid in a 3:2:2 ratio with PEI in a final ratio of 1 DNA:3 PEI. 1 mg / mL culture of plasmid was used for transfection in 0.5 million cells / mL serum medium. Antibody was harvested 5 days after expression by clarification of medium by centrifugation and filtration. Purification was performed by batch binding and elution using Protein A affinity batch chromatography (MabSelect® SuRe, GE Healthcare Life Sciences, Uppsala, Sweden). Resin was added at a ratio of 1 mL resin for every 100 mL of supernatant and batch bound for up to 4 hours. Supernatant was loaded onto a disposable column, drained by gravity and washed with 20 column volumes of PBS. Antibody was eluted with 20 column volumes of 20 mM citric acid, 125 mM NaCl, 50 mM sucrose pH 3.2. The eluted IgG protein was adjusted to pH 5.5 with 1 M sodium citrate.As a final polishing step, preparative size exclusion chromatography was performed using a Hi Load 16 / 60 Superdex 200 grade column (GE Healthcare Life Sciences, Uppsala, Sweden) in a buffer composed of 20 mM citric acid, 125 mM NaCl, 50 mM sucrose pH 5.5.

[0327] The constructs were checked for identity and purity by determining the intact mass using HPLC-MS and assessing the overall monomeric heterodimer by analytical size-exclusion chromatography. The resulting mass spectra were deconvoluted using MaxENT to determine the intact mass of the bispecific antibody constructs. The percentage of monomeric heterodimer was determined by analytical SEC HPLC. The resulting chromatograms were compared to those run on the same day for gel filtration standards (Bio-Rad, Hercules, California).

[0328] The binding specificity of the anti-ASGPR-PCSK9 bispecific antibody was examined by affinity assay, which showed high affinity for the target. As shown in Figure 2A-B, immobilized hASBPR1 or full-length hPCSK9 incubated with the anti-ASGPR-PCSK9 bispecific antibody showed high affinity. In addition, immobilized full-length hPCSK9 complexed with the anti-ASGPR-PCSK9 bispecific antibody was found to bind ASGPR with high affinity, as did immobilized hASBPR1 complexed with the anti-ASGPR-PCSK9 bispecific antibody incubated with full-length hPCSK9 (Figure 2C-D). In addition, pharmacokinetic studies of the anti-ASGPR-PCSK9 bispecific antibody in LDLR knockout mice showed enhanced clearance of PSCK9 in the presence of the antibody at doses of 0.1, 0.3, and 1 mg / kg (Figure 3A-B).

[0329] Example 3: Preparation and analysis of GalNAc-labeled anti-PCSK9 antibodies An anti-PCSK9 antibody labeled with a trimeric N-acetylgalactosamine (GalNAc) moiety was designed according to the schematic in Figures 20A and 20B and prepared as described in Examples 10 and 11. This PCSK9 antibody was based on the anti-PCSK9 antibody described in Chan et al. (2009) Proc Natl Acad Sci USA 106(24):9820-9825.

[0330] The binding specificity of GalNAc-labeled anti-PCSK9 antibody was evaluated by direct binding assays with ASGPR1 and PSCK9, respectively (FIGS. 5A-5B), and analysis of ASGPR1 binding to the complex of GalNAc-labeled anti-PCSK9 antibody with immobilized PCSK9 (FIG. 5C). As shown, GalNAc-labeled anti-PCSK9 antibody exhibits high affinity for both ASGPR and PSCK9. In addition, pharmacokinetic studies of GalNAc-labeled anti-PCSK9 antibody in LDLR knockout mice demonstrated enhanced clearance of PCSK9 from plasma in the presence of the antibody at doses of 0.3, 1, and 3 mg / kg (FIGS. 6A-6B).

[0331] Example 4: Preparation and analysis of GalNAc-labeled anti-ApoB antibodies Anti-ApoB antibody labeled with a trimeric N-acetylgalactosamine (GalNAc) moiety was prepared as follows: Anti-ApoB antibody (6.7 mg / mL) in 20 mM sodium phosphate buffer (pH 7.5) / 150 mM NaCl / 20 mM EDTA was mixed with a 5 mM solution of TCEP and reduction was allowed to proceed for 1.5 hours at 40° C. for a final antibody concentration of 5.0 mg / mL. After 1.5 hours, the reduction mixture was diluted with 20 mM sodium phosphate buffer (pH 7.5) / 150 mM NaCl / 20 mM EDTA and cooled to 22° C. A 12.0 mg / mL (4.83 mM) solution of trimer GalNAc (see compound 8 in FIG. 20A) described in Example 10 in DMF was prepared by dissolving 1.26 mg (512 nmol) of trimer GalNAc reagent in 106 μL of DMF. The trimer GalNAc reagent was added to the reduced anti-ApoB antibody solution, resulting in a final concentration of 5% DMF and a final antibody concentration of 4.0 mg / mL. The conjugation reaction was incubated at 22° C. for 22 hours, at which point the reaction mixture was buffer exchanged into PBS by ultrafiltration (Vivaspin20, 10 kDa MWCO, 3×) and gel filtration using an Illustra NAP-10 column. The eluted conjugate was sterile filtered (0.22 μM) and then characterized by size exclusion chromatography, LC-MS, and SDS-PAGE.

[0332] To examine the binding specificity, a mixture of human serum spiked with purified LDL (1 mL; 75% PBS, 25% serum, 100 μg LDL, protease inhibitor cocktail) was first stripped of protein A and G binding elements and then incubated with control antibody, anti-ApoB antibody, or GalNAc-labeled anti-ApoB antibody. Each mixture was then precipitated using a commercially available protein A / G agarose kit and subjected to immunoprecipitation with apoB100 IgG and Western blotting (Figure 7), which showed that the GalNAc-labeled anti-ApoB antibody exhibited affinity for apoB.

[0333] Example 5: Preparation and analysis of GalNAc-labeled anti-PCSK9 Fab fragments An anti-PCSK9 Fab fragment (see FIG. 8) labeled with a trimeric N-acetylgalactosamine (GalNAc) moiety was designed ( FIG. 8 ) and prepared as follows: This anti-PCSK9 Fab fragment was based on the anti-PCSK9 antibody described in Chan et al. (2009) Proc Natl Acad Sci USA 106(24):9820-9825.

[0334] Anti-PCSK9 Fab fragment (3.92 mg, 0.084 μmol) in 500 μL Tris (0.1M pH 7.4) was first activated by mixing with 1,3-dichloroacetone (6.65 μL, 4.19 μmol) in DMSO at 4° C. The mixture was stirred for 2 min at 4° C., then TCEP.HCl (11.98 μL, 0.838 μmol) in water was added dropwise. The resulting mixture was stirred for 20 h at 20° C., then passed three times consecutively through a Zeba 7K MWCO spin column pre-equilibrated with succinate buffer (0.2M pH 4.5) to obtain the modified anti-PCSK9 Fab. LCMS ESI: 46892.

[0335] To conjugate the activated GalNAc moiety to the activated PCSK9 Fab fragment, a solution of modified anti-PCSK9 Fab (3.92 mg, 0.084 μmol) in 0.5 mL of succinate buffer (0.2 M, pH 4.5) was added at room temperature to the GalNAc moiety (N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-(aminooxy)-3, 6,9,12-tetraoxapentadecane-15-amido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide (int-D1); 57 mg / mL solution in DMSO 98 μL, 5.61 mg, 3.34 μmol) was added. The mixture was stirred at 20° C. for 2 days and then concentrated using an Amicon ultra 30K MWCO centrifugal filter with PBS as wash buffer to obtain the desired GalNAc-labeled anti-PCSK9 Fab conjugate (330 μL, 9.7 mg / mL, 3.2 mg, 78% over two steps). LCMS ESI: 48608. FIG. 9 shows the SAS PAGE of the final product, where lane 1 corresponds to anti-PCSK9 Fab, lane 2 corresponds to anti-PCSK9 FAb (reduced), lane 3 corresponds to GalNAc-labeled anti-PCSK9 Fab, lane 4 corresponds to GalNAc-labeled anti-PCSK9 Fab (reduced), and lane 5 is a protein ladder standard.

[0336] Binding studies with both immobilized PCSK9 and immobilized ASGPR1 demonstrate high affinity of GalNAc-labeled anti-PCSK9 Fab (Figures 10A-B). Additionally, in vivo studies in mice demonstrated enhanced clearance of PCSK9 by administration of GalNAc-labeled anti-PCSK9 Fab (Figures 11A-B).

[0337] Example 6: Preparation and analysis of GalNAc-labeled anti-MICA Fab fragments A series of anti-MICS Fab fragments labeled with trimeric N-acetylgalactosamine (CalNAc) moieties were designed and prepared as follows.

[0338] Anti-MICA Fab fragment (0.72 mg, 0.015 μmol) in 160 μL Tris (0.1 M pH 7.4) was first activated by mixing with 1,3-dichloroacetone (2.4 μL, 0.756 μmol) in DMSO at 4° C. The mixture was stirred for 2 min at 4° C., then TCEP.HCl (2.162 μL, 0.151 μmol) in water was added dropwise. The resulting mixture was stirred for 4 h at 20° C., then passed three times consecutively through a Zeba 7K MWCO spin column pre-equilibrated with succinate buffer (0.2 M pH 4.5) to obtain modified anti-MICA FAb fragments 1, 2, and 3. LCMS ESI-activated Fab fragments 1, 2, and 3: 47239, 46824, and 47498, respectively.

[0339] To conjugate the activated GALNAc moiety to the activated MICA Fab fragment, a solution of each modified anti-MICA Fab fragment (0.6 mg, 0.013 μmol) in 150 μL of succinate buffer (0.2 M, pH 4.5) was added to the GalNAc moiety (N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(1-(aminooxy)-3-acetamido)propyl ... ,6,9,12-tetraoxapentadecane-15-amido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide (int-D1); 36 mg / mL solution in DMSO 23.5 μL, 0.845 mg, 0.503 μmol) was added. The mixture was stirred at 20° C. for 16 hours and then concentrated using an Amicon ultra 30K MWCO centrifugal filter with PBS as washing buffer to obtain GalNAc-labeled anti-MICA Fab fragment. Fab conjugate 1: (125 μL, 2.10 mg / mL, 0.26 mg, 36% over two steps). LCMS ESI: 48901. Fab conjugate 2: (125 μL, 1.57 mg / mL, 0.20 mg, 27% over two steps). LCMS ESI: 48485. Fab conjugate 3: (125 μL, 3.25 mg / mL, 0.41 mg, 56% over two steps). LCMS ESI: 49159.

[0340] Binding studies with both immobilized ASGPR and immobilized MICA demonstrate the high affinity of the GalNAc-tagged anti-MICA Fab fragment (FIGS. 12A-C and Table 7 below).

[0341] [Table 20]

[0342] In addition, in vivo studies in mice demonstrated enhanced clearance of MICA by administration of GalNAc-labeled anti-MICA Fab fragments compared to vehicle (black) or isotype control (anti-lysozyme in red) (Figures 13A-13B; 2 pM in blue and 69 pM in green).

[0343] Example 7: Preparation and analysis of GalNAc-labeled LDLR The extracellular domain of the LDLR protein labeled with a trimeric N-acetylgalactosamine (GalNAc) moiety was designed and prepared as follows: The extracellular domain of the LDLR protein has one of the following amino acid sequences:

[0344] LDLR(LDLR193(193-720)(N515Q)(N657Q)_SrtA_CHis_pRS5a) [ka]

[0345] LDLR reduced type (LDLR193(193-720)(N515Q)(N657Q)_CHis_pRS5a) [ka]

[0346] The purified LDLR protein was then conjugated with GalNAc as follows: A solution of LDLR (5.75 mg, 0.096 μmol) in 1.2 mL of Tris buffer (0.1 M pH 8) was added N,N'-(10-((3-((3-(5-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-10-(2-(2 -(2-aminoacetamido)acetamido)acetamido)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide; 76 μL of 40 mg / mL in DMSO, 3.03 mg, 1.91 μmol) was added, followed by the addition of Sortase A (0.311 mg, 0.014 μmol) in 76 μL of Tris buffer (0.1 M pH 8). The mixture was stirred for 16 hours at 20° C. and then purified by preparative size exclusion chromatography (HiLoad 16 / 600 Superdex 200 pg). The desired fractions were combined and concentrated using an Amicon ultra 30K MWCO centrifugal filter to give GalNAc-labeled LDLR conjugate (300 μL, 7.69 mg / mL, 2.3 mg, 39%). LCMS ESI (after deglycosylation with PNGase F): 61849.

[0347] FIG. 14 shows an SDS-PAGE of the reaction profile, where lane 1 corresponds to LDL starting material, lane 2 corresponds to LDLR starting material (reduced), lane 3 corresponds to GalNAc-labeled LDLR, lane 4 corresponds to GalNAc-labeled LDLR (reduced), and lane 5 is a protein ladder standard.

[0348] Binding studies with both immobilized PCSK9 and immobilized ASGPR1 demonstrate the high affinity of GalNAc-tagged anti-LDLR (FIGS. 15A-B).

[0349] Example 8: Preparation and analysis of GalNAc-labeled PCSK9 PCSK9 protein labeled with a trimeric N-acetylgalactosamine (GalNAc) moiety was designed and prepared as follows: PCSK9 protein was prepared as described below.

[0350] A full-length cDNA encoding human PCSK9 corresponding to GenBank Accession # NM_174936 was cloned into the mammalian expression vector pcDNA3.1(-)Neo (Invitrogen). The cDNA contained a Kozak (CACC) sequence immediately preceding the start codon, and the 3' end contained a sequence encoding a C-terminal Gly-Gly linker, followed by an Avi-tag and a 6x Histidine-tag (SEQ ID NO: 47). HEK293T cells were grown in suspension culture in a humidified atmosphere at 37°C and 8% CO2 using FreeStyle 293 Expression Medium (Thermo-Fisher Scientific). For 1L transfection, 1x10 6Cells / mL were grown in 934 mmL of FreeStyle 293 medium in a 2.5 L baffled shake flask. Transfections were performed using polyethylenimine (PEI) and FreeStyle 293 medium as follows: Mixture A, containing 1 mg of pcDNA3.1(-)Neo-hPCSK9-Avi-His or pcDNA3.1(-)Neo-mPCSK9-Avi-His plasmid in 33 mL of medium, and Mixture B, containing 3 mL of PEI (1 mg / mL) in 33 mL of medium, were incubated at room temperature for 5 minutes, respectively, and then the mixtures were passed through a 0.2 μm syringe filter. Mixture B was then gently added to Mixture A, and after 15 minutes of incubation at room temperature, the transfection mixture (69 mL) was added to the HEK293T cells. The cells were grown at 37°C and 8% CO2 with shaking at 100 rpm. 72 hours after transfection, the cells were harvested by centrifugation at 3000x for 30 minutes, and the culture supernatants were collected and purified by filtration through a 0.22u...

Claims

1. Formula (I): A Ｇ -L-R Ｇ (I) or a pharma- ceutically acceptable salt thereof, During the ceremony, A Ｇ is a protein moiety that binds to an extracellular target, Ｇ is an antibody or fragment thereof, a receptor or fragment thereof, or an antigenic protein or fragment thereof; L is absent or is a linker; and R Ｇ is a moiety that binds to a membrane-bound receptor associated with a degradative pathway, The membrane-bound receptor is a mannose-6-phosphate receptor (M6PR) or an insulin-like growth factor 2 receptor (IGF2R), and the R Ｇ is a small molecule, a carbohydrate or carbohydrate derivative molecule, or an antibody or fragment thereof that binds to M6PR and / or IGF2R; or The membrane-bound receptor is an asialoglycoprotein receptor (ASGPR), and the R Ｇ is a small molecule, a carbohydrate or carbohydrate derivative molecule, or an antibody or fragment thereof that binds to ASGPR; A bifunctional compound, or a pharma- ceutically acceptable salt thereof.

2. A Ｇ is an antibody or a fragment thereof, or a pharma- ceutically acceptable salt thereof.

3. 3. The bifunctional compound of claim 2, or a pharma- ceutically acceptable salt thereof, wherein the antibody is a full-length antibody, a multispecific antibody, a monospecific antibody, a bispecific antibody, or a fragment thereof.

4. The antibody fragments include Fab, Fab', F(ab') ２ , F(ab) ２ 3. The bifunctional compound of claim 2, comprising a fragment of a polypeptide chain (cDNA), a variable fragment (Fv), a domain antibody (dAb), a single domain antibody, or a single chain variable fragment (scFv), or a pharma- ceutically acceptable salt thereof.

5. 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the extracellular target is a pathogenic autoantibody or a fragment thereof.

6. The above A Ｇ The bifunctional compound according to claim 5 , or a pharma- ceutically acceptable salt thereof, comprising an antigen protein or a fragment thereof that recognizes the pathogenic autoantibody.

7. The antigen protein or a fragment thereof is selected from the group consisting of thrombospondin type 1 moiety disintegrin and metalloproteinase, member 13 (ADAMTS13), steroidogenic cytochrome P450 enzyme 21-hydroxylase, N-methyl-d-aspartate (NMDA) receptor, red blood cells, anti-smooth muscle antibody (ASMA), actin, platelets, signal recognition particle (SRP), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), myosin, sperm, and amylase. Alpha 2, type XVII collagen (col17), kallikrein 13, type VII collagen (col7), myeloperoxidase (MPO), type IV collagen, proteinase 3 (PR3), thyrotropin receptor (TSHR), thyroglobulin, thyroid peroxidase (TPO), thyroglobulin, thyroid peroxidase (TPO), platelets, myeloperoxidase (MPO), muscle nicotinic acetylcholine receptor, muscle specific kinase (MuSK), low density lipoprotein receptor protein 4 (LRP4), myo 7. The bifunctional compound of claim 6, wherein the bifunctional compound is selected from the group consisting of sucrose, beta 1 adrenergic receptor, adenine nucleotide translocase, aquaporin-4, myelin oligodendrocyte glycoprotein (MOG), heat shock protein 90 (HSP90), heat shock protein A5 (HSPA5), desmoglein-3, parietal cells, mitochondria, phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain containing 7A (THSD7A), cyclic citrullinated protein, RNA binding protein (Ros), La, double-stranded DNA (dsDNA), angiotensin II type 1 receptor (AT1R), endothelin-1 type A receptor (ETAR), insulin, glutamic acid decarboxylase, and protein tyrosine phosphatase, or a pharma- ceutical acceptable salt thereof.

8. A Ｇ The bifunctional compound of claim 1 , or a pharma- ceutically acceptable salt thereof, wherein:

9. 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the extracellular target is a protein, a pathogenic target, or a non-protein.

10. 10. The bifunctional compound of claim 9, or a pharma- ceutically acceptable salt thereof, wherein the extracellular target is a protein selected from a soluble protein or a membrane-associated protein.

11. 10. The bifunctional compound of claim 9, or a pharma- ceutically acceptable salt thereof, wherein the extracellular target is a non-protein selected from a lipoprotein, a liposome, a nucleic acid, a toxin, a viral particle, or a cell.

12. 11. The bifunctional compound of claim 10, or a pharma- ceutically acceptable salt thereof, wherein the soluble protein comprises an antibody, a soluble receptor, a secreted protein, a growth factor, a cytokine, a hormone, or an enzyme.

13. 11. The bifunctional compound of claim 10, or a pharma- ceutically acceptable salt thereof, wherein the soluble protein is proprotein convertase subtilisin / kexin type 9 (PCSK9), complement factor H-related protein 3 (CFHR3), MICA (MHC class I chain-related gene A), or apolipoprotein-B (Apo-B).

14. 11. The bifunctional compound of claim 10, or a pharma- ceutically acceptable salt thereof, wherein the membrane-associated protein is a type I, type II, or multipass membrane protein, or a glycosylphosphatidylinositol (GPI)-anchored membrane-associated protein.

15. 12. The bifunctional compound of claim 11, or a pharma- ceutically acceptable salt thereof, wherein the lipoprotein is lipoprotein receptor (LPR) or lipoprotein(a) (Lp(a)). Claim 16: The extracellular target is proprotein convertase subtilisin / kexin type 9 (PCSK9), tumor necrosis factor receptor 1 (TNFR1), interleukin 1 receptor (IL1R), low density lipoprotein, apolipoprotein B (ApoB), lipoprotein(a) (Lp(a)), apolipoprotein C3 (ApoCIII), angiopoietin-like 3 (ANGPTL3), angiopoietin-like 4 (ANGPTL4), angiopoietin-like 8 (ANGPTL8), factor 11, growth differentiation factor 15 (GDF15), lipoprotein lipase (LPL ... -leukin 1-beta (IL1β), interleukin 17 (IL17), complement factor B, complement factor D, myeloperoxidase (MPO), immunoglobulin E (IgE), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), interleukin 7 (IL7), interleukin 12A (IL12A), interleukin 23 (IL23), tumor necrosis factor A (TNFA), microtubule-associated protein tau (MAPT), complement factor H-related protein 3 (FHR3), tissue inhibitor of metalloproteinases 1 (TIMP1), ape Phosphorus, bone morphogenetic protein 6 (BMP6), bone morphogenetic protein 9 / growth differentiation factor 2 (BMP9 / GDF2), colony stimulating factor 1 receptor (CSF-1), erythropoietin (EPO), interleukin 5 (IL5), milk fat globule-EGF factor 8 protein (MFGE8), thymic stromal lymphopoietin (TSLP), thrombospondin (TSP), complement component 5 (C5), C-X-C motif chemokine ligand 10 (CXCL10), fibroblast growth factor 23 (FGF23), insulin-like growth factor 1 (IGF1), interleukin 10 (IL10 ), interleukin 13 (IL13), interleukin 2 (IL2), interleukin 6 (IL6), vascular endothelial growth factor A (VEGF-A), adenosine deaminase 2 (ADA2), soluble urokinase-type plasminogen activator receptor (suPAR), transforming growth factor beta 1 (TGF-β1), progranulin, alpha-synuclein, toxins, poisons, HBV soluble antigens, viral antigens, prion proteins, scFvs, AAV, and anti-AAV antibodies, or a pharma- ceutically acceptable salt thereof.

17. The pathogenic target is associated with or causes a harmful or unwanted effect in a sample, cell, or subject; and / or The pathogenic target is present in a sample, cell, or subject at an expression or activity level that produces a harmful or unwanted effect.

10. The bifunctional compound of claim 9, or a pharma- ceutically acceptable salt thereof.

18. 10. The bifunctional compound of claim 9, or a pharma- ceutically acceptable salt thereof, wherein the pathogenic target is a pathogenic autoantibody or fragment thereof, a cell surface receptor, or a neurological target.

19. 19. The bifunctional compound of claim 18, or a pharma- ceutically acceptable salt thereof, wherein the cell surface receptor is selected from TNF receptor 1 (TNFR1), interleukin-1 receptor (IL1R), PD-L1, epidermal growth factor receptor (EGFR), or transferrin.

20. 20. The bifunctional compound of claim 18, or a pharma- ceutically acceptable salt thereof, wherein the neurological target is a tau protein or aggregate, or an immuno-oncological target.

21. 21. The bifunctional compound of claim 20, or a pharma- ceutically acceptable salt thereof, wherein the immuno-oncological target is progranulin.

22. R Ｇ 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein: R1 is a binding moiety for M6PR or IGF2R, a binding moiety for ASGPR, or an ASGPR ligand.

23. The method of claim 22, wherein R comprises a binding moiety to ASGPR; 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the binding moiety to ASGPR is ASGPR1 or ASGPR2.

24. R Ｇ is represented by formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, During the ceremony, R １ は、-CN、-CH ２ -CN、-C≡CH、-CH ２ -N ３ 、-CH ２ -NH ２ 、-CH ２ -N(R ４ )-S(O) ２ -R ５ 、-CH ２ -CO ２ H、-CO ２ H、-CH ２ -OH、-CH ２ -SH、-CH=CH-R ５ 、-CH ２ -R ５ 、-CH ２ -S-R ５ 、-CH ２ -N(R ４ )-R ５ 、-CH ２ -N(R ４ )-C(O)-R ５ 、-CH ２ -N(R ４ )-C(O)-O-R ５ 、-CH ２ -N(R ４ )-C(O)-N(R ４ )-R ５ 、-CH ２ -O-R ５ 、-CH ２ -O-C(O)-R ５ 、-CH ２ -O-C(O)-N(R ４ )-R ５ 、-CH ２ -O-C(O)-O-R ５ 、CH ２ -S(O)-R ５ 、-CH ２ -S(O) ２ -R ５ 、-CH ２ -S(O) ２ -N(R ４ )-R ５ 、-C(O)-NH ２ 、-C(O)-O-R ５ 、-C(O)-N(R ４ )-R ５ or aryl or heteroaryl, wherein the aryl or heteroaryl is R ５ or R １ is -Z-X-Y, where X is a linker or a drug delivery system, Y is absent or a ligand selected from the group consisting of a small molecule, an amino acid sequence, a nucleic acid sequence, an antibody, an oligomer, a polymer, a gene-derived material, a liposome, a nanoparticle, a dye, and a fluorescent probe, or a combination thereof, and Z is absent or is -C≡C-, -CH═CH-, -CH ２ --, --CH ２ -O-, -C(O)-N(R ４ ) -, -CH ２ -S-, CH ２ -S(O)-, -CH ２ -S (O) ２ --, --CH ２ -S (O) ２ -N(R ４ )-, -C(O)-O-, -CH ２ -N(R ４ ) -, -CH ２ -N(R ４ )-C(O)-,-CH ２ -N(R ４ ) -S(O) ２ --, --CH ２ -N(R ４ )-C(O)-O-,-CH ２ -N(R ４ )-C(O)-N(R ４ ) -, -CH ２ -OC(O)-, -CH ２ -O-C(O)-N(R ４ ) -, -CH ２ -O-C(O)-O-, or aryl or heteroaryl, wherein the aryl or heteroaryl is R ５ and optionally substituted with R ２ is -OH, -N ３ , -N(R ３ ) ２ , -N(R ３ )-C(O)-R ３ , -N(R ３ )-C(O)-N(R ３ ) ２ , -N(R ３ )-C(O)-OR ３ , tetrazole, or triazole, and the tetrazole and triazole are R ３ optionally substituted with; R １ Ga-CH ２ When it is —OH, R ２ is -N ３ , -N(R ３ ) ２ , -N(R ３ )-C(O)-R ３ , -N(R ３ )-C(O)-N(R ３ ) ２ , -N(R ３ )-C(O)-OR ３ , tetrazole, or triazole, and the tetrazole and triazole are R ３ and optionally substituted with Each R ３ are independently -H, -(C １ ~C ５ ) alkyl, halo-substituted (C １ ~C ５ ) alkyl, or (C ３ ~C ６ ) cycloalkyl, and the alkyl or cycloalkyl is —CH ２ The - group is -O-, -S-, and -N(R ４ )-, and the alkyl may be replaced by a heteroatom selected from -CH ３ is -N(R ４ ) ２ , -OR ４ , and -S(R ４ wherein said heteroatom groups are separated by at least two carbon atoms; Each R ４ are independently -H, -(C １ ~C ２０ ) alkyl, or (C ３ ~C ６ ) cycloalkyl, wherein said alkyl or cycloalkyl has 1 to 6 -CH groups separated by at least 2 carbon atoms. ２ The - group is -O-, -S-, or -N(R ４ )-, and the alkyl may be replaced by -CH ３ is -N(R ４ ) ２ , -OR ４ , and -S(R ４ wherein said heteroatom groups are separated by at least two carbon atoms; said alkyl and cycloalkyl may be substituted with 1 to 6 halo atoms; and Each R ５ are independently -H, (C ３ ~C ２０ ) cycloalkyl, or (C １～ C ２０ is an alkyl group, and 1 to 6 -CH of said alkyl or cycloalkyl are separated by at least 2 carbon atoms. ２ The - group is -O-, -S-, or -N(R ４ )-, and the alkyl may be replaced by -CH ３ is -N(R ４ ) ２ , -OR ４ , and -S(R ４ wherein said heteroatom groups are separated by at least two carbon atoms; said alkyl and cycloalkyl may be substituted with 1 to 6 halo atoms; 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof.

25. R Ｇ teeth, 【Chemistry 2】 wherein n is 1, 2, or 3; 【Chemistry 3】 (Wherein, n is 2 or 3) is selected from; and R Ｇ is attached to L at the terminal amine; 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof.

26. R Ｇ teeth, 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 wherein R Ｇ 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein * indicates the point of attachment to L.

27. R Ｇ 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said bifunctional compound comprises an M6PR or IGF2R binding moiety and said extracellular target is TNFR1, IL1R, progranulin, tau, MUC5B, TREM2, or EGFR.

28. 2. The bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein L comprises a heteroalkylene moiety.

29. A pharmaceutical composition comprising the bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient.

30. 11. A method of targeting an extracellular target in a cell for degradation or reduction in the level and / or activity of the extracellular target, comprising contacting the cell with a bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof.

31. 1. A pharmaceutical composition for use in a method of targeting an extracellular target for degradation or reduction in the level and / or activity of the extracellular target in a subject, comprising: The pharmaceutical composition comprises the bifunctional compound of claim 1, or a pharma- ceutical acceptable salt thereof, The method comprises administering to the subject a bifunctional compound of claim 1, or a pharma- ceutically acceptable salt thereof. Pharmaceutical compositions.

32. A bispecific antibody or fragment thereof that binds to the asialoglycoprotein receptor (ASGPR) and proprotein convertase subtilisin / kexin type 9 (PCSK9).

33. 33. A pharmaceutical composition comprising the bispecific antibody or fragment thereof of claim 32 and a pharma- ceutically acceptable excipient.

34. A nucleic acid molecule encoding the bispecific antibody or fragment thereof described in claim 32.

35. A method for targeting PCSK9 in a subject or sample for degradation or reduction of PCSK9 levels and / or activity, comprising contacting the sample with a bispecific antibody or fragment thereof described in claim 32.

Images