Bifunctional molecules for lysosomal targeting and related compositions and methods

Abstract

To provide bifunctional molecules for lysosomal targeting.SOLUTION: Provided are bifunctional molecules that include a first moiety that specifically binds a cell surface molecule or extracellular molecule, and a second moiety that specifically binds a lysosomal targeting molecule.SELECTED DRAWING: Figure 19

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 782,19, filed December 19, 2018. No. 3, and U.S. Provisional Patent Application No. 62 / 932,347, filed November 7, 2019. No. 6,399,433, filed on Oct. 13, 2003, which claims the benefit of US Pat. No. 6,399,433, filed on Oct. 13, 2003, and which are incorporated herein by reference in their entireties. Can be enjoyed.

[0002] Statement of government support This invention was made possible by National Institutes of Health under contracts CA227942, GM059907 This invention was made with government support under the U.S. Pat. No. 6,336,313, and GM123636. The Company has certain rights. Summary of the Invention

[0003] A first portion that specifically binds to a cell surface or extracellular molecule and a lysosomal targeting molecule. and a second moiety that specifically binds to the . Bifunctional molecules are provided, e.g. For example, the expression of cell surface and extracellular molecules (e.g., proteins) via the endosomal / lysosomal pathway Compositions and kits containing bifunctional molecules, such as Also provided are methods of using the bifunctional molecules. Methods of making the bifunctional molecules are also provided. can be. [Brief description of the drawings]

[0004] [Figure 1] FIG. 1 is a schematic diagram of a bifunctional molecule and its use, according to one embodiment of the present disclosure. [Diagram 2]A scheme for synthesizing mannose-6-phosphate N-carboxyanhydride, according to one embodiment. This route allows access to mannose-6-phosphate glycans, for example linked to serine residues, for use as monomers in N-carboxyanhydride polymerization. [Diagram 3]

[0023] Figure 1 shows a scheme for the synthesis of mannose-6-phosphonate N-carboxyanhydrides according to one embodiment. This route allows access to mannose-6-phosphonate glycans, for example linked to serine residues, for use as monomers in N-carboxyanhydride polymerization. The phosphonate group is a hydrolytically stable variant of the phosphate group that has previously been shown to have superior serum stability compared to mannose-6-phosphate glycans. [Figure 4] A scheme for synthesizing mannose-6-carboxylate N-carboxyanhydride according to one embodiment. This route allows access to mannose-6-carboxylate glycans, for example linked to serine residues, for use as monomers in N-carboxyanhydride polymerization. The carboxylate group is a hydrolytically stable variant of the phosphate group that has previously been shown to have superior serum stability compared to mannose-6-phosphate glycans. Mannose-6-carboxylate glycans have previously shown a relative binding affinity of 0.3 for cation-independent M6PR (CIM6PR) compared to mannose-6-phosphate glycans. The ability to chemically tune receptor-ligand interactions allows for greater control over biological applications and reduces off-target binding events. [Diagram 5]A scheme for synthesizing mannose-6-acrylate N-carboxyanhydride according to one embodiment. This route allows access to mannose-6-acrylate glycans, for example linked to serine residues, for use as monomers in N-carboxyanhydride polymerization. The acrylate group is a hydrolytically stable variant of the phosphate group that has previously been shown to have superior serum stability compared to mannose-6-phosphate glycans. Mannose-6-acrylate glycans have previously shown a relative binding affinity of 0.7 for CIM6PR compared to mannose-6-phosphate glycans. The ability to chemically tune receptor-ligand interactions allows for greater control over biological applications and reduces off-target binding events. [Figure 6] 1 is a scheme for the synthesis of glucose-6-phosphonate N-carboxyanhydrides according to one embodiment. This route allows access to glucose-6-phosphonate glycans, for example linked to serine residues, for use as monomers in N-carboxyanhydride polymerization. Glucose-6-phosphonate residues have significantly weaker binding affinity to CIM6PR compared to mannose-containing glycans. [Figure 7] A scheme for synthesizing mannose-6-phosphonate isothiocyanate according to one embodiment. This route allows access to mannose-6-phosphonate isothiocyanate (M6Pn-ITC), which can be directly conjugated, for example, to lysine residues in proteins. Conjugation of multiple M6Pn-ITCs to multiple amino acids (e.g., lysines) within a given protein allows multivalent display of M6Pn glycans. [Figure 8]FIG. 1 is a diagram of a general NCA polymerization scheme for synthesizing scaffolds for displaying M6P ligands, according to one embodiment. Copolymers with other amino acid-derived NCAs are similarly easily synthesized, providing access to a multitude of polymers with various structures and compositions bearing multiple M6P ligand residues. These materials are then deprotected to provide the complete polypeptide / glycan structure. [Figure 9] 1 is a scheme for solid-phase peptide synthesis of mannose-6-phosphonate peptide oligomers, according to one embodiment. As shown, scaffolds for displaying M6P ligands can also be synthesized using solid-phase peptide synthesis, starting from amino acids such as M6P, M6Pn, etc. This synthetic route allows for greater control of polypeptide length and composition compared to the NCA polymerization route, and does not require special synthesis conditions compared to NCA polymerization-derived materials. [Figure 10] Schematic of an experiment (top) and fluorescence imaging results (bottom) showing that biotin-capped functionalized M6Pn polymers can mediate the translocation of extracellular neutravidin-647 (NA 647, a protein that strongly binds biotin) from the extracellular space to lysosomes for degradation. Colocalization of both the protein and the lysosomal staining dye is observed. [Figure 11] Data demonstrating that several cell lines exhibit NA647 uptake in an M6Pn polymer-dependent manner. Given these results, it is predicted that any cell line that possesses M6PR (e.g., CIM6PR) will enable shuttling of proteins to lysosomes in this manner and is not limited to the cell lines tested in this study. [Figure 12] Data demonstrating that non-denaturing gel polyacrylamide gel electrophoresis (PAGE) can be used to monitor the functionalization of proteins (including antibodies) with M6P polymers. In this example, the protein of interest was first labeled with a reactive alkyne (e.g., bicyclo[6.1.0]nonyne, BCN), followed by functionalization by incubation with an azide-containing polymer (bio-orthogonal copper-free strain-promoted click reaction). [Figure 13] Schematic (top) and fluorescence imaging data (bottom) showing that poly(M6Pn)-labeled antibodies can shuttle their binding partners to lysosomes. In this example, mouse IgG-488 was incubated with an anti-mouse IgG antibody bearing a poly(M6Pn) tag, resulting in colocalization of both the protein and the lysosomal staining dye (merge). [Figure 14] Schematic (top) and data (bottom) showing that poly(M6Pn)-labeled antibodies can shuttle their binding partners to intracellular compartments. In this example, recombinant human apoE4 was incubated with a mouse-derived anti-human apoE4 antibody, an anti-mouse IgG antibody, or an anti-mouse antibody carrying a poly(M6Pn) tag. Significantly more uptake is observed with the M6Pn-containing secondary antibody. [Figure 15] Data demonstrating that poly(M6Pn)-labeled antibodies can shuttle their binding partners for degradation. In this example, EGFR degradation was assessed by incubating cells with cetuximab bearing an M6Pn tag. For all cell lines tested, loss of total EGFR is observed compared to cetuximab or cetuximab bearing the pseudopolymer (GalNAc). EGF is a positive control for EGFR degradation. Lane 1: control. Lane 2: EGF (100 ng / mL, 1 hour, + control). Lane 3: cetuximab. Lane 4: cetuximab-GalNAc conjugate. Lane 5: cetuximab-M6P conjugate (long). Lane 6: cetuximab-M6P conjugate (short). Percentage of control was calculated by densitometry. [Figure 16] Data showing that poly(M6Pn)-labeled antibody fragments can shuttle their binding partners to lysosomes. In this example, EGFR degradation was assessed by incubating cells with a cetuximab-derived Fab portion bearing an M6Pn tag. Loss of total EGFR is observed compared to cetuximab Fab alone or cetuximab Fab(GalNAc) with a pseudopolymer. [Figure 17] Data demonstrating that poly(M6Pn)-tagged antibodies can shuttle their binding partners for degradation. In this example, degradation of CD71 (transferrin receptor) was assessed by incubating cells with a primary mouse-derived antibody against CD71, an anti-mouse IgG antibody, or an anti-mouse antibody with a poly(M6Pn) tag. The system containing the M6P tag results in significantly more degradation. [Figure 18] Data demonstrating that poly(M6Pn)-labeled antibody fragments can shuttle their binding partners for degradation. In this example, PDL1 degradation was assessed by incubating cells with anti-PDL1 antibody or anti-PDL1 antibody bearing an M6P tag. Degradation is only observed with M6P-labeled anti-PDL1 antibody. [Figure 19] Schematic of targeted extracellular proteolysis using bifunctional molecules, bispecific antibodies, in this example against CIM6PR and a given target, which are released from the target at the reduced pH of the endosome. This strategy allows the given bispecific antibody to cycle with the receptor, delivering cargo and target sequentially to the lysosome without degradation of the antibody. [Figure 20]1 is a schematic diagram of a lysosomal targeting molecule to which the second portion of a bifunctional molecule may bind, according to an embodiment of the present disclosure. In this example, the lysosomal targeting molecule is the asialoglycoprotein receptor (ASGPR), which is expressed substantially exclusively on liver cells (e.g., hepatocytes). As shown on the right, the ASGPR constitutively recycles between the plasma membrane and endosomes, thereby carrying extracellular glycoproteins into the cell for degradation in lysosomes. On the bottom left, an exemplary bifunctional molecule is shown, which includes a first portion that is an antibody (e.g., an antibody that binds to a molecule expressed on the surface of a liver cell or present in the extracellular space of a liver cell) and a second portion that includes a multivalent ASGPR ligand for binding to the ASGPR. In this example, the second portion includes a polymer of N-acetylgalactosamine (GalNAc), specifically, the poly(GalNAc-co-Ala) polymer shown. [Figure 21] Schematic and data showing the transfer of a targeting molecule into HEPG2 (hepatocellular carcinoma) cells using a bifunctional molecule comprising a first moiety (in this example, an antibody) that binds to the targeting molecule and a second moiety that comprises a GalNAc-containing polymer as shown in FIG. [Figure 22] Schematic and data demonstrating efficient cellular uptake via ASGPR in HUH7 (hepatocellular carcinoma) cells. [Figure 23] Schematic and data showing efficient degradation of EGFR in HEP3B (hepatocellular carcinoma) cells using a bifunctional molecule comprising cetuximab (first moiety) conjugated to a second moiety comprising a GalNAc-containing polymer as shown in Figure 20. Also shown is EGFR degradation data for a conjugate comprising cetuximab (first moiety) conjugated to a second moiety comprising an M6PR ligand. [Figure 24]Data showing efficient degradation of EGFR in HEPG2 cells using a bifunctional molecule comprising cetuximab (first moiety) conjugated to a second moiety comprising a GalNAc-containing polymer as shown in Figure 20. Also shown is EGFR degradation data for a conjugate comprising cetuximab (first moiety) conjugated to a second moiety comprising an M6PR ligand. [Diagram 25] Data from a time course study assessing EGFR degradation over time in HEP3B cells. [Figure 26] Immunofluorescence data showing that treatment of HEP3B cells with the cetuximab conjugates described above results in the majority of membrane EGFR being degraded, but residual EGFR remains inside the cells. [Figure 27] Western blot data showing the extent of HER2 degradation in HUH7 and HEPG2 cells in the presence of trastuzumab alone or trastuzumab conjugated to a GalNAc-containing polymer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] A first portion that specifically binds to a cell surface or extracellular molecule and a lysosomal targeting molecule. and a second moiety that specifically binds to the . Bifunctional molecules are provided, e.g. For example, the expression of cell surface and extracellular molecules (e.g., proteins) via the endosomal / lysosomal pathway Compositions and kits containing bifunctional molecules, such as Also provided are methods of using the bifunctional molecules. Methods of making the bifunctional molecules are also provided. can be.

[0006] Before the bifunctional molecules, compositions, kits and methods of the present disclosure are described in more detail, The active molecules, compositions, kits and methods are not limited to the specific embodiments described. It should be understood that the bifunctional molecules, compositions, The scope of the kits and methods is limited only by the appended claims, and therefore the present specification The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood that this is not intended to

[0007] When a range of values ​​is given, any value between the upper and lower limits of the range, unless the context indicates otherwise, Unless expressly indicated, each intervening value to the tenth of the unit of the lower limit and its stated value Any other stated or intervening value within the range is contemplated as a bifunctional molecule, composition, kit, or method. It is understood that the upper and lower limits of these smaller ranges are within the scope of the present invention. These may also be included in smaller ranges, and these are also not specifically excluded within the stated ranges. Apart from the limitations noted above, the bifunctional molecules, compositions, kits and methods are encompassed. If the stated range includes one or both of the limits, either or Ranges excluding both are also included in the bifunctional molecules, compositions, kits and methods.

[0008] Certain ranges are presented herein with the numerical values ​​preceded by the term "about." The term "about" means any number that is preceded by or near the exact number that it precedes. It is used herein to provide literal support for numbers that are equal or approximate. In determining whether a number is near or approximate to a particular recited number, the proximity or Approximate but unlisted numbers are not necessarily specifically listed unless specifically recited in the context in which they are presented. The number may be substantially equivalent to the number.

[0009] Unless otherwise defined, all technical and scientific terms used herein are The term "functional molecule," "composition," "kit," and "method" have the same meaning as commonly understood by those skilled in the art to which they pertain. Any bifunctional molecule, composition similar or equivalent to those described herein. The bifunctional molecules, compositions, kits and methods are also useful for carrying out or testing the bifunctional molecules, compositions, kits and methods. Representative exemplary bifunctional molecules, compositions, kits and methods that can be used in is explained here.

[0010] All publications and patents cited herein are hereby incorporated by reference in their entirety. All such claims are hereby incorporated by reference as if specifically and individually indicated to be incorporated by reference. Disclose or disclose related methods and / or materials incorporated in the document and where the publications are cited. No. 6,313,635, filed on Dec. 13, 2003. The present application is hereby incorporated by reference for the purposes of describing the invention as disclosed herein. The citation of a publication is for its validity prior to the filing date of the application. The publication dates provided are for disclosure of actual publication dates which may need to be independently verified. The bifunctional molecules, compositions, kits and methods may differ from the publication date. This document should not be construed as an admission that there are no prior rights.

[0011] As used in this specification and the appended claims, the singular forms "a," "an," and " and "the" are intended to include plural referents unless the context clearly dictates otherwise. It should be understood that the claims may be drafted to exclude any optional element. Please further note that this statement is not intended to be construed as a substitute for the recitation of claim elements. Literal language for the use of exclusive terminology such as ", " "only" and the like or the use of "negative" limitations It is intended to serve as evidence.

[0012] Bifunctional molecules, compositions, and kits described in the context of separate embodiments for clarity And certain features of the method may also be provided in combination in a single embodiment. Conversely, bifunctional molecules, which have been described in the context of a single embodiment for the sake of brevity, may be The various features of the compositions, kits and methods may be used separately or in any suitable subcombination. All combinations of the embodiments are encompassed by the present disclosure. Specifically encompassed herein are processes and methods in which such combinations can be performed. and / or any and all combinations to the extent that they include compositions are individually and expressly disclosed. Moreover, any such variables may be incorporated herein by reference in any manner whatsoever. All of the subcombinations listed are also included within the bifunctional molecules, compositions, and kits of the invention. Each such subcombination is specifically encompassed by the methods and compositions of the present invention and each such subcombination is individually encompassed by the methods and compositions of the present invention. and are hereby disclosed as if expressly disclosed herein.

[0013] As will be apparent to those of skill in the art upon reading this disclosure, each of the components described and illustrated herein may be Each of the embodiments may be combined with any of the other embodiments without departing from the scope or spirit of the disclosed methods. The individual features of the embodiments may be easily separated or combined. Any recited method has elements and features. Or any other order which is logically possible.

[0014] bifunctional molecule The present disclosure provides a method for the preparation of a lysosomal protein comprising administering to a subject a first portion that specifically binds to a cell surface or extracellular molecule and a second portion that specifically binds to a lysosomal protein. and a second moiety that specifically binds to a targeting molecule, Certain non-limiting embodiments of functional molecules are described.

[0015] As summarized above, the bifunctional molecules of the present disclosure are capable of binding specific to cell surface or extracellular molecules. In some embodiments, the first portion specifically binds to a cell surface molecule. A "cell surface molecule" is, for example, a molecule that is inserted into the cell membrane or that specifically binds to a cell. Contains a membrane-spanning domain (e.g., a membrane tethering domain or a transmembrane domain) The term refers to molecules that are bound to the cell membrane in order to bind to the cell membrane. Cell surface molecules are molecules that are attached to the cell membrane in the endosome / lysosome. The target molecule may be any cell surface molecule for which targeted degradation via a cellular pathway is desired. In this case, the cell surface molecule is a cell surface receptor. The cell surface receptor of interest is not limited to However, there are also stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, and hormone receptors. receptors, receptor tyrosine kinases, and receptors of the epidermal growth factor receptor (EGFR) family Human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor (FGFR) family receptors, vascular endothelial growth factor receptor (VEGFR) family receptors Receptor, Platelet-derived growth factor receptor (PDGFR) family receptor, rearran Receptors of the ged during transfection (RET) receptor family , receptors of the Eph receptor family, discoidin domain receptor (DDR) family and mucin proteins (e.g., MUC1). In one embodiment, the cell surface molecule is CD71 (transferrin receptor). Cell surface receptors include T cell receptors, B cell receptors, natural killer (NK) cell receptors, Macrophage receptors, monocyte receptors, neutrophil receptors, dendritic cell receptors, mast cell receptors, The immune cell receptor is selected from a basophil receptor, and an eosinophil receptor.

[0016] In some embodiments, the first portion binds to the target protein other than through a specific molecular interaction. therefore, less susceptible to blocking) and bulk biophysical or aggregation effects. These proteins specifically bind to cell surface molecules that mediate their effects through their binding to such cell surface molecules. Non-limiting examples of molecules are mucins. Examples of mucins include MUC1, MUC16, MU C2, MUC5 AC, MUC4, CD43, CD45, GPIb, etc. Not limited to these.

[0017] In some embodiments, when the first portion specifically binds to a cell surface molecule, the cell A cell surface molecule is one that is present on a cancer cell. A "cancer cell" is, for example, a cell that is characterized by abnormal cell growth. , abnormal cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth capacity, immunocompromised state Tumor growth and / or development, and / or cell morphology in non-human animal models of the disease Characterized by one or more of the following: the ability to promote any suitable indicator of transformation. "Cancer cell" as used herein refers to a cell that exhibits a neoplastic cell phenotype and is capable of being detected by a may be used interchangeably with "tumor cell," "malignant cell," or "cancerous cell," and may refer to the development of a solid tumor, semi-solid tumor, or tumor. Solid tumors, hematological malignancies (e.g., leukemia cells, lymphoma cells, myeloma cells, etc.), primary In some embodiments, the present invention relates to a cancer cell, including a cancer cell, a tumor, a metastatic tumor, or the like. The cell surface molecule is a tumor-associated or tumor-specific antigen.

[0018] In certain embodiments, when the first portion specifically binds to a cell surface molecule, the cell surface molecule is In some embodiments, the cell surface molecule is present on an immune cell. Cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells , on immune cells selected from mast cells, basophils and eosinophils. In the present specification, the term "cell surface molecule present on immune cells" refers to an inhibitory immune receptor. Inhibitory immunoreceptors are receptors present on immune cells that negatively regulate the immune response. Examples of inhibitory immune receptors that can be inhibited according to the methods of the present disclosure include those Non-limiting examples include CD200R, CD300a (IRp60, mouse MAIR-I), C D300f(IREM-1), CEACAM1(CD66a), FcyRIIb, ILT -2 (LIR-1, LILRB1, CD85j), ILT-3 (LIR-5, CD85k) , LILRB4), ILT-4(LIR-2, LILRB2), ILT-5(LIR-3 , LILRB3, mouse PIR-B), LAIR-1, PECAM-1 (CD31), P Ig superfamily members, including ILR-α (FDF03), SIRL-1, and SIRP-α The inhibitory immune receptors that can be inhibited according to the methods of the present disclosure include the inhibitory immune receptors of the IL-1 family. Further examples of immunoreceptors include sialic acid-binding Ig-like lectin (Siglec) receptors. Examples of the proteins that can be inhibited according to the method of the present disclosure include Siglec7 and Siglec9. Additional examples of inhibitory immune receptors that may be impaired include, but are not limited to, CLEC4 A(DCIR), Ly49Q, and MICL. For details on sexual immune receptors, see, for example, Steevels et al. (2011) The results can be found in J. Immunol. 41(3):575-587. In one embodiment, the cell surface molecule present on the immune cell is a ligand for an inhibitory immune receptor. In certain aspects, the cell surface molecule present on the immune cell is an immune checkpoint molecule. Non-limiting examples of immune checkpoint molecules to which the first portion can specifically bind include: These include PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT and B7. Family members are mentioned.

[0019] As summarized above, the bifunctional molecules of the present disclosure are capable of binding specific to cell surface or extracellular molecules. In some embodiments, the first moiety specifically binds to an extracellular domain. An "extracellular molecule" is a soluble molecule that specifically binds to the soluble molecule. Extracellular molecules are those that are present on the outside of the cell membrane of the neighboring cell. The molecule may be any extracellular molecule for which targeted degradation via the inoculum pathway is desired. In the present invention, the extracellular molecule is a ligand for a cell surface receptor. The bonds include growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) etc.), cytokines (e.g., interleukins, interferons, tumor necrosis factors ( TNF), transforming growth factor β (TGF-β) (such cytokines (including any specific subtypes), hormones, etc. In certain embodiments, the first portion specifically binds to apolipoprotein E4 (ApoE4). To combine.

[0020] In some embodiments, the first portion specifically binds to an extracellular molecule, and the extracellular molecule is , antibodies, for example, that specifically bind to cell surface molecules or different extracellular molecules. In some embodiments, the antibody is an autoantibody. Non-limiting examples of autoantibodies include ribosomal proteinases (RIPs) and ribosomal proteinases (RIPs). equine factor (RF), antinuclear antibody (ANA), antineutrophil cytoplasmic antibody (ANCA), anti-double chain DNA (anti-dsDNA), anti-centromere antibody (ACA), anti-histone antibody, cyclic citrulline Phosphopeptide antibodies (CCP), soluble nuclear antigen antibodies (e.g., anti-SS-A(Ro) and Anti-SS-B(La), anti-RNP, anti-Jo-1, anti-Sm, Scl-70), cardiolipin Antibodies, beta-2 glycoprotein 1 antibodies, antiphospholipid antibodies (APA), lupus anticoagulant factor ( LA), diabetes-related autoantibodies, anti-tissue transglutaminase (anti-tTG), anti-gliagi Anti-thyroid antibodies (AGA), intrinsic factor antibodies, parietal cell antibodies, thyroid autoantibodies (e.g., anti-TPO, TSH receptor antibody), smooth muscle antibody (SMA), antimitochondrial antibody (AMA), liver mitochondria Chlosome type 1 antibody (anti-LKM-1), anti-glomerular basement membrane (GBM), acetylcholine receptor Examples include AChR antibodies.

[0021] In some embodiments, the first portion is a secreted protein that accumulates in a disease (e.g., α-synuclein), cholesterol carriers (e.g., ApoB), infectious disease toxins ( AB toxin, ESAT-6), infectious particles (e.g., whole viruses, whole bacteria, etc. ), coagulation factors (e.g., factor IX), or targets for any FDA-approved antibody that binds to an extracellular molecule. any chemokine or cytokine (e.g., IL-1, -1), mediators of sepsis or chronic inflammation, protein hormones (e.g. (e.g., insulin, ACTH, etc.), protein mediators of mood disorders, energy Protein mediators of homeostasis (e.g., leptin, ghrelin, etc.) present in the bloodstream Protein allergens present in the body, or antibodies against such allergens (e.g., peptide - in the case of nut allergies), protein toxins (e.g. snake venom hyaluronidase, etc.) It specifically binds to extracellular molecules such as

[0022] In certain embodiments, the first portion specifically binds to a cell surface or extracellular molecule and In some embodiments, the cell surface or extracellular molecule is a mutant protein. The inhibitory molecule causes the shuttling of mutant proteins into lysosomes, where they bind to major histocompatibility genes. and its loading onto a major histocompatibility complex (MHC) (e.g., MHCCI or MHCII). In this regard, it is believed that the immune system may recognize the mutant protein by stimulating the immune system's immune system. The bifunctional molecule binds an antibody specific for a mutated undesirable protein (e.g., KIT). Finds use in making.

[0023] "Specifically binds" means that the first and second moieties bind to their respective targets, e.g. About 10 5 M -1 or higher affinity or K a (i.e., specific binding in units of 1 / M This means that the first bond is bonded to the second bond via the equilibrium association constant of the interaction. and the second portion delivers about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 1010 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 More than K a "High affinity" binding is at least 10 7 M -1 , few At most 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 ,at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or it Exceeding K a Alternatively, affinity is the ratio of a specific binding interaction to a specific binding interaction in units of M. The equilibrium dissociation constant (K D ) (e.g., 10 -5 M~10 -13 M or less) In certain embodiments, specific binding can be defined as when the first and second moieties each About 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 More than M Below, or about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K below M D Binding of the first and second moieties to their respective targets Affinity can be determined using conventional techniques, e.g., competitive ELISA (enzyme-linked immunosorbent assay). , equilibrium dialysis, surface plasmon resonance (SPR) techniques (e.g., as outlined by the manufacturer) General procedure using BIAcore 2000 instrument), radioimmunoassay, etc. This can be more easily determined.

[0024] The first part is a cell surface molecule that is targeted for degradation via the endosomal / lysosomal pathway. Or it can be any type of moiety that is capable of binding to an extracellular molecule. In the present invention, the first moiety is a polypeptide, a ligand (e.g., a ligand for a cell surface receptor). , where their cell surface receptors are targeted for degradation), aptamers, nanoparticles, The second moiety is selected from a lysosomal targeting molecule, a nanoparticle, and a small molecule. In certain embodiments, the second moiety is a polypeptide. tides, ligands (e.g., ligands for lysosomal targeting molecules), aptamers, nanoparticles, and small molecules.

[0025] In some embodiments, when a portion of a bifunctional molecule is a polypeptide, the portion is The terms "antibody" and "immunoglobulin" refer to antibodies of any isotype or is an immunoglobulin (e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG 4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., heavy chain polypeptides and an antibody composed of a tetramer consisting of two dimers of a light chain polypeptide; a single-chain antibody; A cell surface molecule or an extracellular molecule (in the case of the first moiety) or a lysosomal targeting molecule (in the case of the second moiety) Antibody fragments (e.g., whole antibodies or single chain fragments) that retain specific binding to the antibody Antibody fragments) (including but not limited to Fv, single chain Fv (scFv), Fab, F(ab')2 , Fab'(scFv')2, diabodies, and nanobodies); chimeric antibodies; Monoclonal antibodies; fully human antibodies; humanized antibodies (e.g., humanized whole antibodies, humanized antibody fragments, etc.) and fusion proteins or fragments comprising an antigen-binding portion of an antibody and a non-antibody protein. or fragments thereof. The antibody may be detectably labeled, for example, with an in vivo imaging agent. Antibodies can be modified with other moieties, such as polyethylene glycol (PEG). The antibody can be further conjugated to a fusion to an antibody Fc region (or a fragment thereof), PEG, etc. Conjugates to, for example, increase the serum half-life of the antibody upon administration to a subject. The present invention may find use in

[0026] "Small molecule" means a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. The molecules are not made up of repeating molecular units such as those found in polymers.

[0027] As summarized above, the second moiety is a lysosomal targeting molecule. As used herein, a "lysosomal targeting molecule" refers to a molecule that specifically binds to the lysosomal cule. When bound by the second portion of the bifunctional molecule, the bifunctional molecule and its first portion form a It shuttles bound cell surface or extracellular molecules to lysosomes in the cell. , a cell surface molecule. Bifunctional molecules and cellular Cell surface or extracellular molecules are degraded by lysosomal enzymes, e.g., acid hydrolases. In this way, the bifunctional molecule binds to the cell surface molecule bound by the first portion. or extracellular molecules for degradation, and that targeting has implications for research and clinical practice. It is used in a variety of in vitro and in vivo applications, including floor applications.

[0028] The second moiety may bind to any suitable lysosomal targeting molecule. Non-limiting examples of the receptor include mannose-6-phosphate receptor (M6PR), sortilin ( sortilin), folate receptor, ASPGR, IFITM3, endosomal / lysosomal pathway molecules (e.g., LIMP-1, LIMP-2), and the like.

[0029] In some embodiments, the lysosomal targeting molecule to which the second moiety is attached is a mannoprotein. The M6PR is present throughout the body's tissues and acts as a receptor for myosin-6-phosphate. Delivery of monose-6-phosphate (M6P)-tagged cargo (e.g., acid hydrolase) to the Golgi compartment and transports M6PR from the extracellular space to lysosomes. For more information on M6PR, see e.g. For example, Gary-Bobo et al. (2007) Curr. Med. Chem. 14:2945 -2953;Das et al.(2016)ACS Macro Lett.5:8 09-813, and others. Examples of M6PR to which a second moiety can be attached The cation-dependent human M Cation-independent M6 provided as 6PR and UniProtKB-P11717 PR (also called insulin-like growth factor 2 receptor (IGF2R)). The ison-independent mannose 6-phosphate receptor binds mannose 6-phosphate (M6 P) Ligands for proteins such as IGF-II, retinoic acid, and plasminogen Its main function is to bind the M6P enzyme and transport it to the lysosome. However, various extracellular M6P glycoproteins, such as the latent TGFβ precursor, Urokinase-type plasminogen activator receptor, granzyme B, growth factors, herpes It can also regulate the activity of viruses, etc.

[0030] In certain embodiments, when the lysosomal targeting molecule is M6PR, the second moiety is M6P Anti-M6PR antibodies are available, including MOB-1772. z Recombinant anti-human M6PR antibody (Creative Biolabs), EPR6599; 2G11, MEM-238, EPR6599 and EPR7691 anti-M6PR antibodies (Ab cam) etc.

[0031] In some embodiments, when the lysosomal targeting molecule is M6PR, the second part The molecule comprises one or more M6PR ligands. In certain embodiments, the molecule comprises one or more M The 6PR ligand comprises one or more mannose-6-phosphates (M6Ps), , M6P has the following structure: [ka]

[0032] Alternatively, or in addition, the one or more M6PR ligands may be one or more includes multiple M6P analogs. An "M6P analog" is one that is not M6P but is a derivative of M6PR. It refers to a molecule that binds to the 6P recognition site. Some M6P analogs that have ester, sulfonate or malonate groups are It shows higher affinity and stronger stability in human serum than P itself. Features have been shown to be important for the binding of M6P to M6PR. The hydroxyl group at the 2-position of the aryl ring is axial, and strong binding to M6PR is observed. The distance between the negative charge and the pyranose ring is also important in the recognition of M6P by M6PR. Suitable analogs generally have a specific function against M6P in order to efficiently bind to M6PR. The single negative charge should be isosteric relative to the binding site of M6PR. Although the phosphorus atom is sufficient to allow the binding, two phosphorus atoms are not necessary to ensure recognition. The presence of a negative charge (as in the malonate and phosphonate isosteric analogs of M6P) has been shown to be beneficial for binding to M6PR. In some embodiments , when the one or more M6PR ligands include one or more M6PR analogues, The M6PR analogs or analogs may be substituted with one or more phosphonate M6P analogs (M6P n), malonate M6P analogues, carboxylate M6P analogues, sulfonate M6Ps analogs, acrylate M6P analogs, and the like. In some embodiments, one or more The M6PR analogs of the formula (I) may be one or more phosphonate M6P analogs having the structure M6Pn), where M + is any counter cation or hydrogen atom): [ka]

[0033] In some embodiments, one or more M6PR analogs have the following structure: + is any counter cation or hydrogen atom) Carboxylate M6P analogues include: [ka]

[0034] In some embodiments, one or more M6PR analogs have the following structure: + is any counter cation or hydrogen atom), P analogues include: [ka]

[0035] Recognition of M6P and M6P Analogs by M6PR and Bifunctional Molecules of the Disclosure Further details regarding M6P analogs that may be utilized can be found, for example, in Gary-Bobo et al. 2007) Curr. Med. Chem. 14:2945-2953, and Jean Jean et al. (2008) Bioorg Med Chem Lett.18(23):6 240-3, the disclosure of which is incorporated by reference in its entirety for all purposes. This is incorporated herein by reference.

[0036] In certain embodiments, when the second portion comprises one or more M6PR ligands, the second The portion may be 1-1000 M6PR ligands, e.g., 1-750, 1-500, 1- 250 pieces, 1~100 pieces, 1~75 pieces, 1~50 pieces, 1~40 pieces, 1~30 pieces, 1~20 pieces 1, 1 to 10 (e.g., 1 to 6), or 1 to 5 M6PR ligands. In some embodiments, when the second portion comprises one or more M6PR ligands: The second part is 10-50 pieces, 15-45 pieces, 20-40 pieces, or 25-35 pieces of M6 In certain embodiments, the second moiety comprises one or more M6PR ligands. If the second part contains 5 or more, 10 or more, 20 or more, 30 or more, 40 or more Above, 50 or more, 75 or more, 100 or more, 250 or more, 500 or more, 750 or more or more than 1000 M6PR ligands.

[0037] In some embodiments, when the second portion comprises one or more M6PR ligands The second portion displays one or more M6PR ligands (e.g., Such bifunctional molecules and their polymer scaffolds. One example of use is shown diagrammatically in Figure 1. In this example, the bifunctional molecule comprises an antibody as a first moiety. The antibodies bind either a target extracellular molecule (shown on the left) or a target cell surface molecule (shown on the right). The antibody can bind to either M6P ligand (denoted as "6P" in Figure 1). The displayed M6P ligand is conjugated to a polymer scaffold that displays the ligand. When the target binds to the cell surface M6PR, the M6PR binds the bifunctional molecule (and the bound target) (Target molecule) to the lysosome for degradation.

[0038] The second portion comprises a polymer scaffold that displays one or more M6PR ligands. In some cases, the polymer scaffold is a glycopolymer that includes one or more M6PR ligands. By way of example, the glycopolymer may be functionalized with one or more M6PR ligands. Glycoproteins containing one or more amino acids (e.g., natural and / or unnatural amino acids) When the glycopolymer is a glycoprotein, the glycoprotein may be an N- It can be a glycoprotein derived from a carboxyanhydride (NCA). Ring-opening polymerization of NCA monomers (ROP) are a class of synthetic and polypeptide compounds with a wide range of useful physical properties. In some embodiments, polymerization is a well-studied route to obtain a hybrid. is metal-catalyzed. It is suitable for large-scale synthesis of α-amino acid N-carboxyanhydrides. The approach is, for example, as described in Semple et al. (2016) Synthetic Commun. ics 47(1):53-61.

[0039] Exemplary Approach for Synthesizing Mannose-6-phosphate N-Carboxyanhydride The schematic diagram is shown in Figure 2. An exemplary approach for this purpose is shown diagrammatically in Figure 3. An exemplary approach for synthesizing carboxyanhydrides is shown generally in Figure 4. An exemplary approach for synthesizing 6-acrylate N-carboxyanhydride is shown in FIG. The schematic diagram for the synthesis of glucose-6-phosphonate N-carboxyanhydride is shown in An exemplary approach is shown diagrammatically in Figure 6. Mannose-6-phosphonate isothiocyanate An exemplary approach for synthesizing the esters is shown generally in FIG.

[0040] In certain embodiments, the bifunctional molecules of the present disclosure are capable of inhibiting the proliferation and proliferation of liver cells, e.g., hepatocytes (hepatocellular carcinoma ( A second antibody that specifically binds to a lysosomal targeting molecule expressed on the surface of HCC cells. A lysosomal target expressed on the surface of a liver cell to which the second moiety can bind. A non-limiting example of an activating molecule is the asialoglycoprotein receptor (ASGPR) (which binds glycoproteins from the blood). This receptor is a hepatic receptor that mediates the clearance of oxidative stress receptors (ASGR1 and ASGR2). Two proteins encoded by ASGR1 and ASGR2, asialoglycoprotein receptor 1 and and 2 (ASGR1 (UniProtKB-P07306-human) and ASGR2 (U niProtKB-P07307-human). ASGPR is a desialic acid Asialoglycoproteins are glycoproteins with exposed galactose and galactosamine residues. Receptors located on liver cells remove target glycoproteins from the circulation. ASGPR is highly expressed on the surface of hepatocytes, several human cancer cell lines, and liver cancers. will be done.

[0041] When the lysosomal targeting molecule is ASGPR, a suitable second moiety is an anti-ASG These include, but are not limited to, PR antibodies, ASGPR ligands, etc. According to an embodiment, such second moiety comprises one or more ASGPR ligands. Suitable ASGPR ligands include one or more N-acetylgalactosamines (Ga InNAc), one or more galactoses, one or more glucoses, and In certain embodiments, the combination of Such a second portion may comprise 1 to 1000 ASGPR ligands, for example 1 to 750, 1 to 200, 500 pieces, 1~250 pieces, 1~100 pieces, 1~75 pieces, 1~50 pieces, 1~40 pieces, 1~3 0, 1-20, 1-10 (e.g., 1-6), or 1-5 ASGPRs In some embodiments, the second portion comprises one or more ASGPRs. When the second portion contains a ligand, the second portion may contain 10 to 50, 15 to 45, 20 to 40, or In certain embodiments, the second moiety comprises 1 or more ASGPR ligands. or multiple ASGPR ligands, the second portion may comprise 3 or more, 5 or more, 10 or more, or more, 20 or more, 30 or more, 40 or more, 50 or more, 75 or more, 100 or more, 250 or more, 500 or more, 750 or more, or 1000 or more ASGPR regans Includes do.

[0042] According to some embodiments, the lysosomal targeting molecule is ASGPR and the second moiety When comprises one or more ASGPR ligands, the second moiety comprises one or more A In one non-limiting example, the second In certain embodiments, such a second moiety comprises a polymer comprising GalNAc. The molecule contains poly(GalNAc-co-Ala), the structure of which is provided below and in FIG. can be. [ka]

[0043] In certain embodiments, the lysosomal targeting molecule is ASGPR and the second moiety is one or more In the case where the second portion contains one (monovalent), two (bivalent) or multiple ASGPR ligands, the second portion may contain one (monovalent), two (bivalent) or multiple ASGPR ligands. , three (trivalent) or more than four ASGPR ligands, e.g., GalNAc, galactose Dendrimer bases containing ASGPR ligands independently selected from glucose and glucose For example, according to some embodiments, the second moiety is a monovalent, divalent or trivalent The GalNAc-containing dendrimer scaffold that may be used includes the following trivalent GalNAc-containing dendrimer scaffolds: A non-limiting example of a polymer scaffold is the following (referred to herein as Tri-GalNAc derivative): called Ndrima): [ka]

[0044] According to some embodiments, the second moiety is a monovalent, divalent or trivalent galactose-containing Non-limiting examples of trivalent galactose-containing dendrimer scaffolds that can be used include A typical example is the following (herein referred to as Tri-Gal dendrimer): [ka]

[0045] According to some embodiments, the second portion is a lysosome expressed on the surface of a liver cell. The first portion specifically binds to a hepatocyte (hepatocyte targeting molecule, e.g., ASGPR). The antibody specifically binds to a cell surface molecule expressed on human hepatocellular carcinoma (HCC) cells. Non-limiting examples of suitable cell surface molecules include growth factor receptors. Growth factor receptors of interest These include epidermal growth factor receptor (EGFR), C-Met, insulin-like growth factor 1 receptor ( IGF1R), fibroblast growth factor receptor 4 (FGFR4), HER2, and platelet-derived These include, but are not limited to, plasma membrane growth factor receptor (PDGFR).

[0046] In certain embodiments, the bifunctional molecule binds a growth factor on the surface of a hepatocellular carcinoma (HCC) cell. It is used to degrade the HCC (e.g., to treat HCC in an individual with HCC). in vivo), where the bifunctional molecule is capable of binding to EGFR, c-Met, IGF1R, FGFR4 or a first portion that specifically binds to HER2, and a second portion that specifically binds to ASGPR. (e.g., the second moiety is GalNAc, galactose and / or glutamine) The scaffold may comprise a scaffold (e.g., a polymer scaffold) that comprises an ASGPR ligand such as course.

[0047] According to some embodiments, the bifunctional molecule is expressed on fibrotic liver cells. They are used to degrade cell surface molecules (e.g., growth factors) that are targeted to the cell surface (e.g., in vivo when administered to an individual with liver fibrosis, where the bifunctional molecule promotes fibrosis. A first portion that specifically binds to a cell surface or extracellular protein (e.g., PDGFR) a second moiety that specifically binds to the ASGPR (e.g., the second moiety comprises a Ga One or more ASGPRs, such as lNAc, galactose and / or glucose The ligand may include a scaffold (eg, a polymer scaffold).

[0048] In certain embodiments, the bifunctional molecule binds to a cell surface molecule in the presence of only the first moiety or enhances degradation of cell surface or extracellular molecules relative to degradation of extracellular molecules According to some embodiments, the bifunctional molecule comprises only the first or second moiety. The degradation of cell surface or extracellular molecules in the presence of Enhances the degradation of extracellular molecules. In this context, "enhancing degradation" means that, under the same conditions, The cell surface or extracellular molecule is degraded in the presence of the bifunctional molecule, leaving only the first moiety alone. not decompose in the presence of the first moiety or the second moiety alone; or Under the same conditions, the cell surface molecule or extracellular molecule is expressed in the presence of the first moiety alone or in the presence of the first moiety alone. to a greater extent than would be decomposed in the presence of the first moiety or the second moiety alone. This means that a cell surface molecule or an extracellular molecule is degraded in the presence of and is decomposed in the presence of the first moiety alone or in the presence of the first moiety or the second moiety alone. If the compound is decomposed in the presence of a bifunctional molecule to a greater extent than would be expected, the decomposition In the presence of the active molecule, the or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5 .5x or more, 6x or more, 6.5x or more, 7x or more, 7.5x or more, 8x or more, 8.5x or more The concentration may be 9 or more, 9.5 or more, or 10 or more.

[0049] The bifunctional molecules of the present disclosure exhibit a reduced degradation of the cell surface molecule compared to the degradation of the cell surface molecule in the presence of the first moiety alone. Non-limiting examples of enhancing degradation of cell surface molecules are provided in Example 4 of the Experimental Section below. degradation of the cell surface molecule compared to degradation of the cell surface molecule in the presence of the first moiety alone. An example of a bifunctional molecule that does not enhance is provided in Example 5.

[0050] Those of skill in the art will appreciate that the bifunctional molecule of interest can be synthesized in the presence of the first moiety alone or in the presence of either the first moiety or degradation of the cell surface molecule or extracellular molecule in the presence of the second moiety alone. It can be readily determined whether the enzyme enhances the degradation of a molecule or an extracellular molecule. Non-limiting examples of suitable approaches to facilitate such determinations are provided in the Experimental Section below. Available in.

[0051] The bifunctional molecules of the present disclosure can be in any suitable format. In the method, the first portion is a polypeptide, the second portion is a polypeptide, and the bifunctional molecule is The product is a fusion protein that includes a first portion fused to a second portion. In other embodiments, the first portion can be fused directly to the second portion, e.g., a spacer domain. is placed between the first and second parts, indirectly to the second part When the bifunctional molecule is a fusion protein, the nucleic acid encoding the bifunctional molecule can be fused to the An expression vector comprising such a nucleic acid is also provided by the present disclosure. Also provided are cells (e.g., host cells) that contain any of the nucleic acids and / or expression vectors. Methods for producing such cells are also provided, which may include, for example, the use of suitable cell transfection methods. Transfection of nuclei using the transfection protocol and transfection reagents disclosed herein The method includes introducing either the acid and / or the expression vector into a cell. In the case of fusion proteins, methods of making the bifunctional molecules are also provided by the present disclosure. Such methods include culturing cells of the present disclosure under conditions in which the bifunctional molecule is expressed within the cells. This may include:

[0052] Other suitable formats of the bifunctional molecules of the present disclosure include conjugates. Thus, in some embodiments, the bifunctional molecules of the present disclosure are conjugated to a second moiety. In certain embodiments, the first moiety is an antibody and the second moiety is a The first moiety specifically binds to M6PR. By way of example, the first moiety may be an antibody and the second moiety may be an The molecule may bind to one or more M6PR ligands (e.g., one or more M6P and / or may comprise a polymer scaffold containing / displaying an M6P analogue, In some such embodiments, the second mer scaffold is conjugated to an antibody. The moiety comprises one or more amino acids functionalized with one or more M6PR ligands. A method for producing such a conjugate is also provided, The method includes conjugating a first moiety to a second moiety. The first moiety is an antibody. and the second moiety is a glycopolymer described herein. Schematically shown in FIG. 12. In some embodiments, the method includes: For example, a first moiety may be conjugated to a polypeptide. In the case of a peptide (e.g., an antibody), conjugating can be achieved by conjugating a preselected portion of the first moiety. The method may include site-specifically conjugating a second moiety to the selected amino acid. In certain embodiments, the preselected amino acid is at the N-terminus or C-terminus of the first portion. In other embodiments, the preselected amino acid is located within the first portion, i.e., In some embodiments, the preselected amino acid is between the N-terminal amino acid and the C-terminal amino acid. The selected amino acid is a non-natural amino acid. Non-limiting examples of unnatural amino acids that can be provided in the first and / or second moieties include: dihydrogen, alkyne, alkene, aminooxy, hydrazine, aldehyde (e.g., formyl Glycine, e.g., SMART ag(trademark) technology), nitrones, nitrile oxides, cyclopropenes, norbornenes, isopropyl alcohols, A functional group selected from socianilide, aryl halide and boronic acid functional groups. Non-natural amino acids that can be incorporated and selected to provide the desired functional groups. Acids are known and can be used for example as described in Maza et al. (2015) Bioconjug Chem. 26 ( 9):1884-9;Patterson et al.(2014)ACS Chem .Biol.9:592-605;Adumeau et al.(2016)Mol. Imaging Biol.(2):153-65, and others.

[0053] In some embodiments, conjugating the first moiety to the second moiety comprises Conjugating the second moiety to a glycan on the first moiety, or vice versa. Such methods involve modifying one or more glycans of a first moiety to provide a second moiety. In one non-limiting example, the first moiety may include providing a functional group that can be bonded to the first moiety. N-glycans of (e.g., antibodies) can be modified to aldehyde groups by periodate oxidation. , which can then be functionalized with a second moiety, such as aminooxy M6Pn.

[0054] When the bifunctional molecule is a conjugate, the conjugation of the first moiety to the second moiety To facilitate gating, one or more linkers may be used. Non-limiting examples of linkers include ester linkers, amide linkers, maleimides or Maleimide-based linkers, valine-citrulline linkers, hydrazone linkers, N -Succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, Chuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, vinylsulfone-based linkers, tetraethylene glycol, etc. Linkers including, but not limited to, polyethylene glycol (PEG), Linkers containing propanoic acid, linkers containing caproic acid, and any combination thereof In certain embodiments, the linker is a linker that is capable of binding to a neutral pH (bloodstream pH 7.3) ~7.5), but is stable at low pH (pH 5 .0-6.5) and upon internalization into lysosomes (pH 4.5-5.0), Chemically labile linkers, such as acid cleavable linkers, are The linkers include hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, These include, but are not limited to, ester-based linkers. The linker may be an enzyme-labile linker, e.g., stable in the bloodstream but not in a target cell. During internalization of the target cell (e.g., cancer cell), e.g., lysosomal proteases in the lysosomes Enzyme-labile phosphorylations that undergo enzymatic cleavage by enzymes (such as cathepsins or plasmin) Enzyme-labile linkers include linkers containing peptide bonds, e.g., dipeptides. tide-based linkers, such as valine-citrulline linkers, e.g., maleimidocaproy Lu-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, valyl -alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker. Examples of linkers include, but are not limited to, chemically labile linkers, enzymatically labile linkers, Cleavable and non-cleavable linkers are known in the art and are described, for example, in Ducry & Stump (201 0) This is described in detail in Bioconjugate Chem. 21:5-13.

[0055] A number of strategies for conjugating the first and second moieties via a linker are available. For example, the first moiety can be a carboxyl group that covalently bonds the linker to the first moiety. where the linker acts as a "chemical handle" for the second moiety. ". Also, by way of example, the second portion may have a functional group capable of reacting with a linker. The linker may be derivatized by covalent attachment to a second moiety, where the linker is The functional group on the linker can react with the "chemical handle" of moiety 1. The functional groups can be varied and selected based on compatibility with the chemical handles of the first or second moieties. According to one embodiment, the chemical handle can be a non-natural amine having a chemical handle. In some embodiments, the hydroxyl group is provided by incorporating a hydroxyl group into the first or second moiety. Conjugation of the first and second moieties is by alkyne-azide cycloaddition. It is.

[0056] Other suitable formats for the bifunctional molecules of the present disclosure include bispecific antibodies. For example, the bifunctional molecules of the present disclosure can be characterized in that the first portion (e.g., the first Fab arm) is attached to a cell surface. a second portion (e.g., a second Fab arm) that specifically binds to a molecule or extracellular molecule; It may be a bispecific antibody that specifically binds to a lysosomal targeting molecule (e.g., M6PR). A schematic diagram of such a bispecific antibody is shown in Figure 19. In some embodiments, Bispecific antibodies are released from their targets in the reduced pH of the endosome. The strategy allows a given bispecific antibody to cycle with the receptor without degradation of the antibody. This allows for sequential delivery of cargo and target to the lysosome. Approaches for making bifunctional molecules are known. For example, bispecific antibodies. In this case, the bispecific antibody is a "knobs-into-hole" antibody. The KIH technology can be used to generate heterodimerized ribozymes. To facilitate this, CH3 Strategies for designing and producing KIHs are known, e.g. For example, Xu et al. (2015) MAbs 7(1):231-42; Carter et al. (200 1)J.Immunol.Methods 248(1-2):7-15;Ridgwa (1996) Protein Eng. 9(7):617-2; and Merck Ant et al. (1998) Nat. Biotechnol. 16(7):677-81 The following are listed.

[0057] composition As summarized above, the present disclosure provides a composition. The composition comprises a selenium-containing bifunctional molecule as described above. Any of the bifunctional molecules of the present disclosure, including any of the bifunctional molecules described in the Section The present invention may include any of the following, which are incorporated herein by reference but will not be repeated for the sake of brevity: stomach.

[0058] In certain aspects, the composition comprises a bifunctional molecule of the present disclosure present in a liquid medium. The medium may be an aqueous liquid medium such as water, a buffer solution, etc. One or more additives, e.g., salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N- (2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) , 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethane Sulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MO PS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TA In addition, protease inhibitors, glycerol, etc. may be present in such compositions. good.

[0059] Pharmaceutical compositions are also provided. The pharmaceutical compositions include any of the bifunctional molecules of the present disclosure, and and a pharma- ceutical acceptable carrier. A pharmaceutical composition generally comprises a therapeutically effective amount of a bifunctional molecule. A "therapeutically effective amount" refers to a dosage sufficient to produce a desired result, e.g., a beneficial or or a desired therapeutic (including prophylactic) outcome, e.g., a first portion of the bifunctional molecule specifically binds Individuals with a cell proliferation disorder (e.g., cancer) associated with a cell surface or extracellular molecule that An effective amount means an amount sufficient to result in a decrease in cell proliferation in a subject, or the like, at one or more doses. Multiple doses may be administered.

[0060] The bifunctional molecules of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More specifically, the bifunctional molecule is combined with a suitable pharma- ceutically acceptable excipient or diluent. By combining the ingredients, it can be formulated into a pharmaceutical composition, such as tablets, capsules, powders, granules, etc. The pharmaceutical composition can be formulated into a solid, semi-solid, or gaseous form such as a pharmaceutical preparation.

[0061] Formulations of the bifunctional molecules of the present disclosure suitable for administration to an individual (e.g., suitable for human administration) include: Test substances that are generally sterile and that have no contraindications for administration to an individual according to the chosen route of administration. The composition may be free of exothermic pyrogens or other contaminants.

[0062] In pharmaceutical dosage forms, the bifunctional molecules may be used alone or in combination with other pharma- ceutical active compounds. The following methods and excipients can be used in any suitable association and combination: Agents are merely examples and are in no way limiting.

[0063] For oral formulations, lactose, mannitol, corn starch or potato Traditional additives such as starch, crystalline cellulose, cellulose derivatives, gum arabic, Binders such as yeast starch or gelatin, corn starch, potato starch or disintegrants such as sodium carboxymethylcellulose, magnesium stearate or lubricants such as talc, and optionally diluents, buffers, wetting agents, preservatives and flavorings. Bifunctional molecules can be used alone or in combination to produce tablets, powders, granules or capsules. or in combination with suitable additives.

[0064] Bifunctional molecules are derived from vegetable oils or other similar oils, synthetic fatty acid glycerides, and higher fatty acid Dissolved, suspended or in aqueous or non-aqueous solvents such as esters or propylene glycol. or emulsifying the mixture, and, if desired, adding a solubilizing agent, an isotonicity agent, a suspending agent, an emulsifying agent, Conventional additives such as stabilizers and preservatives can be used to formulate the composition into an injectable preparation.

[0065] The pharmaceutical composition may be in liquid form, in lyophilized form, or in liquid form reconstituted from a lyophilized form. The lyophilized formulation should be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a dried composition involves adding a certain amount of purified water (typically the water used during lyophilization). The method involves adding back the amount of the solution (equivalent to the volume removed), but this is not sufficient to produce a pharmaceutical composition for parenteral administration. A solution containing an antimicrobial agent may also be used.

[0066] The aqueous formulation of the bifunctional molecule may be prepared in a pH buffer solution, for example, from about 4.0 to about 8.0, for example, about 4. The pH of the solution may be adjusted to a range of about 0.5 to about 7.5, for example about 5.0 to about 7.0. Examples of buffers suitable for the pH include phosphate buffers, histidine buffers, citrate buffers, These include succinate buffers, acetate buffers and other organic acid buffers. The buffer concentration can be, for example, , about 1 mM to about 100 mM, or about 5 mM to about 100 mM, depending on the buffer and the desired tonicity of the formulation. It may be about 50 mM.

[0067] How to use As summarized above, methods of using the bifunctional molecules of the present disclosure are also provided. In an embodiment, the method comprises the step of: This includes the use of any of the following techniques, which are incorporated herein but are described for brevity: Do not repeat.

[0068] In certain embodiments, methods for degrading cell surface or extracellular molecules are provided. Such methods involve the use of lysosomal targeting molecules to target cell surface or extracellular molecules to the lysosome for degradation. The cell surface or extracellular molecule is shuttled to the bifunctional molecule of the present disclosure under conditions that shuttle the molecule to the inoculum. Such methods are used in a variety of applications. In some embodiments, the method is carried out in vitro (e.g., in a tube, cell culture plate, or in a cell culture vessel). In other embodiments, the method may be performed in a laboratory (e.g., in a cell) and used, for example, in testing and / or research applications. In some embodiments, the method is performed in vivo (e.g., in an individual to whom the bifunctional molecule is administered). and are used, for example, in clinical / therapeutic applications.

[0069] In some embodiments, a therapeutically effective amount of any bifunctional molecule or any Methods are provided that include administering the pharmaceutical composition to an individual in need thereof. Any individual can be treated according to the subject method. Generally, such subjects are referred to as "mammals" or are "mammals", and these terms include animals from the orders Carnivora (e.g., dogs and cats), Rodentia ( mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees, It is used broadly to describe organisms within the class Mammalia, including humans, monkeys, and apes. In an embodiment, the individual is a human.

[0070] In some embodiments, an effective amount of the bifunctional molecule (or a pharmaceutical composition comprising the same) comprises: Alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents When administered in one or more doses (e.g., in combination therapy), the bifunctional molecule or or a pharmaceutical composition, as compared to symptoms in the individual in the absence of treatment. The symptoms of a medical condition (e.g., cancer, neurodegenerative disorders, etc.) are reduced by at least about 5%, at least about 10%, %, at least about 15%, at least about 20%, at least about 25%, at least about 30 %, at least about 40%, at least about 50%, at least about 60%, at least about 70 %, at least about 80%, at least about 90% or more.

[0071] In certain embodiments, the individual is diagnosed with amyloid-β deposits in the brain (e.g., Alzheimer's disease). ) or neurodegeneration characterized by tau protein deposition in the brain (e.g., tauopathy) apoE4 (e.g., apoE4 expressed from the ε4 allele of the APOE4 gene) , and targeted degradation of apoE4 to treat neurodegenerative disorders in individuals.

[0072] In some embodiments, a therapeutically effective amount of any bifunctional molecule or any The present invention provides a method for treating cancer comprising administering said pharmaceutical composition to an individual having cancer. According to the method, a first portion of the bifunctional molecule is a cell surface segment that at least contributes to cancer in the individual. The bifunctional molecules are used to specifically bind to molecules or extracellular molecules, and to bind to cell surface molecules or extracellular molecules. The targeted degradation of the exomolecule treats cancer in an individual. In certain embodiments, the first moiety is a cell surface molecules on cancer cells, ligands for cell surface molecules on immune cells, and ligands for cell surface molecules on immune cells. Ligands for cell surface molecules on cells, inhibitory immunoreceptors, and ligands for inhibitory immunoreceptors The antibody specifically binds to a molecule selected from the group consisting of nucleotides.

[0073] In certain embodiments, the individual is diagnosed with a tumor, such as a solid tumor, semi-solid tumor, primary tumor, metastatic tumor, or the like. In some embodiments, the individual has a cancer characterized by the presence of breast cancer, melanoma, lung cancer, or endometrial cancer. Colorectal cancer, prostate cancer, glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia (e.g., acute bone marrow cancer, Myeloid leukemia (AML)) Liver cancer (e.g., hepatocellular carcinoma (HCC), e.g., primary or recurrent HCC, non-Hodgkin's lymphoma, pancreatic cancer, thyroid cancer, any combination thereof, and and any subtype thereof.

[0074] In some embodiments, the individual is diagnosed with certain liver diseases, including hepatocellular carcinoma (HCC), (including but not limited to) and the method is for treating the disease. In certain embodiments, the individual has HCC and the first portion is a cell surface antigen on the individual's HCC cells. In certain embodiments, the first moiety binds to the surface molecule and the second moiety binds to the ASGPR. binds to a tumor-promoting protein on the HCC cells of the individual. The tumor-promoting protein is a growth factor on HCC cells. Typical examples include EGFR, C-Met, IGF1R and FGFR4. Bifunctional molecules such as The second portion may include any of the second portions that are combined with the first portion.

[0075] In certain embodiments, the individual has liver fibrosis and the method comprises treating liver fibrosis. For example, according to some embodiments, the individual has liver fibrosis, The first portion binds to a cell surface molecule on the individual's fibrotic liver cells, and the second portion binds to ASGPR. In certain embodiments, the first moiety binds to a fibrosis-promoting polypeptide on fibrotic liver cells of the individual. According to some embodiments, the fibrosis-promoting protein binds to a fibrotic A growth factor on liver cells. A non-limiting example of such a growth factor is PDGFR. Such bifunctional molecules may be used in combination with ASGPR, as described elsewhere herein. The second moiety may comprise either

[0076] In any method of using the bifunctional molecules of the present disclosure, in certain embodiments, The molecule is a relative degradation of the cell surface or extracellular molecule in the presence of the first moiety alone. In effect, the bifunctional molecules of the present disclosure enhance the degradation of cell surface or extracellular molecules. In any method using the method, according to some embodiments, the bifunctional molecule comprises a first compared to degradation of the cell surface or extracellular molecule in the presence of only the moiety or the second moiety. In contrast, it enhances the degradation of cell surface or extracellular molecules. Further details are provided in the Bifunctional Molecules section above and incorporated herein, but for brevity only. The description will not be repeated in order to

[0077] "Treat," "treating," or "treatment" "Treatment" refers to the treatment of a medical condition in an individual (e.g., a cell proliferative disorder, e.g., cancer, ) and improvement is at least an improvement in symptoms related to the medical condition being treated. The term "treatment" is used broadly to refer to any parameter that affects the outcome, such as at least a reduction in the magnitude of a symptom. Thus, treatment also involves treating an individual's medical condition, or at least identifying the medical condition. to prevent the patient from further suffering from symptoms that indicate a medical condition or at least The associated symptoms are completely suppressed, e.g., prevented from occurring or stopped, For example, this includes discontinued situations.

[0078] In certain aspects, the present disclosure is a method of enhancing antibody-dependent cellular cytotoxicity (ADCC). and administering a bifunctional molecule or pharmaceutical composition of the present disclosure to an individual in need of ADCC. In some embodiments, the first portion of the bifunctional molecule comprises an inhibitory In certain embodiments, the first functional group of the bifunctional molecule specifically binds to an immune receptor or its ligand. Part 1 is PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, or or members of the B7 family of immune checkpoint molecules.

[0079] The bifunctional molecules or pharmaceutical compositions can be used in in vivo and ex vivo methods, as well as in systemic and Any available method and route suitable for drug delivery, including intravenous and topical routes of administration, may be used. The conventional and pharma- ceutical acceptable routes of administration include nasal Intracavitary, intramuscular, intratracheal, subcutaneous, intradermal, topical, intraocular, intravenous, intraarterial, nasal, oral, and and other enteral and parenteral routes of administration. In some embodiments, administration is by parenteral administration. The route of administration may vary depending on the bifunctional molecule and / or the desired effect. The bifunctional molecule or drug can be tailored or adjusted as needed. The pharmaceutical compositions may be administered in a single dose or in multiple doses. In some embodiments, the bifunctional molecule or pharmaceutical composition is administered intravenously. The functional molecule or pharmaceutical composition can be, for example, administered intravenously for systemic delivery (e.g., intravenous infusion) or The drug is administered by injection into a localized area or into a topical site.

[0080] kit As summarized above, the present disclosure also provides kits. In some embodiments, the subject kits include The bifunctional molecules of the present disclosure (the bifunctional molecules described in the Bifunctional Molecules section above) are The present invention includes any of the above-mentioned functional molecules, which are incorporated herein by reference, but are not repeated here for the sake of brevity. and a cell surface molecule or an extracellular molecule to which the first portion specifically binds. In certain embodiments, the instructions include instructions for using the bifunctional molecule to degrade a molecule. The antibody may be used to detect cell surface molecules or cellular functions in vitro, e.g., for diagnostic and / or testing purposes. In other embodiments, the instructions are for, e.g., clinical / therapeutic use. For example, to degrade cell surface or extracellular molecules in vivo. For example, administering any of the bifunctional molecules or pharmaceutical compositions of the present disclosure to an individual in need thereof and instructions for administering the therapeutically active molecule or pharmaceutical composition. Such kits may contain a fixed amount of the bifunctional agent present in unit dose, e.g., ampoules, or in multidose form. In certain embodiments, the kit may comprise a functional molecule or pharmaceutical composition. One or more (e.g., two or more) unit doses (e.g., , ampoules).

[0081] The term "unit dose" as used herein refers to a single dose for human and animal subjects. It refers to physically discrete units suitable for dosage, each unit being sufficient to produce a desired effect. The amount of the unit dose is calculated based on the amount of the particular The functional molecule, the effect to be achieved, and the pharmacodynamics associated with the bifunctional molecule in an individual. In yet another embodiment, the kit includes a bifunctional molecule or pharmaceutical It may comprise a single multiple dose of the composition.

[0082] In another embodiment, any of the glycopolymers of the present disclosure (as described in the Bifunctional Molecules section above) can be used. The present invention includes any of the glycopolymers described herein, which are incorporated herein but are listed for brevity purposes. (without repeating the procedure), and for conjugating the glycopolymer to a molecule of interest. Kits containing instructions are provided for attaching glycopolymers to a molecule of interest. In some embodiments, the molecule of interest may further comprise a reagent for conjugating the molecule. The polypeptide is a polypeptide. Non-limiting examples of such polypeptides include antibodies. In certain embodiments, the molecule of interest is a cellular component as described in the bifunctional molecule section above. Specific to cell surface or extracellular molecules, including either cell surface or extracellular molecules No. 6,399,992, which is incorporated herein by reference, but for the sake of brevity will not be repeated.

[0083] The components of the kit may be in separate containers, or multiple components may be in a single container. May be present.

[0084] The instructions included in the kit may be recorded on a suitable recording medium. For example, the instructions may be written on paper or The instructions may be printed on a substrate such as a plastic or plastic. The may be present in the kit as a package insert or on the kit container or on a component thereof. It may be present on the label (i.e., associated with the packaging or inner packaging). In other embodiments, the instructions are stored on a portable flash drive, DVD, CD-ROM, M, electronic storage data file residing on a suitable computer-readable storage medium such as a diskette In yet other embodiments, the actual instructions are not present in the kit. However, if there is a means to obtain instructions from a remote source, for example via the Internet, An example of this embodiment is a method for viewing instructions and / or The kit includes a web address where the user can download the instructions, as well as The means for obtaining the instructions is recorded on a suitable substrate.

[0085] Glycopolymers, monomers and methods for producing same The present disclosure also provides glycopolymers. In some embodiments, the glycopolymers of the present disclosure The copolymer comprises a polymer scaffold and one or more mannose units attached to the polymer scaffold. and M6PR ligands. and / or the M6PR ligand is one of those described in the Bifunctional Molecules section above It may be either, which is incorporated herein but is not repeated for brevity. For example, the glycopolymer may be functionalized with one or more M6PR ligands. The glycoprotein may be a glycoprotein containing one or more amino acids. In certain embodiments, the glycoprotein may be one or more non-cyano anhydrides (NCAs). The M6PR ligand comprises one or more mannose-6-phosphates (M6Ps). Alternatively, or in addition, the one or more M6PR ligands may include one or more M6 P analogs, e.g., any M6P analogs described herein, e.g., one or more In some embodiments, the compound includes a number of mannose-6-phosphonates (M6Pn). The polymer scaffold can have 1-50 M6PR ligands, e.g., 1-40, 1-30, 1- 20, 1-10 (e.g., 1-6), or 1-5 M6PR ligands. In certain embodiments, the polymer scaffold comprises 10-50, 15-45, 20-40, or In certain embodiments, the polymer scaffold comprises 5 or more M6PR ligands. It comprises 10 or more, 20 or more, 30 or more, or 40 or more M6PR ligands.

[0086] Methods for producing the glycopolymers of the present disclosure are also provided. In some embodiments, the methods include: The preparation of the glycopolymer comprises polymerization. The polymerization may be by NCA polymerization, An example of this is shown diagrammatically in FIG.

[0087] In certain embodiments, the method of making a glycopolymer comprises the step of: In other embodiments, such methods include attaching one or more The study involved synthesizing a polymer scaffold from monomers functionalized with multiple M6PR ligands. For example, the scaffold may comprise one or more peptides functionalized with one or more M6PR ligands. The synthesis can be carried out from monomers of the formula (I) and the synthesis is by solid phase synthesis. The synthesis scheme is shown in Figure 9.

[0088] In a related aspect, the disclosure provides a monomer. The monomer comprises one or more mannose units. In certain embodiments, the compound is functionalized with a methylsulfate-6-phosphate receptor (M6PR) ligand. The monomers are amino acids. In some embodiments, the monomers are unnatural amino acids. The one or more M6PR ligands may be one or more mannose-6-phosphate (M Alternatively or in addition, one or more M6PR links may be included. The ligand may be one or more M6P analogs, such as any of the M6P analogs described herein. P analogues may be included, for example, mannose-6-phosphonate (M6Pn).

[0089] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following embodiments: .

[0090] 1. A bifunctional molecule comprising: a first portion that specifically binds to a cell surface or extracellular molecule; and a second portion that specifically binds to a lysosomal targeting molecule.

[0091] 2. Relative to the degradation of the cell surface or extracellular molecule in the presence of the first moiety alone. or the bifunctional molecule of embodiment 1, which enhances the degradation of a cell surface molecule or an extracellular molecule.

[0092] 3. The method of claim 1 or 2, wherein the first portion specifically binds to a cell surface molecule. The bifunctional molecule described herein.

[0093] 4. The bifunctional molecule of embodiment 3, wherein the cell surface molecule is a cell surface receptor.

[0094] 5. The bifunctional molecule of embodiment 4, wherein the cell surface receptor is a growth factor receptor.

[0095] 6. The method of any one of embodiments 1 to 5, wherein the cell surface molecule is present on a cancer cell. Functional molecules.

[0096] 7. The method according to embodiment 6, wherein the cell surface molecule is a tumor-associated or tumor-specific antigen. Functional molecules.

[0097] 8. The method of any one of embodiments 1 to 7, wherein the cell surface molecule is present on an immune cell. Bifunctional molecules.

[0098] 9. Immune cells include natural killer (NK) cells, macrophages, monocytes, neutrophils, and dendritic cells. cytocytes, T cells, B cells, mast cells, basophils and eosinophils; 9. The bifunctional molecule of embodiment 8.

[0099] 10. The bifunctional molecule of embodiment 8, wherein the cell surface molecule is an inhibitory immunoreceptor.

[0100] 11. The bifunctional antibody of embodiment 10, wherein the cell surface molecule is a ligand for an inhibitory immunoreceptor. functional molecule.

[0101] 12. The bifunctional antibody of embodiment 8, wherein the cell surface molecule is an immune checkpoint molecule. molecule.

[0102] 13. Immune checkpoint molecules include PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and B7 family members. 13. The bifunctional molecule of embodiment 12.

[0103] 14. The bifunctional molecule of embodiment 1, wherein the first portion specifically binds to an extracellular molecule. .

[0104] 15. The bifunctional peptide according to embodiment 14, wherein the extracellular molecule is a ligand for a cell surface receptor. molecule.

[0105] 16. The bifunctional molecule of embodiment 15, wherein the extracellular molecule is a growth factor.

[0106] 17. The bifunctional antibody of embodiment 15, wherein the extracellular molecule is a cytokine or chemokine. functional molecule.

[0107] 18. The bifunctional molecule of embodiment 14, wherein the extracellular molecule is an antibody.

[0108] 19. The bifunctional molecule of embodiment 18, wherein the antibody is an autoantibody.

[0109] 20. The method of embodiment 18 or 20, wherein the antibody specifically binds to a cell surface molecule or an extracellular molecule. 20. The bifunctional molecule of embodiment 19.

[0110] 21. The first moiety is selected from the group consisting of polypeptides, ligands, aptamers, nanoparticles and small molecules. 21. The bifunctional molecule of any one of the preceding embodiments, selected from the group consisting of:

[0111] 22. The bifunctional molecule of embodiment 21, wherein the first portion is a polypeptide.

[0112] 23. The bifunctional molecule of embodiment 22, wherein the first portion is an antibody.

[0113] 24. The antibody is an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv' 24. The bifunctional molecule of embodiment 23, which is a ribozyme, a ribozyme-binding domain ...

[0114] 25. The second moiety is selected from the group consisting of polypeptides, ligands, aptamers, nanoparticles and small molecules. 25. The bifunctional molecule of any one of the preceding embodiments, selected from the group consisting of:

[0115] 26. The lysosomal targeting molecule is the mannose-6-phosphate receptor (M6PR). 26. The bifunctional molecule of any one of embodiments 1 to 25.

[0116] 27. As described in embodiment 26, wherein the second portion comprises one or more M6PR ligands. Bifunctional molecules.

[0117] 28. One or more M6PR ligands are selected from the group consisting of one or more mannose-6-phosphoryl 28. The bifunctional molecule of embodiment 27, comprising an acid (M6P).

[0118] 29. The one or more M6PR ligands include one or more M6P analogues. 29. A bifunctional molecule according to embodiment 27 or embodiment 28.

[0119] 30. One or more M6P analogs are linked to one or more mannose-6-phosphonates. 30. The bifunctional molecule of embodiment 29, comprising a carboxylate (M6Pn).

[0120] 31. The method of any one of embodiments 27 to 30, wherein the second portion comprises 1 to 500 M6PR ligands. 31. The bifunctional molecule of any one of Forms 30.

[0121] 32. A polymer in which the second portion displays one or more M6PR ligands. 32. The bifunctional molecule of any one of embodiments 27 to 31, comprising a scaffold.

[0122] 33. The polymer scaffold is a glycopolymer containing one or more M6PR ligands. 33. The bifunctional molecule of embodiment 32.

[0123] 34. The glycopolymer is one or more functionalized with one or more M6PR ligands. 34. The bifunctional molecule of embodiment 33, wherein said GAG is a glycoprotein comprising a plurality of amino acids.

[0124] 35. The glycoprotein is an N-carboxyanhydride (NCA)-derived glycoprotein. 35. The bifunctional molecule of embodiment 34.

[0125] 36. The method of any one of the preceding claims, wherein the lysosomal targeting molecule is expressed on the surface of liver cells. 25. A bifunctional molecule according to any one of claims 24 to 25.

[0126] 37. The bifunctional antibody of embodiment 36, wherein the lysosomal targeting molecule is expressed on the surface of liver cells. functional molecule.

[0127] 38. The endothelial targeting molecule is a hepatocellular carcinoma (HCC) cell, a fibrotic liver cell, or its The bifunctional molecule of embodiment 36 or embodiment 37, which is expressed on both surfaces.

[0128] 39. The lysosome targeting molecule is the asialoglycoprotein receptor (ASGPR). 39. The bifunctional molecule according to any one of embodiments 36 to 38.

[0129] 40. The method of embodiment 39, wherein the second portion comprises one or more ASGPR ligands. The bifunctional molecule described above.

[0130] 41. One or more ASGPR ligands are one or more N-acetylglucosamines. 41. The bifunctional molecule of embodiment 40, comprising GalNAc.

[0131] 42. One or more ASGPR ligands contain one or more galactoses , embodiment 40 or embodiment 41.

[0132] 43. The one or more ASGPR ligands include one or more glucoses. 43. The bifunctional molecule of any one of embodiments 40 to 42.

[0133] 44. The method according to any one of embodiments 40 to 500, wherein the second portion comprises 1 to 500 ASGPR ligands. 44. The bifunctional molecule of any one of Forms 43.

[0134] 45. The method according to claim 1, wherein the second portion comprises a polymer comprising one or more ASGPR ligands. 45. The bifunctional molecule according to any one of embodiments 40 to 44.

[0135] 46. ​​The method of claim 45, wherein the second portion comprises poly(GalNAc-co-Ala). Bifunctional molecules.

[0136] 47. The second moiety comprises a monovalent, divalent or trivalent GalNAc-containing dendrimer scaffold. 42. The bifunctional molecule of embodiment 41.

[0137] 48. The method of embodiment 47, wherein the second portion comprises a trivalent GalNAc-containing dendrimer scaffold. 2. A bifunctional molecule according to claim 1.

[0138] 49. The second moiety comprises a monovalent, divalent or trivalent galactose-containing dendrimer scaffold. 43. The bifunctional molecule of embodiment 42.

[0139] 50. The method of embodiment 49, wherein the second portion comprises a trivalent galactose-containing dendrimer scaffold. The bifunctional molecule described above.

[0140] 51. An embodiment in which the first portion specifically binds to a cell surface molecule expressed on hepatocytes. 51. The bifunctional molecule of any one of embodiments 36 to 50.

[0141] 52. The bifunctional molecule of embodiment 51, wherein the cell surface molecule is a growth factor receptor.

[0142] 53. Growth factor receptors include epidermal growth factor receptor (EGFR), C-Met, and insulin. Insulin-like growth factor 1 receptor (IGF1R), fibroblast growth factor receptor 4 (FGFR4), and and platelet-derived growth factor receptor (PDGFR), The bifunctional molecule described herein.

[0143] 54. The first moiety is a polypeptide and the second moiety is a polypeptide, and is bifunctional. 10. The method of claim 1, wherein the molecule is a fusion protein comprising a first portion fused to a second portion. 54. The bifunctional molecule according to any one of embodiments 53 to 53.

[0144] 55. The bifunctional peptide according to embodiment 54, wherein the first moiety is directly fused to the second moiety. molecule.

[0145] 56. The method of embodiment 54, comprising a spacer domain between the first and second portions. The bifunctional molecule described above.

[0146] 57. A bifunctional molecule, Cell surface or extracellular molecules The embodiment from embodiment 1 is a bispecific antibody that specifically binds to a lysosomal targeting molecule. 57. The bifunctional molecule of any one of embodiments 56.

[0147] 58. A bifunctional molecule comprising a conjugate comprising a first moiety conjugated to a second moiety. 54. The bifunctional molecule of any one of the preceding embodiments, which is a conjugate.

[0148] 59. The bifunctional molecule of embodiment 58, wherein the first portion is an antibody.

[0149] 60. The second moiety defined in any one of embodiments 27 to 35, 60. A bifunctional molecule according to embodiment 58 or embodiment 59.

[0150] 61. Any of the embodiments 40 to 50, including a second moiety. 58 or 59. A bifunctional molecule according to embodiment 58 or 59.

[0151] 62. A nucleic acid encoding a bifunctional molecule according to any one of embodiments 54 to 57.

[0152] 63. An expression vector comprising the nucleic acid according to embodiment 62.

[0153] 64. A cell comprising the nucleic acid of embodiment 62 or the expression vector of embodiment 63.

[0154] 65. A nucleic acid according to embodiment 62 or an expression vector according to embodiment 63 is introduced into a cell. 65. A method of producing the cell of embodiment 64, comprising introducing

[0155] 66. The method of embodiment 58 or 59, comprising conjugating the first moiety to the second moiety. 60. A method for making a bifunctional molecule according to embodiment 59.

[0156] 67. Conjugating comprises site-specifically conjugating a first moiety to a second moiety. 67. The method of embodiment 66, comprising gating.

[0157] 68. The first portion comprises a polypeptide, and conjugation is performed to bind a second portion site-specifically conjugating to a preselected amino acid of the first portion; 68. The method of embodiment 67.

[0158] 69. Embodiment 6, in which the preselected amino acid is at the N-terminus or C-terminus of the first portion. 8. The method according to claim 8.

[0159] 70. The method of embodiment 68, wherein the preselected amino acid is within the first portion. .

[0160] 71. The method of any one of embodiments 68 to 7, wherein the preselected amino acid is an unnatural amino acid. 10. The method according to any one of claims 1 to 0.

[0161] 72. Any one of embodiments 66 to 71, wherein the first moiety is an antibody. How to.

[0162] 73. The second part is as defined in any one of embodiments 27 to 35. The method of any one of embodiments 66 to 72, wherein

[0163] 74. The second part is as defined in any one of embodiments 40 to 50. The method of any one of embodiments 66 to 72, wherein

[0164] 75. The conjugation is by alkyne-azide cycloaddition. The method according to any one of embodiments 66 to 74.

[0165] 76. A method for degrading a cell surface molecule or an extracellular molecule, comprising: Lysosomal targeting molecules target cell surface or extracellular molecules to lysosomes for degradation. The method of claim 1, wherein the cell surface molecule or extracellular molecule is detected under conditions of A method comprising contacting a bifunctional molecule according to any one of claims 1 to 5.

[0166] 77. The bifunctional molecule is capable of binding to a cell surface molecule or an extracellular molecule in the presence of only the first moiety. 76. Enhancement of degradation of cell surface or extracellular molecules relative to degradation. How to.

[0167] 78. The method according to embodiment 76 or embodiment 77, which is carried out in vitro.

[0168] 79. The method of embodiment 76 or embodiment 77, which is carried out in vivo.

[0169] 80. A pharmaceutical composition comprising: A bifunctional molecule according to any one of embodiments 1 to 61; and a pharma- ceutically acceptable carrier.

[0170] 81. The pharmaceutical composition according to embodiment 80, wherein the composition is formulated for parenteral administration.

[0171] 82. A pharmaceutical composition according to embodiment 80 or embodiment 81, for use in an individual in need thereof. The method comprises administering to

[0172] 83. An effective amount of a pharmaceutical composition according to embodiment 80 or embodiment 81 is administered to an individual with cancer. 23. A method of treating cancer comprising administering

[0173] 84. The first portion comprises a cell surface molecule on a cancer cell; a ligand for a cell surface molecule on a cancer cell; Cell surface molecules on immune cells, ligands for cell surface molecules on immune cells, inhibitory immune receptors, and a ligand of an inhibitory immunoreceptor, The method of embodiment 83.

[0174] 85. The individual has hepatocellular carcinoma (HCC), and the first portion is a cell surface on an HCC cell of the individual. 85. The method of claim 83 or 84, wherein the first moiety binds to a molecule and the second moiety binds to an ASGPR. The method described.

[0175] 86. The method of embodiment 85, wherein the first moiety binds to a growth factor on the individual's HCC cells. How to.

[0176] 87. The first portion is selected from the group consisting of EGFR, C-Met, IGF1R, and FGFR4. 91. The method of embodiment 90, wherein the growth factor is selected from the group consisting of:

[0177] 88. The second part is as defined in any one of embodiments 36 to 50. The method of any one of embodiments 85 to 87, wherein

[0178] 89. A method for enhancing antibody-dependent cellular cytotoxicity (ADCC), comprising: administering to an individual having the disease a pharmaceutical composition according to embodiment 80 or embodiment 81. .

[0179] 90. A method for enhancing the immunogenicity of cancer in an individual, comprising the steps of: 82. A method comprising administering to an individual the pharmaceutical composition of embodiment 81.

[0180] 91. The first part is selected from the group consisting of inhibitory immunoreceptors and ligands of inhibitory immunoreceptors. The method according to embodiment 89 or embodiment 90, which specifically binds to a molecule selected from .

[0181] 92. The method of any one of embodiments 82 to 91, wherein administering is by parenteral administration. The method according to any one of the above.

[0182] 93. The bifunctional molecule is capable of binding to a cell surface molecule or an extracellular molecule in the presence of only a first moiety. 82. Enhancement of degradation of cell surface or extracellular molecules relative to degradation of 92. The method of any one of embodiments 91 to 91.

[0183] 94. A bifunctional molecule according to any one of embodiments 1 to 61; Instructions for degrading a cell surface or extracellular molecule to which the first portion specifically binds and, including, a kit.

[0184] 95. The instructions are for degrading cell surface or extracellular molecules in vitro. The kit of embodiment 94.

[0185] 96. The instructions are for degrading a cell surface molecule or an extracellular molecule in vivo. The kit of embodiment 94,

[0186] 97. The bifunctional molecule according to any one of embodiments 1 to 61 or embodiment 80. Or a pharmaceutical composition according to embodiment 81, instructions for administering the bifunctional molecule or pharmaceutical composition to an individual in need thereof; and Including the kit.

[0187] 98. The bifunctional molecule or pharmaceutical composition is present in one or more unit doses. 98. The kit of claim 97.

[0188] 99. The bifunctional molecule or pharmaceutical composition is present in two or more unit doses, embodiment 97. The kit according to claim 1,

[0189] 100. A polymer scaffold; One or more mannose-6-phosphate receptors (M6PRs) attached to a polymer scaffold A glycopolymer comprising:

[0190] 101. A glycopolymer comprising one or more M6PR ligands functionalized thereon. or a glycoprotein comprising a plurality of amino acids. .

[0191] 102. The glycoprotein is an N-carboxyanhydride (NCA) derived glycoprotein. 102. The glycopolymer of embodiment 101.

[0192] 103. One or more M6PR ligands are linked to one or more mannose-6-lysine The glycoprotein according to any one of embodiments 100 to 102, comprising glyceryl phosphate (M6P). Copolymer.

[0193] 104. One or more M6PR ligands include one or more M6P analogues , A glycopolymer according to any one of embodiments 100 to 103.

[0194] 105. One or more M6P analogues may be linked to one or more mannose-6-phosphoryl The glycopolymer of embodiment 104, comprising a carboxylate (M6Pn).

[0195] 106. The polymer scaffold of any one of embodiments 100 to 500 M6PR ligands. 106. The glycopolymer according to any one of embodiments 105 to 105.

[0196] 107. A glycopolymer according to any one of embodiments 100 to 106. A method of manufacturing, comprising the steps of: Attaching one or more M6PR ligands to a polymer scaffold; or Polymer scaffolds are synthesized from monomers functionalized with one or more M6PR ligands. The method of claim 1,

[0197] 108. The scaffold comprises one or more M6PR ligands functionalized with one or more M6PR ligands. The method according to embodiment 107, wherein the polymerization from monomers is by solid phase synthesis. Method of posting.

[0198] 109. Glycopolymers are glycoprotein polymers and can be synthesized using solid-phase peptide synthesis. 109. The method of embodiment 108, wherein the compound is synthetic.

[0199] 110. A glycopolymer according to any one of embodiments 100 to 106, and instructions for conjugating the glycopolymer to a molecule of interest. to.

[0200] 111. The method according to claim 1, further comprising the step of conjugating the glycopolymer to a molecule of interest. 111. The kit according to embodiment 110.

[0201] 112. The method according to embodiment 110 or embodiment 111, wherein the molecule of interest is a polypeptide. Kit included.

[0202] 113. The kit according to embodiment 112, wherein the polypeptide is an antibody.

[0203] 114. An embodiment in which the molecule of interest specifically binds to a cell surface molecule or an extracellular molecule. A kit described in any one of embodiments 110 to 113.

[0204] 115. Functionalized with one or more mannose-6-phosphate receptor (M6PR) ligands Monomers that have been substituted.

[0205] 116. The monomer according to embodiment 115, wherein the monomer is an amino acid.

[0206] 117. The monomer according to embodiment 115, wherein the monomer is an unnatural amino acid.

[0207] 118. One or more M6PR ligands are linked to one or more mannose-6-lysine The compound according to any one of embodiments 115 to 117, comprising carboxylic acid (M6P). Mah.

[0208] 119. The one or more M6PR ligands include one or more M6P analogues; The monomer according to any one of embodiments 115 to 118.

[0209] 120. One or more M6P analogs may be one or more mannose-6-phosphoryl The monomer of embodiment 119, comprising a carboxylate (M6Pn). EXAMPLES

[0210] The following examples are offered by way of illustration and not by way of limitation.

[0211] experiment Example 1 - Mannose-6-phosphate polymers shuttle cargo to lysosomes In this example, a biotin cap ("first portion" as used herein - see FIG. 10) was ) and M6Pn polymer (the "second portion" as used herein). ) (see FIG. 10) was tested to confirm that the bifunctional molecule enters the extracellular space for degradation. NeutrAvidin-647 (NA647) - a strong biotin-binding protein that binds to lysosomes We determined whether the protein could mediate the translocation of lysosomal proteins. The bifunctional molecule is indeed a neutral marker, as colocalization of both with the neutral dye is observed. Fluorescence imaging demonstrating that avidin-647 can mediate trafficking to lysosomes The sizing results (below) are provided.

[0212] Next, various cell lines were tested as described above. Figure 11 shows that several cell lines were Data are presented demonstrating that NA 647 is uptaken in a 6Pn polymer-dependent manner. Considering these results, any cell line that has M6PR (e.g., CIM 6PR) shuttling cell surface and extracellular molecules into lysosomes in this way. and is not expected to be limited to the cell lines tested in this study.

[0213] Example 2 - M6Pn-conjugated antibodies shuttle targets to lysosomes In this example, the first moiety is an antibody that binds to a particular target of interest and the second moiety is A bifunctional molecule, an M6Pn-containing glycoprotein, was tested.

[0214] FIG. 13 shows that poly(M6Pn)-labeled antibodies shuttle their binding partners to lysosomes. A schematic (top) and fluorescence imaging data (bottom) are presented demonstrating that the In this example, mouse IgG-488 was used with an anti-mouse antibody bearing a poly(M6Pn) tag. Incubation with IgG antibody colocalizes both protein and lysosomal staining dyes It was merged.

[0215] FIG. 14 shows that poly(M6Pn)-labeled antibodies shuttle their binding partners to intracellular compartments. A schematic (top) and further data (bottom) are provided showing that the In this example, recombinant human apoE4 was combined with a mouse-derived anti-human apoE4 antibody, anti-mouse The cells were incubated with an IgG antibody or an anti-mouse antibody with a poly(M6Pn) tag. Significantly more uptake is observed with the M6Pn-containing secondary antibody.

[0216] FIG. 15 shows that poly(M6Pn)-labeled antibodies can shield their binding partners for degradation. Further data is provided demonstrating that M6P can be used to Incubation of cells with n-tagged cetuximab results in EGFR degradation For all cell lines tested, total EGFR loss was significantly reduced with cetuximab or This is observed in comparison with cetuximab with the pseudopolymer (GalNAc). Lane 1: Control. Lane 2: EGF (100 ng / mL, 1 h, + control). Lane 3: Setukima Lane 4: Cetuximab-GalNAc conjugate. Lane 5: Cetuximab- Lane 6: Cetuximab-M6P conjugate (short). The percentage of control was calculated by densitometry.

[0217] FIG. 16 shows that poly(M6Pn)-labeled antibody fragments target their binding partners for degradation. Data is provided to demonstrate that the M6Pn shuttle can be used in this example. By incubating cells with the Fab portion derived from cetuximab bearing the E GFR degradation was assessed. Total EGFR loss was assessed by cetuximab Fab alone or pseudopolymer. This is observed in comparison to cetuximab Fab(GalNAc), which has

[0218] FIG. 17 shows that poly(M6Pn)-labeled antibodies can shield their binding partners for degradation. Further data are provided demonstrating that CD7 Primary mouse-derived antibody against 1, anti-mouse IgG antibody, or poly(M6Pn) tagged The cells were incubated with an anti-mouse antibody that targets CD71 (transfected The degradation of the M6P tag-containing system resulted in significantly more degradation. vinegar.

[0219] FIG. 18 shows that poly(M6Pn)-labeled antibodies can shield their binding partners for degradation. Further data is provided demonstrating that M6P can be used to Incubating the cells with tagged anti-PDL1 antibody or anti-PDL1 antibody PDL1 degradation was evaluated by. Degradation was observed only with M6P-labeled anti-PDL1 antibody. .

[0220] Example 3 - ASGPR ligands shuttle cargo to lysosomes in hepatocytes Some current therapeutic approaches suffer from off-target effects. The present invention relates to a method for treating liver-related diseases, such as liver cancer and liver fibrosis, comprising administering to the liver a The targeted degradation of the asialoglycoprotein receptor (ASGPR) and Scavenger receptors, called scavenger receptors, are expressed exclusively or nearly exclusively in liver cells (hepatocytes) As shown diagrammatically in Figure 20 (left), ASGPR is expressed in the plasma membrane and endothelial cells. ASGPR constitutively recycles between the lysosome and the nucleosome for degradation. Transports extracellular glycoproteins into cells. Hepatocytes using a bifunctional molecule comprising one moiety and a second moiety comprising an ASGPR ligand The use of this receptor to degrade extracellular or membrane proteins is described herein. This is demonstrated in writing.

[0221] In the ASGPR-related examples herein, N-acetylgalactosamine (GalNAc ), in particular the poly(GalNAc-co-Ala) polymer shown in FIG. 20 (bottom left). The target molecule to be degraded is conjugated to a second moiety that contains a polymer. Bifunctional molecules containing antibodies were tested.

[0222] Bifunctional molecules containing ASGPR ligands indeed deliver cargo to lysosomes in hepatocytes. To assess whether a compound can be synthesized, the assay shown in FIG. Anti-mouse I conjugated to poly(GalNAc-co-Ala) polymer Bifunctional molecules containing IgG antibodies were tested to confirm that the bifunctional molecules inhibited extracellular fluorescently labeled mouse IgG ( We determined whether IgG-AF647 could be shuttled to the intracellular compartment of hepatocytes. In the examples, a bifunctional antibody comprising an anti-mouse IgG antibody conjugated to an M6PR ligand is We also tested the anti-inflammatory molecule. HEPG2 cells (a hepatocellular carcinoma cell line) were treated with 50 nM IgG-AF6 The cells were incubated with 47 and 25 nM of anti-mouse conjugate for 1 hour. The uptake was analyzed by flow cytometry. As shown by the bar graph, An approximately 8-fold increase in cell fluorescence was observed when the lNAc-containing conjugate was used, whereas , the M6Pn-containing conjugate induced a two-fold increase above background. In hepatocytes in which the expression level of ASGPR is higher than that of M6PR, cellular uptake was significantly increased. GalNAc-containing conjugates were more effective in inducing inflammation This demonstrates that.

[0223] FIG. 22 shows the same assay as described for FIG. 21, but with HUH7 cells (another hepatocyte Results of immunohistochemistry in a human ovarian cancer cell line (HUH7 cell line) are shown. Furthermore, when cells were treated with M6PR or ASGPR inhibitors ( monomeric M6P (mM6P) and monomeric GalNAc (mGalNAc), respectively. Incubation controls were also included. When cells were incubated with inhibitors, Incorporation was reduced, and uptake by GalNAc- or M6Pn-containing conjugates was indeed We showed that this is mediated by ASGPR or M6PR, respectively.

[0224] Example 4: Cetuximab-ASGPR ligand conjugate used in hepatocytes Efficient degradation of EGFR in Epidermal growth factor receptor (EGFR) regulates proliferation and angiogenesis in hepatocellular carcinoma (HCC) It is known that EGFR is a marker of HCC. 68% of HCC patients express EGFR on their HCC cells. Transplantation currently remains the best treatment option for patients with HCC and provides a good quality of life. The supply of deceased donor organs is limited. Although antibodies are often used for treatment, HCC cells express receptor tyrosine kinase (EGFR , HER2, HER3, c-Met, IGF1R) heterodimerization (same downstream effector phosphorylation of β-actin receptor β to restore oncogenic signaling via RTK crosstalk Resistance to these treatments develops by promoting cell death through the EGFR-blocking antibody, Seb. Tuximab failed in phase II clinical trials in patients with HCC.

[0225] In this example, an ASGPR ligand (in this example, The antibody was an anti-EGFR antibody conjugated to poly(GalNAc-co-Ala) ( In this example, a bifunctional molecule containing cetuximab inhibits EGFR uptake in HCC cells. The cetuximab-M6PR ligand was evaluated to determine whether it could induce the degradation of M6PR. The deconjugates were also tested.

[0226] HEP3B cells were used in the first experiment. The cells were treated with 10 nM cetuximab conjugate. The cells were incubated with lysate for 48 hours and then lysed for Western blot analysis. The Western blot (shown at the bottom of FIG. 23) shows five lanes: 1) no treatment; EGF (a known down-regulator of EGFR), 3) cetuximab, 4) cetuximab-Ga 1) a cetuximab-M6Pn conjugate; and 5) a cetuximab-M6Pn conjugate. As shown, the cetuximab-GalNAc conjugate efficiently inhibited EGRFR. degradation, and greater EGFR degradation compared to the cetuximab-M6Pn conjugate Vinculin was used as a loading control.

[0227] HEPG2 cells were used in the second experiment. The results are shown in Figure 24. Efficient degradation of EGFR was observed, possibly due to the effects of HEP3B and Due to the relative levels of EGFR in the EGFR-positive and HEPG2 cell lines, cetuximab-GalN The results were similar between the Ac and cetuximab-M6Pn conjugates. On the sides, the relative mRNA levels of these receptors and EGFR in the two cell lines are shown. (Available in public databases). Because EGFR levels are relatively low in Even in the case of tuximab-M6Pn conjugates, most membrane EGFR was degraded. and that the residual EGFR seen in Western blots is internal EGFR. ASGPR levels were also monitored across the different treatments. It was shown that ASGPR levels remained constant throughout the study.

[0228] Next, EGFR degradation in HEP3B cells treated with cetuximab conjugates A time course study was conducted to evaluate the effect of 1 By 2 hours, the cetuximab-GalNAc conjugate reduced EGFR levels by 50% Degradation increases at 48 hours. Cetuximab-M6Pn conjugate did not reduce EGFR levels below 50% at any of the time points tested.

[0229] Immunofluorescence experiments were performed to determine whether the residual EGFR was membrane or intracellular. The results are shown in Figure 26. The cell contours of HEP3B and cetuximab are Showing EGFR localization on the membrane. HEP3B treated with cetuximab-M6Pn conjugate When HEP3B is transfected with SETUXIMI, part of the EGFR is localized inside the cell and part remains on the membrane. When treated with the β-GalNAc conjugate, EGFR was barely observed on the membrane. However, the majority of EGFR is intracellular. The gates degrade most of the membrane-bound EGFR and show the residual ( Approximately 30%) EGFR appears to be an intracellular EGFR.

[0230] Example 5: HER2 mediated by trastuzumab alone and trastuzumab conjugates 2 decomposition] In this example, trastuzumab alone or in the presence of the GalNAc-containing polymer shown in FIG. In the presence of conjugated trastuzumab ("Trastuzumab-GalNAc") We assessed the extent of HER2 degradation in HUH 7 and HEPG2 cells at 100 ng / mL. 7 and HEPG2 cells were treated with 10 nM trastuzumab or trastuzumab-GalNA c conjugate and incubated for 48 h, then for Western blot analysis The Western blot in Figure 27 shows three lanes for each cell line: 1) treatment 2) without trastuzumab, and 3) with trastuzumab-GalNAc. The figures show the average percentage of HER2 relative to control in each cell line. As shown in Fig. 1, trastuzumab alone or GalNAc-containing polypeptides were significantly inhibited in both cell lines. There was no statistical difference in HER2 degradation in the presence of trastuzumab conjugated to a lipar. Therefore, the trastuzumab-GalNAc conjugate did not inhibit trastuzumab. The HER2 degradation was not enhanced by either ribozyme or ribozyme alone compared with that in the presence of ribozyme alone.

[0231] Thus, the foregoing merely illustrates the principles of the present disclosure. Although not explicitly described or illustrated herein, the principles of the present invention are embodied herein and the spirit and It will be understood that various configurations may be devised that fall within the scope of the present invention. All examples and conditional phrases recited in the specification are consistent with the principles of the present invention and the principles of the inventors. The primary purpose is to aid the reader in understanding concepts contributed to the advancement of the art. and should not be construed as a limitation to such specifically recited examples and conditions. The present invention is also described with reference to the principles, aspects, and embodiments thereof. All statements herein encompass both structural and functional equivalents thereof. Additionally, such equivalents include both currently known equivalents and future developments. Both the equivalents, i.e. any development element that performs the same function regardless of structure, are also included. Accordingly, the scope of the present invention is intended to encompass all of the exemplary embodiments shown and described herein. It is not intended to be limited to the illustrative embodiments.

Claims

1. A bifunctional molecule, a first portion that specifically binds to a cell surface or extracellular molecule; and a second portion that specifically binds to a lysosomal targeting molecule.

2. compared to degradation of the cell surface or extracellular molecule in the presence of the first moiety alone. In contrast, the bifunctional molecule of claim 1 enhances the degradation of the cell surface or extracellular molecule. 。

3. The method of claim 1 or claim 2, wherein the first portion specifically binds to a cell surface molecule. Bifunctional molecules.

4. The bifunctional molecule of claim 3 , wherein the cell surface molecule is a cell surface receptor.

5. The bifunctional molecule of claim 4 , wherein the cell surface receptor is a growth factor receptor.

6. The bifunctional molecule according to any one of claims 1 to 5, wherein the cell surface molecule is present on a cancer cell. sex molecule.

7. The bifunctional antibody of claim 6, wherein the cell surface molecule is a tumor-associated or tumor-specific antigen. functional molecule.

8. The bifunctional molecule according to any one of claims 1 to 7, wherein the cell surface molecule is present on an immune cell. functional molecule.

9. The immune cells include natural killer (NK) cells, macrophages, monocytes, neutrophils, and dendritic cells. cytocytes, T cells, B cells, mast cells, basophils and eosinophils; The bifunctional molecule of claim 8.

10. The bifunctional molecule of claim 8 , wherein the cell surface molecule is an inhibitory immunoreceptor.

11. The bifunctional polypeptide of claim 10, wherein the cell surface molecule is a ligand for an inhibitory immunoreceptor. molecule.

12. The bifunctional molecule of claim 8, wherein the cell surface molecule is an immune checkpoint molecule. 。

13. The immune checkpoint molecule is PD-1, PD-L1, CTLA4, TIM3, L AG3, TIGIT, and B7 family members. Item 13. The bifunctional molecule according to item 12.

14. The bifunctional molecule of claim 1 , wherein the first portion specifically binds to an extracellular molecule.

15. The bifunctional molecule of claim 14, wherein the extracellular molecule is a ligand for a cell surface receptor. 。

16. The bifunctional molecule of claim 15 , wherein the extracellular molecule is a growth factor.

17. 16. The bifunctional polypeptide of claim 15, wherein the extracellular molecule is a cytokine or a chemokine. molecule.

18. The bifunctional molecule of claim 14 , wherein the extracellular molecule is an antibody.

19. The bifunctional molecule of claim 18 , wherein the antibody is an autoantibody.

20. 20. The antibody specifically binds to a cell surface or extracellular molecule according to claim 18 or 19.

2. A bifunctional molecule according to claim 1.

21. The first portion is selected from the group consisting of polypeptides, ligands, aptamers, nanoparticles and small molecules. The bifunctional molecule according to any one of claims 1 to 20, selected from the group consisting of:

22. 22. The bifunctional molecule of claim 21 , wherein the first portion is a polypeptide.

23. 23. The bifunctional molecule of claim 22, wherein the first moiety is an antibody.

24. The antibody is an IgG, a single chain Fv (scFv), a Fab, a ２ , (scFv') ２ 24. The bifunctional molecule of claim 23, which is a nucleotide sequence or a nanobody.

25. The second portion is selected from the group consisting of polypeptides, ligands, aptamers, nanoparticles and small molecules. The bifunctional molecule of any one of claims 1 to 24, selected from the group consisting of:

26. The lysosomal targeting molecule is a mannose-6-phosphate receptor (M6PR).

26. The bifunctional molecule according to any one of claims 1 to 25.

27. 27. The method of claim 26, wherein the second portion comprises one or more M6PR ligands. Functional molecules.

28. The one or more M6PR ligands are one or more mannose-6-phosphate (M6P).

29. The one or more M6PR ligands include one or more M6P analogues.

29. The bifunctional molecule of claim 27 or claim 28.

30. The one or more M6P analogs may comprise one or more mannose-6-phosphonates.

30. The bifunctional molecule of claim 29, comprising:

31. 31. The method of claim 27, wherein the second portion comprises 1 to 500 M6PR ligands. The bifunctional molecule according to any one of claims 1 to 4.

32. The second portion is a polymer that displays the one or more M6PR ligands. The bifunctional molecule of any one of claims 27 to 31, comprising a scaffold.

33. The polymer scaffold is a glycopolymer comprising the one or more M6PR ligands. The bifunctional molecule of claim 32.

34. The glycopolymer may comprise one or more M6PR ligands functionalized thereon. or a glycoprotein comprising a plurality of amino acids.

35. The glycoprotein is an N-carboxyanhydride (NCA) derived glycoprotein. Item 35. The bifunctional molecule according to item 34.

36. 26. Any of claims 1 to 25, wherein the lysosomal targeting molecule is expressed on the surface of liver cells.

3. The bifunctional molecule according to claim 1.

37. 37. The bifunctional polypeptide of claim 36, wherein the lysosomal targeting molecule is expressed on the surface of a hepatocyte. molecule.

38. The lysosome targeting molecule is a hepatocellular carcinoma (HCC) cell, a fibrotic liver cell, or an 38. The bifunctional molecule of claim 36 or 37, which is expressed on both surfaces.

39. The lysosomal targeting molecule is an asialoglycoprotein receptor (ASGPR). Item 39. The bifunctional molecule according to any one of items 36 to 38.

40. 40. The method of claim 39, wherein the second portion comprises the one or more ASGPR ligands. The bifunctional molecules described above.

41. The one or more ASGPR ligands are one or more N-acetylgalactosidases.

41. The bifunctional molecule of claim 40, comprising samine (GalNAc).

42. the one or more ASGPR ligands comprise one or more galactose; 42. The bifunctional molecule of claim 40 or 41.

43. The one or more ASGPR ligands include one or more glucose.

43. The bifunctional molecule according to any one of claims 40 to 42.

44. 44. Any of claims 40 to 43, wherein the second portion comprises 1 to 500 ASGPR ligands. The bifunctional molecule according to any one of claims 1 to 4.

45. the second portion comprises a polymer comprising the one or more ASGPR ligands; The bifunctional molecule according to any one of claims 40 to 44.

46. 46. ​​The method of claim 45, wherein the second portion comprises poly(GalNAc-co-Ala). Functional molecules.

47. The second portion comprises a monovalent, divalent or trivalent GalNAc-containing dendrimer scaffold. The bifunctional molecule of claim 41.

48. 48. The method of claim 47, wherein the second portion comprises a trivalent GalNAc-containing dendrimer scaffold. The bifunctional molecules described above.

49. The second portion comprises a monovalent, divalent or trivalent galactose-containing dendrimer scaffold. The bifunctional molecule of claim 42.

50. 50. The method of claim 49, wherein the second portion comprises a trivalent galactose-containing dendrimer scaffold. Bifunctional molecules.

51. 3. The method of claim 2, wherein the first portion specifically binds to a cell surface molecule expressed on a hepatocyte.

51. The bifunctional molecule according to any one of claims 6 to 50.

52. The bifunctional molecule of claim 51 , wherein the cell surface molecule is a growth factor receptor.

53. The growth factor receptor is an epidermal growth factor receptor (EGFR), C-Met, insulin-like Fibroblast growth factor 1 receptor (IGF1R), fibroblast growth factor receptor 4 (FGFR4), and Platelet-derived growth factor receptor (PDGFR), Bifunctional molecules.

54. The first portion is a polypeptide, the second portion is a polypeptide, and the second portion is a polypeptide. The functional molecule is a fusion protein comprising the first portion fused to the second portion. A bifunctional molecule according to any one of claims 1 to 53.

55. 55. The bifunctional peptide of claim 54, wherein the first moiety is directly fused to the second moiety. molecule.

56. 55. The method of claim 54, further comprising: The bifunctional molecules described above.

57. The bifunctional molecule is The cell surface molecule or extracellular molecule The lysosomal targeting molecule. The bispecific antibody according to any one of claims 1 to 56, which specifically binds to functional molecule.

58. The bifunctional molecule comprises the first moiety conjugated to the second moiety. The bifunctional molecule of any one of claims 1 to 53, which is a conjugate.

59. 59. The bifunctional molecule of claim 58, wherein the first portion is an antibody.

60. The method according to claim 58 or claim 35, comprising the second part as defined in any one of claims 27 to 35.

60. The bifunctional molecule of claim 59.

61. Claim 58 or claim 59, comprising a second part as defined in any one of claims 40 to 50.

60. The bifunctional molecule of claim 59.

62. A nucleic acid encoding a bifunctional molecule according to any one of claims 54 to 57.

63. 63. An expression vector comprising the nucleic acid of claim 62.

64. 64. A cell comprising the nucleic acid of claim 62 or the expression vector of claim 63.

65. Introducing the nucleic acid of claim 62 or the expression vector of claim 63 into a cell.

65. A method for producing the cell of claim 64, comprising:

66. 58. The method of claim 57, further comprising conjugating the first moiety to the second moiety.

60. A method for making the bifunctional molecule of claim 59.

67. The conjugating step site-specifically conjugates the first moiety to the second moiety.

67. The method of claim 66, comprising conjugating.

68. The first portion comprises a polypeptide, and the conjugating comprises and site-specifically conjugating a moiety to a preselected amino acid of said first moiety.

68. The method of claim 67, comprising:

69. 7. The method of claim 6, wherein the preselected amino acid is at the N-terminus or C-terminus of the first portion.

8. The method according to claim 8.

70. 69. The method of claim 68, wherein the preselected amino acid is within the first portion. 。

71. 71. The method of claim 68, wherein the preselected amino acid is an unnatural amino acid. The method according to claim 5.

72. The method of any one of claims 66 to 71, wherein the first moiety is an antibody.

73. The second part is as defined in any one of claims 27 to 35. 66-72. The method of any one of claims 66 to 72.

74. The second part is as defined in any one of claims 40 to 50. 66-72. The method of any one of claims 66 to 72.

75. 4. The method of claim 3, wherein the conjugating is by alkyne-azide cycloaddition. 66-74. A method according to any one of claims 66 to 74.

76. 1. A method for degrading a cell surface or extracellular molecule, comprising: The lysosomal targeting molecule targets the cell surface or extracellular molecule to the lysosome for degradation. The cell surface molecule or extracellular molecule of any one of claims 1 to 61 is shuttled to the The method of any one of claims 1 to 5, further comprising contacting said compound with the bifunctional molecule of claim 1.

77. The bifunctional molecule binds to the cell surface or extracellular molecule in the presence of only the first moiety. enhances degradation of said cell surface or extracellular molecule relative to degradation of said molecule. The method of claim 76.

78. 78. The method of claim 76 or claim 77, wherein the method is performed in vitro.

79. 78. The method of claim 76 or claim 77, wherein the method is performed in vivo.

80. 1. A pharmaceutical composition comprising: A bifunctional molecule according to any one of claims 1 to 61; and a pharma- ceutically acceptable carrier.

81. 81. The pharmaceutical composition of claim 80, wherein the composition is formulated for parenteral administration.

82. Administering the pharmaceutical composition of claim 80 or claim 81 to an individual in need thereof. The method includes:

83. Administering an effective amount of the pharmaceutical composition of claim 80 or claim 81 to an individual having cancer.

46. ​​A method of treating cancer comprising:

84. The first portion is a cell surface molecule on a cancer cell, a ligand for a cell surface molecule on a cancer cell, an immunoglobulin Cell surface molecules on immune cells, ligands for cell surface molecules on immune cells, inhibitory immune receptors, and and a ligand of an inhibitory immunoreceptor. The method of claim 83.

85. The individual has hepatocellular carcinoma (HCC), and the first portion comprises a cytoplasmic portion of the HCC cell of the individual.

83. The method of claim 82, wherein the first moiety binds to a cell surface molecule and the second moiety binds to an ASGPR.

84. The method according to claim 84.

86. 86. The method of claim 85, wherein the first portion binds to a growth factor on a HCC cell of the individual. How to.

87. The first portion is selected from the group consisting of EGFR, C-Met, IGF1R and FGFR4.

91. The method of claim 90, wherein the antibody binds to a growth factor selected from the group consisting of:

88. The second part is as defined in any one of claims 36 to 50.

88. The method according to any one of claims 85 to 87.

89. A method for enhancing antibody-dependent cellular cytotoxicity (ADCC), comprising administering to an individual in need of ADCC.

82. A method comprising administering a pharmaceutical composition of claim 80 or claim 81 to a

90. 82. A method for enhancing the immunogenicity of cancer in an individual, comprising the steps of: The method comprises administering to an individual the pharmaceutical composition described above.

91. The first portion is selected from the group consisting of an inhibitory immunoreceptor and a ligand of an inhibitory immunoreceptor.

91. The method of claim 89 or claim 90, wherein the antibody specifically binds to a molecule selected from the group consisting of:

92. 92. The method of claim 82, wherein the administering is by parenteral administration. The method described.

93. The bifunctional molecule binds to a cell surface or extracellular molecule in the presence of only the first moiety. enhances degradation of the cell surface or extracellular molecule relative to degradation of 82-91. The method of any one of claims 82 to 91.

94. A kit comprising: A bifunctional molecule according to any one of claims 1 to 61; The first portion is a molecule that specifically binds to a cell surface or extracellular molecule. A kit comprising:

95. The instructions are for degrading the cell surface or extracellular molecule in vitro.

95. The kit of claim 94,

96. The instructions are for degrading the cell surface or extracellular molecule in vivo.

95. The kit of claim 94.

97. A kit comprising: A bifunctional molecule according to any one of claims 1 to 61 or claim 80 or claim 81. A pharmaceutical composition according to claim 81, Instructions for administering said bifunctional molecule or pharmaceutical composition to an individual in need thereof. and, including, a kit.

98. The bifunctional molecule or pharmaceutical composition is present in one or more unit doses.

97. The kit according to claim 97.

99. 98. The method of claim 97, wherein the bifunctional molecule or pharmaceutical composition is present in two or more unit doses. Kit included.

100. A polymer scaffold; One or more mannose-6-phosphate receptors (M6P) attached to the polymer scaffold R) a ligand.

101. The glycopolymer may comprise one or more M6PR ligands functionalized thereon.

101. The glycopolymer of claim 100, which is a glycoprotein comprising a plurality of amino acids.

102. The glycoprotein is an N-carboxyanhydride (NCA) derived glycoprotein. The glycopolymer according to claim 101.

103. The one or more M6PR ligands are one or more mannose-6-phosphate The glycopolymer according to any one of claims 100 to 102, comprising (M6P).

104. The one or more M6PR ligands include one or more M6P analogues. The glycopolymer according to any one of claims 100 to 103.

105. The one or more M6P analogs may comprise one or more mannose-6-phosphonates. The glycopolymer of claim 104, comprising:

106. Claims 100-105, wherein the polymer scaffold comprises 1 to 500 M6PR ligands. The glycopolymer according to any one of claims 1 to 5.

107. A method for producing a glycopolymer according to any one of claims 100 to 106, 、107 conjugating one or more M6PR ligands to said polymer scaffold; or The polymer scaffold is synthesized from monomers functionalized with one or more M6PR ligands. The method of claim 1, further comprising:

108. The scaffold comprises one or more peptides functionalized with the one or more M6PR ligands.

108. The method of claim 107, wherein the polymer is polymerized from monomers and the synthesizing is by solid phase synthesis. The method described.

109. the glycopolymer is a glycoprotein polymer, and the synthesizing is a solid phase peptide 109. The method of claim 108, which is synthetic.

110. A glycopolymer according to any one of claims 100 to 106, and instructions for conjugating the glycopolymer to a molecule of interest. kit.

111. further comprising a reagent for conjugating the glycopolymer to a molecule of interest. The kit of claim 110.

112. 112. The kit of claim 110 or 111, wherein the molecule of interest is a polypeptide.

113. The kit of claim 112, wherein the polypeptide is an antibody.

114. 110. The method of claim 110, wherein the molecule of interest specifically binds to a cell surface molecule or an extracellular molecule. The kit according to any one of claims 1 to 113.

115. Functionalized with one or more mannose-6-phosphate receptor (M6PR) ligands monomer.

116. 116. The monomer of claim 115, wherein the monomer is an amino acid.

117. 116. The monomer of claim 115, wherein the monomer is an unnatural amino acid.

118. The one or more M6PR ligands are one or more mannose-6-phosphate The monomer according to any one of claims 115 to 117, comprising (M6P).

119. The one or more M6PR ligands include one or more M6P analogues. The monomer according to any one of claims 115 to 118.

120. The one or more M6P analogs may comprise one or more mannose-6-phosphonates. The monomer of claim 119, comprising:

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