Bifunctional molecules and related compositions and methods for lysosome targeting

Abstract

Provides a bifunctional molecule. [Solution] A bifunctional molecule is provided, comprising a first part containing a protein that specifically binds to cell surface molecules or extracellular molecules, and a second part containing a scaffold for displaying one or more ligands that specifically bind to an asialoglycoprotein receptor (ASGPR). The bifunctional molecule is used, for example, for targeted degradation of cell surface and extracellular molecules (e.g., proteins) via the endosomal / lysosomal pathway.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is based on U.S. Provisional Patent Application No. 62 / 782,19, filed on December 19, 2018. Patent No. 3, and U.S. Provisional Patent Application No. 62 / 932,347, filed on November 7, 2019. These applications assert the interest of the patent, and these applications are incorporated herein by reference in their entirety. Born.

[0002] Statement of government support This invention is in accordance with Contracts CA227942, GM059907, awarded by the National Institutes of Health. , and was carried out with government support under GM123636. The government has a 1 They have certain rights. [Overview of the Initiative]

[0003] The first part specifically binds to cell surface molecules or extracellular molecules, and the lysosomal targeting molecule A bifunctional molecule is provided, comprising a second part that specifically binds to a certain substance. The bifunctional molecule is, for example, For example, cell surface and extracellular molecules via the endosomal / lysosomal pathway (e.g., tan Used for targeted degradation of proteins. Compositions and kits containing bifunctional molecules, Furthermore, methods using bifunctional molecules are also provided. Methods for producing bifunctional molecules are also provided. It can be done. [Brief explanation of the drawing]

[0004] [Figure 1] This is a schematic diagram of a bifunctional molecule and its use according to one embodiment of the present disclosure. [Figure 2]A scheme for synthesizing mannose-6-phosphate N-carboxyanhydride according to one embodiment. This route provides access to mannose-6-phosphate glycans, for example, that are attached to serine residues, for use as monomers in N-carboxyanhydride polymerization. [Figure 3] A scheme for synthesizing mannose-6-phosphonate N-carboxyanhydride according to one embodiment. This route provides access to mannose-6-phosphonate glycans, for example, bonded to serine residues, for use as monomers in N-carboxyanhydride polymerization. The phosphonic acid group is a hydrolysis-stable phosphate group variant that has been previously 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 provides access to mannose-6-carboxylate glycan, for example, bound to a serine residue, for use as a monomer in N-carboxyanhydride polymerization. The carboxylate group is a hydrolysis-resistant phosphate group variant that has been previously shown to have superior serum stability compared to mannose-6-phosphate glycan. Mannose-6-carboxylate glycan has been previously shown to have a relative binding affinity of 0.3 to cation-independent M6PR (CIM6PR) compared to mannose-6-phosphate glycan. The ability to chemically modulate receptor-ligand interactions allows for finer control of biological applications and a reduction in off-target binding events. [Figure 5]Scheme for synthesizing mannose-6-acrylate N-carboxy anhydride according to one embodiment. This route enables access to, for example, mannose-6-acrylate glycans attached to serine residues for use as monomers in N-carboxy anhydride polymerization. The acrylate group is a variant of the phosphate group that is stable against hydrolysis, which has been previously shown to have excellent serum stability compared to mannose-6-phosphate glycans. Mannose-6-acrylate glycans have previously been shown to have a relative binding affinity of 0.7 for CIM6PR compared to mannose-6-phosphate glycans. The ability to chemically regulate receptor-ligand interactions allows for more precise control of biological applications and reduction of off-target binding events. [Figure 6] Scheme for synthesizing glucose-6-phosphonate N-carboxy anhydride according to one embodiment. This route enables access to, for example, glucose-6-phosphonate glycans attached to serine residues for use as monomers in N-carboxy anhydride polymerization. Glucose-6-phosphonate residues have significantly weaker binding affinity for CIM6PR compared to mannose-containing glycans. [Figure 7] Scheme for synthesizing mannose-6-phosphonate isothiocyanate according to one embodiment. This route enables access to mannose-6-phosphonate isothiocyanate (M6Pn-ITC), which can be conjugated directly to, for example, lysine residues in proteins. Conjugation of multiple M6Pn-ITCs to multiple amino acids (e.g., lysine) within a given protein enables multivalent presentation of M6Pn glycans. [Figure 8]Figure of a general NCA polymerization scheme for synthesizing a scaffold for displaying M6P ligands according to one embodiment. Copolymers with other amino acid-derived NCAs can be synthesized similarly, providing access to a number of polymers with various structures and compositions having multiple M6P ligand residues. These materials are then deprotected to provide the complete polypeptide / glycan structure. [Figure 9] 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 synthetic conditions compared to materials derived from NCA polymerization. [Figure 10] Schematic diagram (top) and fluorescence imaging results (bottom) of an experiment showing that M6Pn polymers functionalized with biotin caps can mediate the transfer of extracellular neutravidin-647 (NA 647: a protein to which biotin binds strongly) from the extracellular space to lysosomes for degradation. Co-localization of both the protein and the lysosome staining dye is observed. [Figure 11] Data demonstrating that several cell lines show uptake of NA647 in an M6Pn polymer-dependent manner. Considering these results, it is predicted that any cell line having M6PR (e.g., CIM6PR) enables shuttling of proteins to lysosomes by this method and is not limited to the cell lines tested in this study. [Figure 12] Data showing 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 incubation with an azide-containing polymer for functionalization (bio-orthogonal copper-free strain-promoted click reaction). [Figure 13] A schematic diagram (top) and fluorescence imaging data (bottom) demonstrate 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 with a poly(M6Pn) tag, and both the protein and lysosomal staining dye were colocalized (merged). [Figure 14] A schematic diagram (top) and data (bottom) demonstrate that poly(M6Pn)-labeled antibodies can shuttle their binding partners into intracellular compartments. In this example, recombinant human apoE4 was incubated with mouse-derived anti-human apoE4 antibody, anti-mouse IgG antibody, or anti-mouse antibody with a poly(M6Pn) tag. Significantly greater uptake was 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 evaluated by incubating cells with cetuximab tagged with M6Pn. For all cell lines tested, loss of total EGFR was observed compared to cetuximab or cetuximab with a 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). Percentages of control were calculated by densitometry. [Figure 16] Data demonstrating that poly(M6Pn)-labeled antibody fragments can shuttle their binding partners to lysosomes. In this example, EGFR degradation was evaluated by incubating cells with cetuximab-derived Fab moieties bearing the M6Pn tag. Loss of total EGFR was observed compared to cetuximab Fab alone or cetuximab Fab with a pseudopolymer (GalNAc). [Figure 17] Data demonstrating that poly(M6Pn)-labeled antibodies can shuttle their binding partners for degradation. In this example, the degradation of CD71 (transferrin receptor) was evaluated by incubating cells with primary mouse-derived antibody against CD71, anti-mouse IgG antibody, or anti-mouse antibody with a poly(M6Pn) tag. Systems containing the M6P tag resulted 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 evaluated by incubating cells with an anti-PDL1 antibody or anti-PDL1 antibody with an M6P tag. Degradation was observed only with the M6P-labeled anti-PDL1 antibody. [Figure 19] A schematic diagram of targeted extracellular protein degradation using a bifunctional molecule, which is a bispecific antibody. In this example, a bispecific antibody against CIM6PR and a given target is used, which detaches from the target at the reduced pH of the endosome. This strategy allows the given bispecific antibody to cycle with the receptor, enabling continuous delivery of cargo and the target to the lysosome without antibody degradation. [Figure 20]This is a schematic diagram of a lysosomal targeting molecule to which a second portion of a bifunctional molecule can bind, according to embodiments of the present disclosure. In this example, the lysosomal targeting molecule is an asialoglycoprotein receptor (ASGPR) substantially exclusively expressed on liver cells (e.g., hepatocytes). As shown on the right, ASGPR is constitutively recycled between the plasma membrane and endosomes, thereby transporting extracellular glycoproteins into the cell for degradation in lysosomes. In the lower left, an exemplary bifunctional molecule is shown, comprising a first portion which is an antibody (e.g., an antibody that binds to molecules expressed on the surface of hepatocytes or molecules present in the extracellular space of hepatocytes) and a second portion which contains a polyvalent ASGPR ligand for binding to ASGPR. In this example, the second portion comprises a polymer of N-acetylgalactosamine (GalNAc), in particular the poly(GalNAc-co-Ala) polymer shown. [Figure 21] Schematic diagram and data illustrating the transfer of a target molecule into HEPG2 (hepatocellular carcinoma) cells using a bifunctional molecule comprising a first portion that binds to the target molecule (in this example, an antibody) and a second portion containing a GalNAc-containing polymer as shown in Figure 20. [Figure 22] Schematic diagram and data demonstrating efficient cell uptake via ASGPR in HUH7 (hepatocellular carcinoma) cells. [Figure 23] Schematic diagram and data showing the efficient degradation of EGFR in HEP3B (hepatocellular carcinoma) cells using a bifunctional molecule containing cetuximab (first part) conjugated to a second part containing a GalNAc-containing polymer, as shown in Figure 20. EGFR degradation data for a conjugate containing cetuximab (first part) conjugated to a second part containing an M6PR ligand are also shown. [Figure 24]Figure 20 shows data demonstrating the efficient degradation of EGFR in HEPG2 cells using a bifunctional molecule containing cetuximab (first portion) conjugated to a second portion containing a GalNAc-containing polymer. EGFR degradation data for a conjugate containing cetuximab (first portion) conjugated to a second portion containing an M6PR ligand are also shown. [Figure 25] Data from a time-series study evaluating EGFR degradation in HEP3B cells over time. [Figure 26] Immunofluorescence data shows that while treatment of HEP3B cells with the above cetuximab conjugate degrades most of the membrane EGFR, residual EGFR remains inside the cell. [Figure 27] Western blot data showing the degree of HER2 degradation in HUH7 and HEPG2 cells, either with trastuzumab alone or in the presence of trastuzumab conjugated to a GalNAc-containing polymer. [Modes for carrying out the invention]

[0005] The first part specifically binds to cell surface molecules or extracellular molecules, and the lysosomal targeting molecule A bifunctional molecule is provided, comprising a second part that specifically binds to a certain substance. The bifunctional molecule is, for example, For example, cell surface and extracellular molecules via the endosomal / lysosomal pathway (e.g., tan Used for targeted degradation of proteins. Compositions and kits containing bifunctional molecules, Furthermore, methods using bifunctional molecules are also provided. Methods for producing bifunctional molecules are also provided. It can be done.

[0006] Before the bifunctional molecules, compositions, kits, and methods described herein are described in more detail, bifunctional Potential molecules, compositions, kits, and methods are not limited to the specific embodiments described. It should be understood that, naturally, this can change. Bifunctional molecules, compositions, The scope of the kit and method is limited only by the appended claims, therefore, this specification The terminology used is intended solely to describe specific embodiments and not to limit them. Please understand that this is not the intended meaning.

[0007] If a range of values ​​is provided, the range between the upper and lower limits of that range, unless otherwise specified in the context. Unless otherwise specified, each intermediary value up to one-tenth of the lower limit unit, and its description. Any other listed or intervening value within the range may be used for bifunctional molecules, compositions, kits, and methods. It should be understood that these are included within the law. The upper and lower limits of these smaller ranges are independent. They may be included in an even smaller range, and these are also particularly excluded from the range described. Apart from the stated limitations, the bifunctional molecules, compositions, kits, and methods are included within the description. If the range includes one or both of the limits, then either of the limits included or The scope that excludes both is also included in bifunctional molecules, compositions, kits, and methods.

[0008] Certain ranges are presented herein with the term “approximately” preceding the numerical value. The term "approximately" refers to the exact number that precedes it, and the approximate value of the term that precedes it. or is used herein to provide literal support for approximate numbers. In determining whether a number is close to or approximates a particular list of numbers, proximity or Numbers that approximate but are not listed are specifically listed in the context in which they are presented. It can be a number that is practically equivalent to the previous number.

[0009] Unless otherwise defined, all technical and scientific terms used herein are two The same meaning as that generally understood by those skilled in the art to belong to functional molecules, compositions, kits, and methods. Having a taste. Any bifunctional molecule or composition similar or equivalent to those described herein. The kits and methods also include the implementation or testing of bifunctional molecules, compositions, kits and methods. These can be used, but are representative exemplary bifunctional molecules, compositions, kits, and methods. This will be explained here.

[0010] All publications and patents referenced herein refer to the respective publications or patents referenced in this specification. This line is included by reference as if specifically and individually indicated to be incorporated by Disclose the relevant methods and / or materials incorporated into the book and cited in the publication. To describe this, it is incorporated herein by reference. Citations of publications prior to the filing date are used. This is a disclosure regarding the publication year, and the publication year provided may require individual verification of the actual publication date. The publication date may differ from that of the publication date for bifunctional molecules, compositions, kits and methods. This should not be interpreted as acknowledging that there is no priority right.

[0011] As used herein and in the appended claims, the singular forms "a", "an", and The noun "the" refers to multiple objects unless explicitly indicated otherwise in the context. Please note that the claims may also be drafted in a way that excludes optional elements. Please note further that, in this way, this statement is "exclusively" in relation to the enumeration of elements of the claims. Literalization of the use of exclusive terms such as "only" or the use of "negative" limitation It is intended to serve as evidence.

[0012] For clarity, bifunctional molecules, compositions, and kits are described in the context of separate embodiments. And certain features of the method may also be provided by combining them in a single embodiment. This is recognized. Conversely, a bifunctional molecule, described in the context of a single embodiment for the sake of brevity. The various features of the composition, kit, and method can be described separately or in any suitable subcombination. It may be provided in the context. All combinations of embodiments are provided by this disclosure. This includes, in this specification, processes and in which such combinations can be implemented. / or any combination to the extent that it encompasses the composition is disclosed individually and explicitly. It is disclosed as if it had been done. Furthermore, embodiments describing such variable elements All listed subcombinations also relate to the bifunctional molecules, compositions, and kits of the present invention. Specifically encompassed by the method and each such subcombination individually And disclosed herein as if explicitly disclosed.

[0013] As will be apparent to those skilled in the art upon reading this disclosure, the individual cases described and illustrated herein Each embodiment may, without departing from the scope or spirit of the methods described herein, be used to describe several other embodiments. Individual features of any embodiment that can be easily separated or combined It has components and characteristics. Any enumerated method is in the order of the enumerated events. Or it can be performed in any other logically possible order.

[0014] bifunctional molecule This disclosure relates to a first portion that specifically binds to cell surface molecules or extracellular molecules, and to lysosomes. The present invention provides a bifunctional molecule comprising a second portion that specifically binds to a target molecule. This describes specific, non-limiting embodiments of functional molecules.

[0015] As summarized above, the bifunctional molecules of this disclosure are specific to cell surface molecules or extracellular molecules. It includes a first portion that binds to a cell surface molecule. In some embodiments, the first portion is a cell surface molecule. It specifically binds to. "Cell surface molecules" are, for example, those inserted into the cell membrane or cells. It has a membrane-spanning domain (e.g., a cell membrane tethering domain or a transmembrane domain). To do so, it refers to molecules bound to the cell membrane. Cell surface molecules are endosomes / lysosomal molecules. Targeted degradation via the cellular pathway may be desired for any cell surface molecule. Therefore, cell surface molecules are cell surface receptors. The target cell surface receptors are limited to... However, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormones Receptors, receptor tyrosine kinases, and the epidermal growth factor receptor (EGFR) family Body (e.g., HER2 (human epidermal growth factor receptor 2)), fibroblast growth factor receptor (FGFR) family receptors, vascular endothelial growth factor receptor (VEGFR) family Receptor, platelet-derived growth factor receptor (PDGFR) family receptor, rearran Receptors of the transfection (RET) receptor family Eph receptor family receptors, discoidin domain receptor (DDR) family Examples include receptors and mucin proteins (e.g., MUC1). In this case, the cell surface molecule is CD71 (transferrin receptor). In certain embodiments, Cell surface receptors include T cell receptors, B cell receptors, natural killer (NK) cell receptors, Macrophage receptor, monocyte receptor, neutrophil receptor, dendritic cell receptor, mast cell receptor, These are immune cell receptors selected from basophil receptors and eosinophil receptors.

[0016] In some embodiments, the first part is not via specific molecular interactions (and Therefore, bulk biophysical activity or aggregation activity (less susceptible to blocking) Through its function, it specifically binds to cell surface molecules that mediate its effect. A non-restrictive example of a molecule is mucin. Examples of mucins include MUC1, MUC16, and MU Examples include C2, MUC5 AC, MUC4, CD43, CD45, and GPIb. This is not limited to these.

[0017] In some embodiments, if the first part specifically binds to cell surface molecules, then the fine Cell surface molecules are present on cancer cells. "Cancer cells" are, for example, cells with abnormal cell growth. Abnormal cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth capacity, immune deficiency Tumor growth and / or development, as well as / or cell shape, in non-human animal models of the state. Characterized by one or more of the ability to promote any appropriate indicator of qualitative transformation. This refers to cells that exhibit a neoplastic cell phenotype and can be diagnosed. "Cancer cells" is used herein to mean This term can be used interchangeably with "tumor cells," "malignant cells," or "cancer cells," and refers to solid tumors, semi-solid tumors, and semi-solid tumors. Solid tumors, hematological malignancies (e.g., leukemia cells, lymphoma cells, myeloma cells, etc.), primary tumors It includes cancer cells such as tumors and metastatic tumors. In some embodiments, it is present on cancer cells. These cell surface molecules are tumor-associated antigens or tumor-specific antigens.

[0018] In certain embodiments, if the first part specifically binds to a cell surface molecule, the cell surface part The offspring are present on immune cells. In some embodiments, the cell surface molecules are T cells. Cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells They are present on immune cells selected from mast cells, basophils, and eosinophils. In certain embodiments The cell surface molecules present on immune cells are suppressive immune receptors. When this happens, "suppressive immune receptors" are receptors present on immune cells that negatively regulate the immune response. It is a body. Examples of inhibitory immune receptors that can be inhibited according to the method of this disclosure include these. Not limited to, but 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 superf Examples include family inhibitory immune receptors. Inhibitory immune receptors that can be inhibited according to the methods disclosed herein. Further examples of disease receptors include sialic acid-binding Ig-like lectin (Siglec) receptors. Examples include Siglec7 and Siglec9. Additional examples of suppressive immune receptors that may be harmed include, but are not limited to, CLEC4. Examples of C-type lectins include A(DCIR), Ly49Q, and MICL. Inhibition For more details on sex immune receptors, see, for example, Steevels et al. (2011) E. It can be found in ur.J.Immunol.41(3):575-587. How many In that configuration, cell surface molecules present on immune cells are ligands for inhibitory immune receptors. In certain aspects, cell surface molecules present on immune cells are immune checkpoint molecules. The first part is a non-limiting example of an immune checkpoint molecule to which it can specifically bind. For example, PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT and B7 The family members are listed.

[0019] As summarized above, the bifunctional molecules of this disclosure are specific to cell surface molecules or extracellular molecules. It includes a first portion that binds to the target. In some embodiments, the first portion is extracellular It binds specifically to the offspring. An "extracellular molecule" is a soluble molecule, and the vicinity of that soluble molecule... This refers to something located outside the cell membrane of a neighboring cell. Extracellular molecules include endosomes / lyso Targeted degradation via somal pathways may be desired for any extracellular molecule. In this case, the extracellular molecule is a ligand for the cell surface receptor. The enzymes include growth factors (for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF)). (etc.), cytokines (e.g., interleukins, interferons, tumor necrosis factor ( TNF), transforming growth factor β (TGF-β) (such cytokines This includes, but is not limited to, hormones (including any specific subtype), etc. In certain embodiments, the first portion specifically binds to apolipoprotein E4 (ApoE4). To combine.

[0020] In some embodiments, the first part specifically binds to an extracellular molecule, and the extracellular molecule Antibodies, for example, antibodies 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: Equine factor (RF), antinuclear antibody (ANA), antineutrophil cytoplasmic antibody (ANCA), anti-double chain DNA (anti-dsDNA), anti-centromere antibody (ACA), anti-histone antibody, cyclic citrate Phosphorylated peptide antibodies (CCPs), soluble nuclear antigen antibodies (e.g., anti-SS-A(Ro) and Anti-SS-B(La), anti-RNP, anti-Jo-1, anti-Sm, anti-Scl-70), cardiolipin Antibodies, beta-2 glycoprotein 1 antibody, antiphospholipid antibody (APA), lupus anticoagulant ( LA), diabetes-related autoantibodies, anti-tissue transglutaminase (anti-tTG), anti-gliazinase Antibodies (AGA), endogenous factor antibodies, parietal cell antibodies, thyroid autoantibodies (e.g., anti-TPO, TSH receptor antibody, smooth muscle antibody (SMA), anti-mitochondrial antibody (AMA), liver Closome type 1 antibody (anti-LKM-1), anti-glomerular basement membrane (GBM), acetylcholine receptor Examples include antimicrobial regurgitation (AChR) antibodies.

[0021] In some embodiments, the first part is a secreted protein that accumulates in disease (for example) For example, α-synuclein, cholesterol carriers (e.g., ApoB), infectious disease toxins ( For example, AB toxin, ESAT-6), infectious particles (for example, whole viruses, whole bacteria, etc.) ), coagulation factors (e.g., factor IX), any FDA-approved antibody that binds to extracellular molecules Target (e.g., TNF-alpha), any chemokine or cytokine (e.g., IL) -1, mediators of sepsis or chronic inflammation), protein hormones (for example) then, insulin, ACTH, etc.), protein mediators of mood disorders, energy homeostatic protein mediators (e.g., leptin, ghrelin, etc.), present in the bloodstream protein allergens, or antibodies against such allergens (e.g., in the case of peanut allergy), protein toxins (e.g., snake venom hyaluronidase, etc.) specifically binds to extracellular molecules such as.

[0022] In certain embodiments, the first portion specifically binds to a cell surface or extracellular molecule, and that cell surface or extracellular molecule is a mutant protein. In some embodiments, the bifunctional molecule causes the shuttling of the mutant protein to the lysosome and promotes its loading onto the major histocompatibility complex (MHC) (e.g., MHC I or MHC II), thereby promoting the recognition of the mutant protein by the immune system. In this regard, bifunctional molecules find use in making antibodies specific for mutant unwanted proteins (e.g., KIT).

[0023] "Specifically binds" means that the first portion and the second portion bind to their respective targets with an affinity of, for example, about 10 M or greater or a K -1 (i.e., the equilibrium association constant of the specific binding interaction in units of 1 / M). In certain embodiments, the first portion and the second portion bind to their respective targets with an affinity of about 10 a M M, 10 6 M -1 M, 10 7 M -1 M, 10 8 M -1 M, 10 9 M -1 M, 1010 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 The above K a They bind together. "High affinity" binding is at least 10 7 M -1 , few Kutomo 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 This refers to a bond at a specific point. Alternatively, affinity is a specific bond interaction measured in units of M. The equilibrium dissociation constant (K D )(For example, 10 -5 M~10 -13 As M (or less) It can be defined. In a particular embodiment, the specific binding is such that the first part and the second part each Approximately 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 This means binding together. Binding of the first and second parts to their respective targets. Affinity can be determined using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay). , equilibrium dialysis, surface plasmon resonance (SPR) technology (for example, outlined by the manufacturer) Using a standard procedure with the BIAcore 2000 instrument, for radioimmunoassays, etc. It can be decided more easily.

[0024] The first part is the cell surface molecule that is targeted for degradation via the endosomal / lysosomal pathway. Or it may be any type of part that can bind to extracellular molecules. (Specific embodiment) So, the first part is polypeptides, ligands (for example, ligands for cell surface receptors) (and in that case, the cell surface receptor is targeted for degradation), aptamer, na It is selected from particles and small molecules. The second part binds to lysosomal targeting molecules. It can be any type of part that can do so. In a particular embodiment, the second part is polyp Tides, ligands (e.g., ligands for lysosome-targeting molecules), aptamers, nanoparticles, And selected from small molecules.

[0025] In some embodiments, if the portion of the bifunctional molecule is a polypeptide, then the portion It is an antibody. The terms "antibody" and "immunoglobulin" refer to any isotype of antibody or This refers to immunoglobulins (e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG)). 4) IgE, IgD, IgA, IgM, etc.), total antibodies (e.g., heavy chain polypeptides and Antibodies composed of a tetramer made up of two dimers of light chain polypeptides; single-chain antibodies; fine Cell surface molecules or extracellular molecules (in the case of the first part) or lysosome-targeting molecules (in the second part) In the case of a partial antibody, the antibody fragment (e.g., whole antibody or single chain) maintains specific binding to the antibody fragment (e.g., whole antibody or single chain). Antibody fragments) (not limited to, but including Fv, single-chain Fv(scFv), Fab, F(ab')2) Chimeric antibodies; including Fab'(scFv')2, diabodies, and nanobodies; Monoclonal antibodies; fully human antibodies; humanized antibodies (e.g., humanized whole antibodies, humanized antibody dilutions) (Such as fragments); and fusion proteins containing the antigen-binding portion of an antibody and a non-antibody protein. or include those fragments. Antibodies can be detected, for example, by in vivo imaging agents. It can be recognized. The antibody is a part of other parts, for example, polyethylene glycol (PEG). It can be further conjugated to the antibody Fc region (or fragment thereof), PEG. Conjugates, for example, increase the serum half-life of antibodies when administered to a subject. It may find uses for this purpose.

[0026] "Small molecules" refer to compounds with a molecular weight of 1,000 atomic mass units (amu) or less. In some embodiments, the small molecules are 750 amu or less, 500 amu or less, and 400 It is less than or equal to amu, less than or equal to 300 amu, or less than or equal to 200 amu. In certain embodiments, subdivision The child is not made from repeating molecular units like those found in polymers.

[0027] As summarized above, the second part is lysosomal targeting molecule It specifically binds to cule. As used herein, "lysosome-targeting molecule" means When bonded by the second part of a bifunctional molecule, the bifunctional molecule and its first part This shuttles the bound cell surface molecules or extracellular molecules to the lysosomes inside the cell. It is a cell surface molecule. During delivery to and internalization of lysosomes, it is a bifunctional molecule and a microclav. Cellular surface molecules or extracellular molecules are broken down by lysosomal enzymes, such as acid hydrolases. In this way, the bifunctional molecule is bound to the cell surface molecule by the first part. Alternatively, extracellular molecules are targeted for degradation, and this targeting is used in research and clinical applications. It is used in a variety of in vitro and in vivo applications, including floor applications.

[0028] The second part can bind to any suitable lysosomal targeting molecule. Examples of non-specific examples of this include mannose-6-phosphate receptors (M6PR), soltirin ( sortilin, folate receptor, ASPGR, IFITM3, endosomal / lysosomal pathway Examples include molecules (e.g., LIMP-1, LIMP-2).

[0029] In some embodiments, the lysosome-targeting molecule to which the second portion binds is manno It is a methyl-6-phosphate receptor (M6PR). M6PRs are present throughout the body's tissues, Nose-6-phosphate (M6P) tagged cargo (e.g., acid hydrolase) in the Golgi compartment It also plays a role in transporting lysosomes from the extracellular space. For more information on M6PR, see the example. For example, Gary-Bobo et al. (2007) Curr. Med. Chem. 14:2945 -2953;Das et al.(2016)ACS Macro Lett.5:8 See 09-813 and others. An example of M6PR in which the second part can be joined. This is provided as UniProtKB-P 20645, a cation-dependent human M Cation-independent M6, offered as 6PR and UniProtKB-P11717. PR (also known as insulin-like growth factor 2 receptor (IGF2R)) is one example. The ion-independent mannose 6-phosphate receptor is located on the cell surface and contains mannose 6-phosphate (M6 P) Ligands such as carrier proteins, IGF-II, retinoic acid, and plasminogen It is a multifunctional protein that binds to lysosomes. Its main function is to bind the M6P enzyme and transport it to lysosomes. The purpose is to deliver various extracellular M6P glycoproteins, such as latent TGFβ precursors. Urokinase-type plasminogen activator receptor, granzyme B, growth factor, herpes It can also regulate the activity of viruses such as Svirus.

[0030] In a particular embodiment, if the lysosome-targeting molecule is M6PR, the second part is M6P This antibody specifically binds to R. Anti-M6PR antibodies are available, such as MOB-1772. z-recombinant anti-human M6PR antibody (Creative Biolabs), EPR6599, 2G11, MEM-238, EPR6599 and EPR7691 anti-M6PR antibodies (Ab Includes cam, etc.

[0031] In some embodiments, when the lysosome targeting molecule is M6PR, the second part Each part contains one or more M6PR ligands. In certain embodiments, one or more M 6PR ligands contain one or more mannose-6-phosphate (M6P) molecules, where The M6P has the following structure: [ka]

[0032] Alternatively, or in addition to that, one or more M6PR ligands may be used, one or more This includes multiple M6P analogues. An "M6P analogue" is an M6P that is not M6P but is an M6PR. This refers to molecules that bind to the 6P recognition site. Phosphonate group, carboxylate group, sulf Several M6P analogs having a phosphate group, sulfonate group, or malonate group are M6 It exhibits higher affinity and stronger stability in human serum than P itself. The characteristics have been shown to be important for the binding of M6P and M6PR. For example, Pyrano The hydroxyl group at position 2 of the ring is axial, and a strong bond to M6PR is observed. It can be inferred that the distance between the negative charge and the pyranose ring is also related to the recognition of M6P by M6PR. It plays a role. Suitable analogues are generally those that bind to M6P efficiently in order to bind to M6PR. It should be isosteric, and a single negative charge is connected to M6PR. It is sufficient to enable the combination, but phosphorus atoms are not necessary to ensure recognition, and two The presence of a negative charge (as in the malonate and phosphonate isoconforms and analogs of M6P) This has been shown to be beneficial for coupling to M6PR. In some embodiments, If one or more M6PR ligands contain one or more M6PR analogs, 1 One or more M6PR analogs are one or more phosphonate M6P analogs (M6P n) Malonate M6P analogues, carboxylate M6P analogues, sulfonate M6P derivatives This includes analogues, acrylate M6P analogues, etc. In some embodiments, one or more. The M6PR analog is one or more phosphonate M6P analogs having the following structure ( M6Pn) includes (wherein M + (where is any countercation or hydrogen atom): [ka]

[0033] In some embodiments, one or more M6PR analogs have the following structure (wherein M + One or more cations (where is any countercation or hydrogen atom) Includes a ruboxylate M6P analog: [ka]

[0034] In some embodiments, one or more M6PR analogs have the following structure (wherein M + One or more malonate M6 having any countercation or hydrogen atom Includes P analogues: [ka]

[0035] Recognition of M6P and M6P analogs by M6PR, and the bifunctional molecules of the present disclosure For more information on M6P analog analogs that may be used, see, for example, Gary-Bobo et al. 2007) Curr. Med. Chem. 14:2945-2953, and Jean Jean et al. (2008) Bioorg Med Chem Lett.18(23):6 It can be seen in 240-3, and its disclosure is by reference in its entirety for all purposes. This specification is incorporated herein.

[0036] In certain embodiments, if the second part includes one or more M6PR ligands, the second The portion contains 1 to 1000 M6PR ligands, for example, 1 to 750, 1 to 500, 1 to 250 pieces, 1~100 pieces, 1~75 pieces, 1~50 pieces, 1~40 pieces, 1~30 pieces, 1~20 pieces It contains 1 to 10 (e.g., 1 to 6) or 1 to 5 M6PR ligands. In some embodiments, if the second portion includes one or more M6PR ligands, The second part consists of 10-50, 15-45, 20-40, or 25-35 M6 The second part comprises a PR ligand. In certain embodiments, the second part comprises one or more M6PR ligands. If it includes, the second part is 5 or more, 10 or more, 20 or more, 30 or more, 40 or less Above, 50 or more, 75 or more, 100 or more, 250 or more, 500 or more, 750 or more The above, or containing 1000 or more M6PR ligands.

[0037] In some embodiments, the second portion may include one or more M6PR ligands. The second part displays one or more M6PR ligands (for example, Includes a polymer scaffold (functionalized by), such a bifunctional molecule and An example of its use is schematically shown in Figure 1. In this example, the bifunctional molecule is an antibody as the first part. It includes. Antibodies target extracellular molecules (shown on the left) or target cell surface molecules (shown on the right). It can bind to either of them. The antibody binds to the M6P ligand (labeled "6P" in Figure 1). It is conjugated onto a polymer scaffolding for display. The displayed M6P rig When the nucleotide binds to cell surface M6PR, M6PR acts as a bifunctional molecule (and the bound target). (Target) molecules are shuttled to lysosomes for degradation.

[0038] The second part includes a polymer scaffold that displays one or more M6PR ligands. In this case, the polymer scaffold is a glycopolymer containing one or more M6PR ligands. This is possible. For example, glycopolymers may be functionalized with one or more M6PR ligands. Sugars containing one or more amino acids (e.g., natural and / or unnatural amino acids) It may be a glycoprotein. If the glycopolymer is a glycoprotein, then the glycoprotein is N- It may be a glycoprotein derived from carboxyanhydride (NCA). Ring-opening polymerization of NCA monomers. (ROP) refers to synthetic polypeptides and polypeptides with a wide range of useful physical properties. • A well-studied pathway that results in hybridization. In some embodiments, polymerization It is metal-catalyzed. Suitable for large-scale synthesis of α-amino acid-N-carboxyanhydrides. The approach is, for example, Semple et al. (2016) Synthetic Commun This is described in ications 47(1):53-61.

[0039] Exemplary approaches for synthesizing mannose-6-phosphate N-carboxyanhydride Figure 2 shows a schematic representation. To synthesize mannose-6-phosphonate N-carboxyanhydride... An exemplary approach is schematically shown in Figure 3. Mannose-6-carboxylate N- An exemplary approach for synthesizing carboxyanhydrides is schematically shown in Figure 4. (Manno) An exemplary approach for synthesizing s-6-acrylate N-carboxyanhydride, Figure 5. This is outlined below. For the synthesis of glucose-6-phosphonate N-carboxyanhydride An exemplary approach is schematically shown in Figure 6. Mannose-6-phosphonate isothiocyanate An exemplary approach for synthesizing nates is schematically shown in Figure 7.

[0040] In certain embodiments, the bifunctional molecule of the present disclosure is used to control liver cells, for example, hepatocytes (hepatocellular carcinoma). The second type of cell specifically binds to lysosomal targeting molecules expressed on the surface of HCC cells. This includes the portion of the second part that can bind to lysosomal targets expressed on the surface of liver cells. A non-limiting example of a molecular molecule is the asialoglycoprotein receptor (ASGPR) (a receptor for glycoconjugates from blood). It is a liver receptor that mediates the removal of denudates. This receptor is related to the ASGR1 gene and Two proteins encoded by ASGR2, asialoglycoprotein receptor 1 and ASGR2 (ASGR1 (UniProtKB-P07306-Human) and ASGR2 (U Contains niProtKB-P07307-human). ASGPR has sialic acid removed. Asialoglycan is a glycoprotein in which galactose and galactosamine residues are exposed. It binds to proteins. Receptors located on liver cells remove target glycoproteins from circulation. ASGPR is highly expressed in hepatocytes, several human cancer cell lines, and on the surface of liver cancer cells. It will be done.

[0041] If the lysosome-targeting molecule is ASGPR, then the appropriate second part would be anti-ASG. This includes, but is not limited to, PR antibodies and ASGPR ligands. Depending on the application, such a second part may contain one or more ASGPR ligands. Suitable ASGPR ligands include one or more N-acetylgalactosamine (Ga INAc), one or more galactoses, one or more glucoses, and Any combination of these is included, but is not limited to them. In a particular embodiment, The second part, for example, contains 1 to 1000 ASGPR ligands, or 1 to 750, 1 to 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 ASGPR units Includes Gund. In some embodiments, the second part is one or more ASGPR If ligands are included, the second portion can be 10-50, 15-45, 20-40, and It contains 25 to 35 ASGPR ligands. In certain embodiments, the second part contains one Or, if it contains multiple ASGPR ligands, the second part is 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, More than 250, more than 500, more than 750, or more than 1000 ASGPR Rigans Includes "do".

[0042] According to some embodiments, the lysosome targeting molecule is ASGPR, and the second part If it contains one or more ASGPR ligands, the second part contains one or more A Includes a scaffold containing an SGPR ligand. In one non-restrictive example, the second The portion comprises a polymer containing GalNAc. In certain embodiments, such a second portion The component contains poly(GalNAc-co-Ala), and its structure is provided below and in Figure 20. It can be done. [ka]

[0043] In a particular embodiment, the lysosome targeting molecule is ASGPR, and the second part has one Or, if it contains multiple ASGPR ligands, the second part is either one (monovalent) or two (divalent). , 3 (trivalent) or 4 or more ASGPR ligands, e.g., GalNAc, galactol Dendrimer feet containing ASGPR ligands independently selected from glucose and benzoyl sulfate. The field includes, for example, according to some embodiments, the second part is monovalent, divalent or trivalent. Includes a GalNAc-containing dendrimer scaffold. Trivalent GalNAc-containing dendrimers that can be used Non-limiting examples of rimmer scaffolding are as follows (in this specification, Tri-GalNAc de (called Ndrimer): [ka]

[0044] According to some embodiments, the second part contains monovalent, divalent, or trivalent galactose. Includes dendrimer scaffolds. No limit on the number of trivalent galactose-containing dendrimer scaffolds that may be used. Typical examples are as follows (referred to herein as Tri-Gal dendrimers): [ka]

[0045] According to some embodiments, the second part is expressed on the surface of liver cells. When it specifically binds to a targeting molecule, such as ASGPR, the first part is hepatocyte (hepatocyte) It specifically binds to cell surface molecules expressed on cells (including cellular carcinoma (HCC) cells). Non-limiting examples of cell surface molecules include growth factor receptors. This includes epidermal growth factor receptor (EGFR), C-Met, insulin-like growth factor 1 receptor ( IGF1R), fibroblast growth factor receptor 4 (FGFR4), HER2, and platelet-derived This includes, but is not limited to, growth factor receptors (PDGFRs).

[0046] In certain embodiments, the bifunctional molecule enhances growth factors on the surface of hepatocellular carcinoma (HCC) cells. It is used to break down (for example, when administering it to an individual with HCC to treat HCC) (In Vivo), here the bifunctional molecules are EGFR, C-Met, IGF1R, FGFR4 Alternatively, it may include a first portion that specifically binds to HER2, and a second portion that is specific to ASGPR. It binds to (for example, the second part is GalNAc, galactose and / or glucose) This may include scaffolds containing ASGPR ligands such as courses (e.g., polymer scaffolds).

[0047] According to some embodiments, the bifunctional molecule is expressed on fibrotic liver cells. Used to break down cell surface molecules (e.g., growth factors) (e.g., in vivo) (When administered to individuals with hepatic fibrosis), here, the bifunctional molecule promotes fibrosis. The first part specifically binds to cell surface or extracellular proteins (e.g., PDGFR). The second part contains a portion that specifically binds to ASGPR (for example, the second part is Ga One or more ASGRs, such as lNAc, galactose, and / or glucose. This may include a ligand-containing scaffold (e.g., a polymer scaffold).

[0048] In certain embodiments, the bifunctional molecule is a cell surface molecule in the presence of only the first part. This enhances the degradation of cell surface molecules or extracellular molecules, relatively speaking, compared to the degradation of extracellular molecules. According to some embodiments, the bifunctional molecule may exist in only one or the other part. Compared to the degradation of cell surface molecules or extracellular molecules under the presence of It enhances the degradation of extracellular molecules. In this context, "enhancing degradation" means that under the same conditions, Cell surface molecules or extracellular molecules are degraded in the presence of bifunctional molecules, and the first part alone Not being decomposed in the presence of the first part or the second part alone, or Under the same conditions, cell surface molecules or extracellular molecules, in the presence of the first part alone or the first To a greater extent than the decomposition in the presence of the first part or the second part alone, the bifunctional component This means that it is degraded in the presence of its offspring. Cell surface molecules or extracellular molecules under the same conditions And it is decomposed in the presence of the first part alone or in the presence of the first part or the second part alone. If the degradation occurs to a greater extent than that in the presence of a bifunctional molecule, then the degradation is bifunctional. In the presence of potential molecules, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times Above, 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 It could be 9 times or more, 9.5 times or more, or 10 times or more.

[0049] The bifunctional molecule of this disclosure, compared to the degradation of cell surface molecules in the presence of the first part alone, A non-limiting example of enhancing the degradation of cell surface molecules is provided in Example 4 of the experimental section below. The degradation of cell surface molecules is compared to the degradation of cell surface molecules in the presence of the first part alone. Examples of bifunctional molecules that do not enhance are provided in Example 5.

[0050] Those skilled in the art will know that the bifunctional molecule of interest is obtained in the presence of the first part alone or the first part or Compared to the degradation of cell surface molecules or extracellular molecules in the presence of the second part alone, the cell surface It is possible to easily determine whether or not the degradation of molecules or extracellular molecules is enhanced. Non-limiting examples of appropriate approaches to facilitate such decisions are shown in the following experimental section. It will be provided at [location / service name].

[0051] The bifunctional molecules of this disclosure may be in any suitable format. Several embodiments So, the first part is a polypeptide, the second part is a polypeptide, and the difunctional part The child is a fusion protein containing the first part fused with the second part. The first part is Part 2 can be directly fused. In other embodiments, part 1 may be, for example, a spacer domain. In the case where it is placed between the first part and the second part, indirectly to the second part They can be fused. If the bifunctional molecule is a fusion protein, the nucleus encoding the bifunctional molecule Acids are also provided by this disclosure. Expression vectors containing such nucleic acids are also provided by this disclosure. Cells (e.g., host cells) containing either the nucleic acid and / or expression vector are also provided. Methods for producing such cells are also provided, which, for example, involve appropriate cell transport. Using the transfection protocol and transfection reagents, the core of the present disclosure This involves introducing either an acid and / or an expression vector into cells. The bifunctional molecule In the case of fusion proteins, methods for constructing bifunctional molecules are also provided in this disclosure. Such a method involves culturing the cells of this disclosure under conditions in which the bifunctional molecule is expressed within the cell. This could include the following.

[0052] Other suitable formats of the bifunctional molecules of this disclosure include conjugates. Therefore, in some embodiments, the bifunctional molecule of the present disclosure is conjugated in the second part. It includes a first part that has been modified. In certain embodiments, the first part is an antibody and the second part It specifically binds to M6PR. For example, the first part may be an antibody, and the second part The minutes include one or more M6PR ligands (e.g., one or more M6P and / or The polymer scaffold may include / display an M6P analog, where the polymer The MAR scaffold is conjugated with an antibody. In some such embodiments, the second The portion consists of one or more amino acids functionalized with one or more M6PR ligands. It is a glycoprotein. A method for producing such a conjugate is also provided. The method involves conjugating the first part into the second part. The first part is an antibody A non-limiting example of the method is that the second part is a glycopolymer described herein. This is schematically shown in Figure 12. In some embodiments, the method is divided into a first part and a second part. This includes site-specific conjugation of a portion. For example, if the first portion is polyp If it contains a cytoplasm (e.g., an antibody), conjugation is performed by pre-selecting the first part. This may also include site-specific conjugation of the second portion to the amino acid. In certain embodiments, the pre-selected amino acids are located at the N-terminus or C-terminus of the first portion. In other embodiments, the pre-selected amino acids are located inside the first portion, i.e., the first portion It is located between the N-terminal amino acid and the C-terminal amino acid. In some embodiments, it is pre-selected. The amino acids are unnatural amino acids. Part 1 is used to facilitate conjugation. Non-exclusive examples of non-natural amino acids that may be provided in part and / or part two include: Zide, alkyne, alkene, aminooxy, hydrazine, aldehyde (e.g., formyl) Glycine, for example, Catalent Pharma Solutions' SMART ag(trademark) technology), nitrone, nitrile oxide, cyclopropene, norbornene, i Having a functional group selected from socyanide, aryl halide, and boronic acid functional groups. These include non-natural amino acids that can be incorporated and selected to provide the desired functional group. Acids are well known, for example, 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. This is described in Imaging Biol. (2):153-65, and elsewhere.

[0053] In some embodiments, conjugating the first part into the second part is This includes conjugating part 2 onto a glycan on part 1, or vice versa. Such a method involves modifying one or more glycans in the first part, so that the second part is connected This may include providing functional groups that can be combined. In one non-limiting example, the first part The N-glycan of an antibody (for example) can be modified into an aldehyde group by periodic acid oxidation. Then, it can be functionalized with a second part, for example, aminooxy M6Pn.

[0054] If a bifunctional molecule is a conjugate, the conjugate of the first part to the second part... One or more linkers may be used to facilitate gates. Non-limiting examples of linkers include ester linkers, amide linkers, maleimides, and These are maleimide-based linkers, valine-citrulline linkers, hydrazone linkers, and N -Succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, S Xynimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) Linker, vinyl sulfone-based linker, tetraethylene glycol However, linkers containing polyethylene glycol (PEG), etc., are not limited to these. Linkers containing ropanoic acid, linkers containing caproleic acid, and any combination thereof. Examples of linkers include those containing [a specific type of linker]. In certain embodiments, the linker has a neutral pH (blood flow pH 7.3). It is stable at ~7.5, but in the weakly acidic endosomes (pH 5) of target cells (e.g., cancer cells) Hydrolysis occurs during internal translocation to lysosomes (pH 4.5-5.0) and 0.0 (0.0-6.5). It is a chemically unstable linker, such as an acid-cleaving linker. Linkers include hydrazone-based linkers, oxime-based linkers, and carbonate-based linkers. This includes, but is not limited to, ester-based linkers. According to a particular embodiment... The linker is an enzyme-unstable linker; for example, it is stable in the bloodstream but not in target cells. During internal migration, the lysosomes of target cells (e.g., cancer cells) contain, for example, lysosomal proteas. Enzyme-unstable phosphorus that is cleaved enzymatically by enzymes (such as cathepsin or plasmin). It is a linker. Enzyme-unstable linkers include linkers containing peptide bonds, for example, dipeptide linkers. Citric acid-based linkers, e.g., valine-citrulline linkers, e.g., maleimidocaproyal Luvaline-citrulline-p-aminobenzyl (MC-vc-PAB) linker, Valyl - Contains alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, etc. These are rare, but not limited to them. Chemically unstable linkers, enzymatically unstable linkers. - And non-cuttable linkers are publicly known, for example, Ducry & Stump (201 0) Bioconjugate is described in detail in Chem. 21:5-13.

[0055] Numerous strategies for conjugating the first and second parts via a linker G is available. For example, the first part covalently bonds the linker to the first part. It may be derivatized by the second part, where the linker is the "chemical handle" of the second part. It has a functional group that can react with ". Also, as an example, the second part has a linker The second part may be derivatized by covalent bonding, where the linker is the It has a functional group that can react with the "chemical handle" of part 1. The active ingredients can vary and are selected based on their compatibility with the chemical handle of the first or second part. It is possible. According to one embodiment, the chemical handle is a non-natural mesh having a chemical handle. This is provided by incorporating the no acid into the first or second part. In some embodiments The conjugation of the first and second parts is by alkyne-azide cycloaddition. That is the case.

[0056] Other suitable formats of the bifunctional molecules of this disclosure include bispecific antibodies. For example, the bifunctional molecule of the present disclosure has a first portion (e.g., a first Fab arm) on the cell surface. It specifically binds to molecules or extracellular molecules, and the second part (e.g., the second Fab arm) It may be a bispecific antibody that specifically binds to lysosome-targeting molecules (e.g., M6PR). A schematic diagram of such a bispecific antibody is shown in Figure 19. In some embodiments, Bispecific antibodies detach from their target at the reduced pH of endosomes. Through this transition, a given bispecific antibody cycles with its receptor without antibody degradation. This enables the continuous delivery of cargo and targets to lysosomes. (Bispecific antibody) Approaches for producing them are well known. For example, if a bifunctional molecule is a bispecific antibody In this case, bispecific antibodies are called "knobs-into-holes". It can be fabricated using the (KIH) approach. The KIH technique involves heterodimerization. CH3 is designed to create either a "knob" or a "hole" in each heavy chain to facilitate this process. This includes manipulating the domain. KIH's design and manufacturing strategies are publicly known, for example. 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 Y et al. (1996) Protein Eng. 9(7):617-2; and Merch Ant et al. (1998) Nat. Biotechnol. 16(7):677-81 The items listed are listed below.

[0057] composition As summarized above, this disclosure provides a composition. The composition comprises the above-mentioned bifunctional molecule Any of the bifunctional molecules of this disclosure, including any of the bifunctional molecules described in the section. It may include any of these, and they are incorporated here, but for brevity, they are not repeated. stomach.

[0058] In certain embodiments, the composition comprises the bifunctional molecules of the present disclosure present in a liquid medium. The medium may be an aqueous liquid medium such as water or a buffer solution. One or more additives, for example, a salt. (For example, NaCl, MgCl2, KCl, MgSO4), buffering agent (Tris buffer, N- (2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) , 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethane Sodium sulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MO PS), N-Tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TA) Even if PS, protease inhibitors, glycerol, etc. are present in such a composition good.

[0059] Pharmaceutical compositions are also provided. A pharmaceutical composition comprises any of the difunctional molecules of the present disclosure, and It contains a pharmaceutically acceptable carrier. Pharmaceutical compositions generally contain a therapeutically effective amount of a bifunctional molecule. Includes. "Therapeutic dose" means a dose sufficient to produce the desired result, for example, a beneficial dose. Or the desired treatment (including prevention) outcome, for example, the first part of the bifunctional molecule is specifically bound. Individuals with cell proliferation disorders (e.g., cancer) related to cell surface molecules or extracellular molecules This means a sufficient amount to cause a decrease in cell proliferation, etc. The effective dose is one or It can be administered in multiple doses.

[0060] The bifunctional molecules of this disclosure can be incorporated into various formulations for therapeutic administration. Specifically, the bifunctional molecule is combined with an appropriate pharmaceutically acceptable excipient or diluent. By combining these ingredients, they can be formulated into pharmaceutical compositions such as tablets, capsules, powders, and granules. It can be formulated into solid, semi-solid, or gaseous formulations.

[0061] Formulations of the bifunctional molecules of this disclosure that are suitable for administration to an individual (for example, suitable for human administration) are: Generally sterile and contraindicated for administration to individuals according to the selected route of administration. It may be in a state that does not contain any heat-generating substances or other contaminants.

[0062] In pharmaceutical dosage forms, difunctional molecules are used alone or in combination with other pharmaceutically active compounds. It can be administered in combination with the following methods and excipients. The agent is merely an example and is by no means limiting.

[0063] In the case of oral formulations, lactose, mannitol, corn starch, or potato Conventional additives such as starch, crystalline cellulose, cellulose derivatives, gum arabic, corn Binders such as starch or gelatin, corn starch, potato starch, etc. or disintegrants such as sodium carboxymethylcellulose, magnesium stearate Lubricants such as talc, and optionally, diluents, buffers, wetting agents, preservatives, and flavoring agents. Using this method, a difunctional molecule alone is used to manufacture tablets, powders, granules, or capsules. Alternatively, it can be used in combination with appropriate additives.

[0064] Difunctional molecules include vegetable oils or other similar oils, synthetic fatty acid glycerides, and higher-order fatty acids. Dissolve or suspend in an aqueous or non-aqueous solvent such as an ester or propylene glycol. Alternatively, by emulsification, and if desired, by adding solubilizers, isotonic agents, suspending agents, emulsifiers, It can be formulated into an injectable preparation using conventional additives such as stabilizers and preservatives.

[0065] The pharmaceutical composition is in liquid form, lyophilized form, or liquid form reconstituted from the lyophilized form. It may be in its original state, and the lyophilized preparation should be reconstituted with sterile solution before administration. A standard procedure for reconstituting a dried composition involves adding a certain amount of pure water (typically during freeze-drying). The process involves adding back the volume equivalent to the volume removed, but this is not related to the production of parenterally administered pharmaceutical compositions. A solution containing an antibacterial agent may be used raw.

[0066] Aqueous formulations of bifunctional molecules are prepared in a pH buffer solution at, for example, about 4.0 to about 8.0, for example, about 4 It can be prepared with a pH of 0.5 to approximately 7.5, for example, in the range of approximately 5.0 to approximately 7.0. Examples of buffers suitable for a given pH include phosphate buffer, histidine buffer, citrate buffer, This includes succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration is, for example, Depending on the desired tonicity of the buffer and formulation, the concentration can be approximately 1 mM to approximately 100 mM, or approximately 5 mM to It could be approximately 50 mM.

[0067] How to use As summarized above, methods using the bifunctional molecules of this disclosure are also provided. In the application method, the method applies the bifunctional molecules described in the section on bifunctional molecules above. This includes using one of the children, which is incorporated here but is written for brevity. Don't repeat that mistake.

[0068] In certain embodiments, a method for degrading cell surface molecules or extracellular molecules is provided. One method involves lysosome-targeting molecules using lysosomes to degrade cell surface molecules or extracellular molecules. Under conditions of shuttle to the osome, cell surface molecules or extracellular molecules of the bifunctional molecules of this disclosure This includes contact with any of the following. Such methods are used for a variety of purposes. In this embodiment, the method is performed in vitro (for example, in a tube, cell culture plate or It is carried out in places such as hulls and used for, for example, testing and / or research purposes. In this case, the method is performed in vivo (for example, in an individual to whom a bifunctional molecule is administered). For example, it is used in clinical / therapeutic applications.

[0069] In some embodiments, a method is provided that includes administering to an individual in need thereof a therapeutically effective amount of any of the bifunctional molecules or any of the pharmaceutical compositions of the present disclosure. A variety of individuals are treatable according to the method. Generally, such subjects are "mammals" or "mammalian," terms that are widely used to describe organisms within the class of mammals, including carnivores (e.g., dogs and cats), rodents ( e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is human.

[0070] In some embodiments, an effective amount of the bifunctional molecule (or a pharmaceutical composition comprising the same) is, when administered alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents ( e.g., in combination therapy), in one or more doses, effective to reduce the symptoms of the individual's medical condition (e.g., cancer, neurodegenerative disorder, etc.) 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 as compared to the symptoms in the individual in the absence of treatment with the bifunctional molecule or pharmaceutical composition.

[0071] In certain aspects, the individual has or is suspected of having a neurodegenerative disorder characterized by amyloid-β deposition in the brain (e.g., Alzheimer's disease ) or tau protein deposition in the brain (e.g., tauopathy), and the first portion of the bifunctional molecule is apoE4 ​(For example, apoE4 expressed from the ε4 allele of the APOE4 gene) specifically binds and the neurodegenerative disorder of an individual is treated by targeted degradation of apoE4.

[0072] In some embodiments, methods are provided that include administering a therapeutically effective amount of any of the bifunctional molecules or any pharmaceutical composition of the present disclosure to an individual having cancer. According to such methods, the first portion of the bifunctional molecule specifically binds to a cell surface molecule or extracellular molecule that at least contributes to the cancer of the individual, and targeted degradation of the cell surface molecule or extracellular molecule using the bifunctional molecule treats the cancer of the individual. In certain embodiments, the first portion specifically binds to a molecule selected from cell surface molecules on cancer cells, ligands of cell surface molecules on cancer cells, cell surface molecules on immune cells, ligands of cell surface molecules on immune cells, inhibitory immune receptors, and ligands of inhibitory immune receptors.

[0073] In certain embodiments, the individual has cancer characterized by the presence of a solid tumor, semi-solid tumor, primary tumor, metastatic tumor, etc. In some embodiments, the individual has cancer selected from breast cancer, melanoma, lung cancer, colorectal cancer, prostate cancer, glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia (e.g., acute myeloid leukemia (AML)), liver cancer (e.g., hepatocellular carcinoma (HCC), e.g., primary or recurrent

[0074] According to some embodiments, the individual has a particular liver disease (including but not limited to hepatocellular carcinoma (HCC)), and the method is for treating that disease. For example In certain embodiments, the individual has HCCs, and the first part is the cell surface on the HCC cells of the individual. The first part is bonded to the surface molecule, and the second part is bonded to ASGPR. In certain embodiments, the first part It binds to tumor-promoting proteins on HCC cells of an individual. According to some embodiments Tumor-promoting proteins are growth factors on HCC cells. Such growth factors are not limited Typical examples include EGFR, C-Met, IGF1R, and FGFR4. Difunctional molecules such as those described elsewhere in this specification are linked to ASGPR. It may include any of the second parts that combine.

[0075] In a particular embodiment, the individual has hepatic fibrosis, and the method treats the hepatic fibrosis. This is for the purpose of... For example, according to some embodiments, an individual has hepatic fibrosis, The first part binds to cell surface molecules on fibrous liver cells of an individual, and the second part binds to ASGPR It binds to. In certain embodiments, the first part promotes fibrosis on fibrous liver cells of an individual. It binds to proteins. According to some embodiments, the fibrosis-promoting protein is fibrous. It is a growth factor on liver cells. A non-specific example of such a growth factor is PDGFR. Such bifunctional molecules are described elsewhere in this specification as ASGPR It may include any of the second parts that are joined together.

[0076] In any method using the difunctional molecules of this disclosure, in certain embodiments, the difunctionality The molecule is relative to the degradation of cell surface molecules or extracellular molecules in the presence of only the first part. Specifically, it enhances the degradation of cell surface molecules or extracellular molecules. Similarly, the bifunctional molecules of this disclosure In any method of use, according to some embodiments, the bifunctional molecule enhances the degradation of cell surface or extracellular molecules relative to the degradation of cell surface or extracellular molecules in the presence of only the first portion or the second portion. Details regarding such enhanced degradation are provided in the section on bifunctional molecules above and are incorporated herein but not repeated for the sake of brevity.

[0077] "Treat", "treating", or "treatment" means at least an improvement in the medical condition of an individual (e.g., a cell proliferative disorder, e.g., cancer ), and improvement is used broadly to refer to a parameter related to the medical condition being treated, e.g., at least a reduction in the magnitude of symptoms. Thus, treatment also means that the medical condition or at least the symptoms characterizing the medical condition are completely suppressed, e.g., prevented from occurring or stopped, such that the individual does not suffer any further

[0078] In certain aspects, the disclosure provides a method of enhancing antibody-dependent cellular cytotoxicity (ADCC) comprising administering to an individual needing ADCC the bifunctional molecule or pharmaceutical composition of the disclosure. In some embodiments, the first portion of the bifunctional molecule specifically binds to an inhibitory immune receptor or its ligand. In certain aspects, the first portion of the bifunctional molecule specifically binds to an immune checkpoint molecule such as PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, or a member of the B7 family. ​​​​​​​​​​​

[0079] The bifunctional molecule or pharmaceutical composition can be used in vivo and ex vivo, and systemically. Use any available method and route suitable for drug delivery, including local administration routes. It may be administered to the individual. Conventional and pharmaceutically acceptable routes of administration include the nose. Intracavitary, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intraocular, intravenous, intraarterial, nasal, oral, This includes other enteral and parenteral administration routes. In some embodiments, administration is parenteral. This is due to the following: The route of administration is combined depending on the bifunctional molecule and / or the desired effect. It can be combined or adjusted as needed. Bifunctional molecules or medical The drug composition can be administered as a single dose or in multiple doses. In some embodiments, In some embodiments, the bifunctional molecule or pharmaceutical composition is administered intravenously. Functional molecules or pharmaceutical compositions are, for example, for systemic delivery (e.g., intravenous injection). Alternatively, it is administered by injection to a local site.

[0080] kit As summarized above, this disclosure also provides a kit. In some embodiments, the target kit The difunctional molecules of this disclosure (the difunctional molecules described in the section on difunctional molecules above) It contains any of the sex molecules, which are incorporated here, but for brevity, the description is repeated. Either of the following (not), and a cell surface molecule or extracellular molecule to which the first part specifically binds. Includes instructions for using a bifunctional molecule to decompose the child. In certain embodiments, the instructions The book is intended for, for example, testing and / or examination purposes, to analyze cell surface molecules or other microorganisms in vitro. It is intended for the degradation of extracellular molecules. In other embodiments, the instructions may be for, for example, clinical / therapeutic use. Therefore, it is intended to degrade cell surface molecules or extracellular molecules in vivo. For example For example, one of the bifunctional molecules or pharmaceutical compositions of this disclosure and a bifunctional molecule to an individual that requires it. A kit is provided that includes instructions for administering a potent molecule or pharmaceutical composition. Unagi Kit is available in a fixed quantity in unit dose form, such as an ampoule or multi-dose form. It may contain a potent molecule or a pharmaceutical composition. Therefore, in certain embodiments, the kit is One or more (e.g., 2 or more) unit doses of a bifunctional molecule or pharmaceutical composition (e.g., It may include (ampoules).

[0081] As used herein, the term "unit dose" refers to a single dose for human and animal subjects. This refers to a physically distinct unit appropriate for the dosage, and each unit is sufficient to produce the desired effect. It contains a predetermined amount of composition, calculated in a specific quantity. The amount of the unit dose is the specific amount used. Functional molecules, the effects to be achieved, and pharmacodynamics related to bifunctional molecules in organisms. It depends on various factors. In further embodiments, the kit may be a bifunctional molecule or a pharmaceutical. The composition may include a single or multiple dosages.

[0082] In other embodiments, any glycopolymer of the present disclosure (as described in the section on difunctional molecules above) It contains any glycopolymer listed, which is incorporated here but is noted for brevity. (Do not repeat the process), and for conjugating glycopolymers to the target molecule A kit including instructions is provided. Such a kit allows you to apply a glycopolymer to the target molecule. The material may further contain reagents for conjugation. In some embodiments, the desired fraction The child is a polypeptide. Non-exclusive examples of such polypeptides include antibodies. In certain embodiments, the molecule of interest is a detailed molecule as described in the section on bifunctional molecules above. Specifically targeting cell surface molecules or extracellular molecules, including either cell surface molecules or extracellular molecules. It is combined and incorporated here, but for the sake of brevity, the description will not be repeated.

[0083] The components of the kit may be in separate containers, or multiple components may be in a single container. It's okay for it to exist.

[0084] The instructions included in the kit can be recorded on a suitable recording medium. For example, the instructions can be on paper. Alternatively, it can be printed on a substrate such as plastic. For example, instructions These are present in the kit as package inserts, or in the kit's container or its components. It may be present on the label (i.e., associated with the packaging or internal packaging). In other embodiments, the instructions are for a portable flash drive, DVD, CD-RO Electronic storage data files located on appropriate computer-readable storage media such as diskettes. It exists as a file. In other embodiments, the actual instructions are not present in the kit. However, for example, there are means to obtain instructions from a remote source via the internet. Provided. One example of this embodiment allows you to view the instructions and / or the instructions This kit includes a web address from which you can download the instructions. The means for obtaining the instructions are recorded on a suitable substrate.

[0085] Glycopolymers, monomers, and methods for producing the same This disclosure also provides glycopolymers. In some embodiments, the G A lycopolymer consists of a polymer scaffold and one or more mannose molecules bound to the polymer scaffold. Contains a 6-phosphate receptor (M6PR) ligand. Glycopolymer, polymer scaffold and / or M6PR ligand is one of the difunctional molecules described in the section above. Either is acceptable, and it will be incorporated here, but for the sake of brevity, the description will be repeated. No. For example, glycopolymers are functionalized with one or more M6PR ligands. It may be a glycoprotein containing one or more amino acids. Glycoproteins are N-carb It may be a glycoprotein derived from xyanhydride (NCA). In certain embodiments, one or more The M6PR ligand contains one or more mannose-6-phosphate (M6P) molecules. Furthermore, one or more M6PR ligands contain one or more M6 P analogues, for example, any M6P analogues described herein, for example, one or more. It contains several mannose-6-phosphonate (M6Pn) compounds. In some embodiments, The polymer scaffold consists of 1 to 50 M6PR ligands, for example, 1 to 40, 1 to 30, 1 to Contains 20, 1-10 (e.g., 1-6), or 1-5 M6PR ligands. In certain embodiments, the polymer scaffolds may be 10-50, 15-45, 20-40, or It contains 25 to 35 M6PR ligands. In certain embodiments, the polymer scaffold has 5 or more Contains 10 or more, 20 or more, 30 or more, or 40 or more M6PR ligands.

[0086] Methods for producing glycopolymers are also provided in the present disclosure. In some embodiments, The preparation of glycopolymers involves polymerization. Polymerization may be carried out by NCA polymerization. An example of this is schematically shown in Figure 8.

[0087] In certain embodiments, the method for producing a glycopolymer involves one or more M6PR-ligans. This includes bonding the do to a polymer scaffold. In other embodiments, such a method involves one or This involves synthesizing polymer scaffolds from monomers functionalized with multiple M6PR ligands. For example, the scaffold is one or more M6PR ligands functionalized with one or more M6PR ligands. It can be synthesized from monomers, and the synthesis is by solid-phase synthesis. Exemplary solid-phase synthesis The scheme is shown in Figure 9.

[0088] In related embodiments, the disclosure provides monomers. A monomer is one or more monomers. It is functionalized with a north-6-phosphate receptor (M6PR) ligand. In certain embodiments, The monomer is an amino acid. In some embodiments, the monomer is a non-natural amino acid. One or more M6PR ligands are present in one or more mannose-6-phosphate (M6PR ligands). It may include 6P). Alternatively, or in addition to it, one or more M6PRs. Gand is one or more M6P analogues, for example, any M6 described herein. P analogues, such as mannose-6-phosphonate (M6Pn), may be included.

[0089] Notwithstanding the attached claims, this disclosure is also defined by the following embodiments. .

[0090] 1. A difunctional molecule, A first portion that specifically binds to cell surface molecules or extracellular molecules, A bifunctional molecule comprising a second portion that specifically binds to lysosome-targeting molecules.

[0091] 2. Relative to the degradation of cell surface molecules or extracellular molecules in the presence of only the first part. The bifunctional molecule of Embodiment 1 enhances the degradation of cell surface molecules or extracellular molecules.

[0092] 3. The first part specifically binds to cell surface molecules, in Embodiment 1 or Embodiment 2. The described bifunctional molecule.

[0093] 4. The bifunctional molecule according to Embodiment 3, wherein the cell surface molecule is a cell surface receptor.

[0094] 5. The bifunctional molecule according to Embodiment 4, wherein the cell surface receptor is a growth factor receptor.

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

[0096] 7. The cell surface molecule is a tumor-associated antigen or a tumor-specific antigen, as described in Embodiment 6. Functional molecules.

[0097] 8. The cell surface molecule is present on an immune cell, as described in any one of Embodiments 1 to 7. Bifunctional molecules.

[0098] 9. Immune cells include natural killer (NK) cells, macrophages, monocytes, neutrophils, and trees. Selected from the group consisting of rhizocytes, T cells, B cells, mast cells, basophils, and eosinophils, The bifunctional molecule described in Embodiment 8.

[0099] 10. The bifunctional molecule according to Embodiment 8, wherein the cell surface molecule is an inhibitory immune receptor.

[0100] 11. The two-cell interface according to Embodiment 10, wherein the cell surface molecule is a ligand for an inhibitory immune receptor. functional molecule.

[0101] 12. The bifunctionality according to 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, A group selected from members of the LAG3, TIGIT, and B7 families. The bifunctional molecule described in application form 12.

[0103] 14. A bifunctional molecule according to Embodiment 1, wherein the first portion specifically binds to an extracellular molecule. .

[0104] 15. The bifunctionality described in Embodiment 14, wherein the extracellular molecule is a ligand for a cell surface receptor. molecule.

[0105] 16. The bifunctional molecule according to Embodiment 15, wherein the extracellular molecule is a growth factor.

[0106] 17. The bicellular molecule according to Embodiment 15, wherein the extracellular molecule is a cytokine or chemokine. functional molecule.

[0107] 18. The bifunctional molecule according to Embodiment 14, wherein the extracellular molecule is an antibody.

[0108] 19. The bifunctional molecule according to Embodiment 18, wherein the antibody is an autoantibody.

[0109] 20. The antibody specifically binds to cell surface molecules or extracellular molecules, as in Embodiment 18 or The bifunctional molecule described in Embodiment 19.

[0110] 21. Is the first part a polypeptide, ligand, aptamer, nanoparticle, or small molecule? A bifunctional molecule according to any one of Embodiments 1 to 20, selected from the group.

[0111] 22. The bifunctional molecule according to Embodiment 21, wherein the first part is a polypeptide.

[0112] 23. The bifunctional molecule according to Embodiment 22, wherein the first part is an antibody.

[0113] 24. Antibodies include IgG, single-chain Fv(scFv), Fab, (Fab)2, (scFv' )2, or a bifunctional molecule according to Embodiment 23, which is a nanobody.

[0114] 25. The second part is a polypeptide, ligand, aptamer, nanoparticle, and small molecule. A bifunctional molecule according to any one of Embodiments 1 to 24, selected from the group.

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

[0116] 27. Embodiment 26, wherein the second part comprises one or more M6PR ligands. A bifunctional molecule.

[0117] 28.1 or more M6PR ligands, 1 or more mannose-6-phosphate A bifunctional molecule according to Embodiment 27, comprising an acid (M6P).

[0118] 29.1 or more M6PR ligands containing one or more M6P analogs, A bifunctional molecule as described in Embodiment 27 or Embodiment 28.

[0119] 30.1 or more M6P analogs, one or more mannose-6-phosphone A bifunctional molecule according to Embodiment 29, comprising (M6Pn).

[0120] 31. Embodiment 27, wherein the second part contains 1 to 500 M6PR ligands. A bifunctional molecule as described in any one of the 30 forms.

[0121] 32. The second part is a polymer that displays one or more M6PR ligands. A bifunctional molecule according to any one of Embodiments 27 to 31, including a scaffold.

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

[0123] 34. Glycopolymers functionalized with one or more M6PR ligands The bifunctional molecule described in Embodiment 33 is a glycoprotein containing multiple amino acids.

[0124] 35. The glycoprotein is a glycoprotein derived from N-carboxyanhydride (NCA). The bifunctional molecule described in application form 34.

[0125] 36. Embodiments 1 to 36 in which the lysosome-targeting molecule is expressed on the surface of liver cells. A bifunctional molecule as described in any one of the 25 items.

[0126] 37. The bifunctional cell according to Embodiment 36, wherein the lysosome-targeting molecule is expressed on the surface of hepatocytes. functional molecule.

[0127] 38. Sosome-targeting molecules target hepatocellular carcinoma (HCC) cells, fibrous liver cells, or their A bifunctional molecule according to Embodiment 36 or Embodiment 37, expressed on both surfaces.

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

[0129] 40. The second part comprises one or more ASGPR ligands, as described in Embodiment 39. A difunctional molecule.

[0130] 41.1 or more ASGPR ligands, one or more N-acetylgalactols A bifunctional molecule according to Embodiment 40, comprising tosamine (GalNAc).

[0131] 42.1 or more ASGPR ligands containing one or more galactoses , the bifunctional molecule described in Embodiment 40 or Embodiment 41.

[0132] 43.1 or more ASGPR ligands containing one or more glucose molecules A bifunctional molecule according to any one of Embodiments 40 to 42.

[0133] 44. Embodiment 40, wherein the second part contains 1 to 500 ASGPR ligands. A bifunctional molecule as described in any one of Form 43.

[0134] 45. The second part comprises a polymer containing one or more ASGPR ligands. A bifunctional molecule according to any one of the embodiments 40 to 44.

[0135] 46. ​​Embodiment 45, in which the second part includes poly(GalNAc-co-Ala) A bifunctional molecule.

[0136] 47. The second part comprises a monovalent, divalent, or trivalent GalNAc-containing dendrimer scaffold. The bifunctional molecule described in Embodiment 41.

[0137] 48. Embodiment 47, in which the second part includes a trivalent GalNAc-containing dendrimer scaffold. The bifunctional molecule described above.

[0138] 49. The second part comprises a monovalent, divalent, or trivalent galactose-containing dendrimer scaffold. The bifunctional molecule described in Embodiment 42.

[0139] 50. Embodiment 49, in which the second part includes a trivalent galactose-containing dendrimer scaffold. A difunctional molecule.

[0140] 51. The first part specifically binds to cell surface molecules expressed on hepatocytes, in an actionable form. A bifunctional molecule according to any one of embodiments 36 to 50.

[0141] 52. The bifunctional molecule according to 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. Growth factor 1 receptor (IGF1R), fibroblast growth factor receptor 4 (FGFR4), and In Embodiment 52, selected from the group consisting of platelet-derived growth factor receptors (PDGFRs), The described bifunctional molecule.

[0143] 54. The first part is a polypeptide, the second part is a polypeptide, and it is difunctional. Embodiment 1 is a fusion protein in which the molecule includes a first portion fused to a second portion. A bifunctional molecule according to any one of the embodiments 53.

[0144] 55. The bifunctionality according to Embodiment 54, wherein the first part is directly fused with the second part. molecule.

[0145] 56. Embodiment 54, which includes a spacer domain between the first and second parts. A difunctional molecule.

[0146] 57. Difunctional molecules, Cell surface molecules or extracellular molecules This is a bispecific antibody that specifically binds to lysosome-targeting molecules, as described in Embodiment 1. A bifunctional molecule as described in any one of the 56 application forms.

[0147] 58. A bifunctional molecule containing a first part conjugated to a second part. A bifunctional molecule according to any one of Embodiments 1 to 53, which is a gate.

[0148] 59. The bifunctional molecule according to Embodiment 58, wherein the first part is an antibody.

[0149] 60. Including a second part defined in any one of Embodiments 27 to 35, A bifunctional molecule as described in Embodiment 58 or Embodiment 59.

[0150] 61. Embodiments including a second part defined in any one of Embodiments 40 to 50. A bifunctional molecule as described in 58 or embodiment 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 described in Embodiment 62.

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

[0154] 65. Introducing the nucleic acid described in Embodiment 62 or the expression vector described in Embodiment 63 into cells A method for producing cells according to Embodiment 64, including the input of a substance.

[0155] 66. Embodiment 58, which includes conjugating the first part into the second part. This is a method for producing a bifunctional molecule as described in Embodiment 59.

[0156] 67. Conjugation is a site-specific conjugation of the first part into the second part. The method according to embodiment 66, which includes gate.

[0157] 68. The first part contains a polypeptide and can be conjugated, while the second part This includes site-specific conjugation of a pre-selected amino acid in the first portion, The method according to Embodiment 67.

[0158] 69. Embodiment 6, in which a pre-selected amino acid is located at the N-terminus or C-terminus of the first portion. The method described in 8.

[0159] 70. The method according to Embodiment 68, wherein a pre-selected amino acid is located inside the first portion. .

[0160] 71. Embodiments 68 to 7, in which the pre-selected amino acids are non-natural amino acids. A method that is one of 0.

[0161] 72. The first part is an antibody, as described in any one of Embodiments 66 to 71. The method.

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

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

[0164] 75. Conjugation occurs through alkyne-azide cycloaddition, implementation The method according to any one of Embodiments 66 to 74.

[0165] 76. A method for degrading cell surface molecules or extracellular molecules, Lysosome-targeting molecules target lysosomes to degrade cell surface molecules or extracellular molecules. Under conditions of churning, cell surface molecules or extracellular molecules are subjected to the conditions of Embodiments 1 to 61. A method comprising contacting a bifunctional molecule described in any one of the following descriptions.

[0166] 77. A bifunctional molecule is a cell surface molecule or an extracellular molecule in the presence of only the first part. Embodiment 76 enhances the degradation of cell surface molecules or extracellular molecules relative to degradation. The method.

[0167] 78. The method of Embodiment 76 or Embodiment 77, which is performed in vitro.

[0168] 79. The method of Embodiment 76 or Embodiment 77, which is performed in vivo.

[0169] 80. A pharmaceutical composition, A bifunctional molecule according to any one of Embodiments 1 to 61, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

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

[0171] 82. The pharmaceutical composition described in Embodiment 80 or Embodiment 81 is used by an individual that requires it. A method including administering to [a specific body part].

[0172] 83. An effective amount of the pharmaceutical composition described in Embodiment 80 or Embodiment 81 for an individual with cancer. A method of treating cancer that involves administering substances.

[0173] 84. The first part is a cell surface molecule on cancer cells, a ligand for a cell surface molecule on cancer cells, Cell surface molecules on immune cells, ligands for cell surface molecules on immune cells, inhibitory immune receptors, And it specifically binds to molecules selected from the group consisting of ligands for inhibitory immune receptors. The method described in Embodiment 83.

[0174] 85. The individual has hepatocellular carcinoma (HCC), and the first part is on the cell surface of the individual's HCC cells. In Embodiment 83 or Embodiment 84, the molecule is bonded, and the second part is bonded to ASGPR. Method of description.

[0175] 86. The first part binds to growth factors on individual HCC cells, as described in Embodiment 85. The method.

[0176] 87. A group in which the first part consists of EGFR, C-Met, IGF1R, and FGFR4. The method according to embodiment 90, wherein the growth factor is selected from the following.

[0177] 88. The second part is defined as any one of Embodiments 36 to 50. The method according to any one of Embodiments 85 to 87.

[0178] 89. A method for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC), which requires ADCC A method comprising administering the pharmaceutical composition described in Embodiment 80 or Embodiment 81 to an individual. .

[0179] 90. A method for enhancing the immunogenicity of cancer in an individual, which is the embodiment 80 or an embodiment A method comprising administering the pharmaceutical composition described in Form 81 to an individual.

[0180] 91. A group in which the first part consists of an inhibitory immune receptor and a ligand for an inhibitory immune receptor. The method according to Embodiment 89 or Embodiment 90, which specifically binds to a molecule selected from the above. .

[0181] 92. The administration method is parenteral administration, as in Embodiments 82 to 91. Any one of the following methods.

[0182] 93. A bifunctional molecule is a cell surface molecule or an extracellular molecule in the presence of only the first part. Embodiment 82 enhances the degradation of cell surface molecules or extracellular molecules relative to degradation. The method according to any one of Embodiments 91.

[0183] 94. A bifunctional molecule according to any one of Embodiments 1 to 61, Instructions for degrading cell surface molecules or extracellular molecules to which the first part specifically binds. A kit that includes [the above].

[0184] 95. The instructions are for the in vitro degradation of cell surface molecules or extracellular molecules. A kit according to one embodiment 94.

[0185] 96. The instructions are for the in vivo degradation of cell surface molecules or extracellular molecules. The kit described in Embodiment 94.

[0186] 97. The bifunctional molecule described in any one of Embodiments 1 to 61 or Embodiment 80 Alternatively, the pharmaceutical composition described in Embodiment 81, Instructions for administering a bifunctional molecule or pharmaceutical composition to an individual requiring it, and A kit that includes this.

[0187] 98. A difunctional molecule or pharmaceutical composition is present in one or more unit doses. The kit described in Form 97.

[0188] 99. Embodiment 97, in which a bifunctional molecule or pharmaceutical composition is present in two or more unit doses. The kit described above.

[0189] 100. Polymer scaffolding and, One or more mannose-6-phosphate receptors (M6PRs) bound to a polymer scaffold. A glycopolymer containing a ligand.

[0190] 101. Glycopolymers functionalized with one or more M6PR ligands The glycopolymer according to Embodiment 100 is a glycoprotein containing multiple amino acids. .

[0191] 102. The glycoprotein is derived from N-carboxyanhydride (NCA). , the glycopolymer described in Embodiment 101.

[0192] 103.1 or more M6PR ligands, 1 or more mannose-6-ligands A glycerin acid (M6P) as described in any one of Embodiments 100 to 102. Copolymer.

[0193] 104.1 or more M6PR ligands containing one or more M6P analogs The glycopolymer according to any one of Embodiments 100 to 103.

[0194] 105.1 or more M6P analogs, one or more mannose-6-phosphorus The glycopolymer according to Embodiment 104, comprising nate (M6Pn).

[0195] 106. Embodiment 100, in which the polymer scaffold contains 1 to 500 M6PR ligands. The glycopolymer according to any one of embodiments 105.

[0196] 107. The glycopolymer described in any one of Embodiments 100 to 106 A method of manufacturing, Attaching one or more M6PR ligands to a polymer scaffold, or Synthesize polymer scaffolds from monomers functionalized with one or more M6PR ligands. A method that includes doing so.

[0197] 108. The scaffold is functionalized with one or more M6PR ligands. The polymerization from monomers and subsequent synthesis are carried out by solid-phase synthesis, as described in Embodiment 107. Method of loading.

[0198] 109. Glycopolymers are glycoprotein polymers, and their synthesis can be performed on solid-phase peptides. The method according to Embodiment 108, which is synthesized.

[0199] 110. A glycopolymer according to any one of Embodiments 100 to 106, A kit containing instructions for conjugating a glycopolymer to the target molecule. to.

[0200] 111. Further contains reagents for conjugating glycopolymers to target molecules. The kit described in Embodiment 110.

[0201] 112. The molecule of interest is a polypeptide, as described in Embodiment 110 or Embodiment 111. A kit containing [something].

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

[0203] 114. Embodiments in which the target molecule specifically binds to cell surface molecules or extracellular molecules. A kit as described in any one of embodiments 110 to 113.

[0204] 115. Functionalized with 1 or more mannose-6-phosphate receptor (M6PR) ligands Modified monomer.

[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 a non-natural amino acid.

[0207] 118.1 or more M6PR ligands, 1 or more mannose-6-ligands A mono acid (M6P) as described in any one of Embodiments 115 to 117 Mar.

[0208] 119.1 or more M6PR ligands containing one or more M6P analogs, A monomer according to any one of Embodiments 115 to 118.

[0209] 120.1 or more M6P analogs, one or more mannose-6-phosphorus The monomer according to Embodiment 119, comprising nate (M6Pn). [Examples]

[0210] The following examples are provided as illustrations, not as limitations.

[0211] experiment [Example 1 - Mannose-6-phosphate polymer shuttles cargo to lysosomes] In this embodiment, a biotin cap (referred to as "Part 1" in this specification - shown in Figure 10) is used. (shown as a triangle) and M6Pn polymer (the "second part" as used herein) A bifunctional molecule containing (see Figure 10) was tested, and the bifunctional molecule was found to be degraded in the extracellular space. Neutraavidin-647 (NA647-biotin) strongly binds from between to lysosomes. We determined whether it could mediate the movement of proteins. Figure 10 shows the proteins and lysososo Co-localization with both the chromium-staining dye and the bifunctional molecule is observed, indicating that the bifunctional molecule is actually neutral. Fluorescent imaging demonstrates that avidin-647 can mediate the translocation to lysosomes. The aging results (below) are provided.

[0212] Next, various cell lines were tested as described above. Figure 11 shows several cell lines in M Data were shown demonstrating the incorporation of NA 647 in a 6Pn polymer-dependent manner. These results suggest that any cell line possessing M6PR (e.g., CIM) is suitable. 6PR) This method allows for the shuttle of cell surface and extracellular molecules to lysosomes. This is expected to enable this, and is not limited to the cell lines tested in this study.

[0213] [Example 2 - M6Pn conjugate antibody shuttles target substances to lysosomes] In this embodiment, the first part is an antibody that binds to a specific target of interest, and the second part is We tested a bifunctional molecule that is a M6Pn-containing glycoprotein.

[0214] Figure 13 shows how poly(M6Pn) labeled antibodies bind their binding partners to lysosomes. A schematic diagram (top) and fluorescence imaging data (bottom) demonstrating that it can be performed are presented. In this example, mouse IgG-488 is provided with an anti-mouse tagged with poly(M6Pn). Incubate with IgG antibody and colocalize both protein and lysosomal staining dye. Merged.

[0215] Figure 14 shows how poly(M6Pn) labeled antibodies bind their binding partners to intracellular compartments. A schematic diagram (above) and further data (below) are provided to show that it can be done. In this example, recombinant human apoE4 is used with a mouse-derived anti-human apoE4 antibody, and an anti-mouse antibody. The antibodies were incubated with IgG antibodies or anti-mouse antibodies with a poly(M6Pn) tag. Significantly greater uptake was observed with secondary antibodies containing M6Pn.

[0216] Figure 15 shows how poly(M6Pn) labeled antibodies shatter their binding partners for degradation. Further data is provided to demonstrate that it is possible to do so. In this example, M6P EGFR degradation by incubating cells with cetuximab containing an n-tag. The following was evaluated: For all cell lines tested, total EGFR loss was observed with cetuximab. This is observed when compared to cetuximab containing a pseudopolymer (GalNAc). EGF This 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). The control percentage was calculated using densitometry.

[0217] Figure 16 shows poly(M6Pn) labeled antibody fragments degrading their binding partners. Data is provided to demonstrate that shuttles can be used. In this example, M6Pn By incubating cells with a cetuximab-derived Fab moiety containing E GFR degradation was evaluated. Total EGFR loss was assessed with cetuximab Fab alone or pseudopolymer. This is observed when compared to cetuximab Fab (GalNAc), which has the following characteristics:

[0218] Figure 17 shows how poly(M6Pn) labeled antibodies shatter their binding partners for degradation. Further data is provided to demonstrate that it is possible to capture. In this example, CD7 A primary mouse-derived antibody, anti-mouse IgG antibody, or poly(M6Pn) tagged antibody against 1. CD71 (transferred) is transferred by incubating cells with anti-mouse antibodies. The degradation of phosphorus receptors was evaluated. Systems containing the M6P tag showed significantly more degradation. vinegar.

[0219] Figure 18 shows how poly(M6Pn) labeled antibodies shatter their binding partners for degradation. Further data is provided to demonstrate that it is possible to do so. In this example, M6P Incubating cells with a tagged anti-PDL1 antibody or anti-PDL1 antibody. PDL1 degradation was evaluated using this method. Degradation was observed only with M6P-labeled anti-PDL1 antibody. .

[0220] [Example 3 - ASGPR ligand shuttles cargo to lysosomes in hepatocytes] Some current treatments suffer from off-target effects. In this example, It can be used to treat liver-related diseases such as liver cancer and liver fibrosis, in the liver Targeted degradation of proteins is described. Asial glycoprotein receptor (ASGPR) and The scavenger receptors, known as [name of receptors], are exclusively or nearly exclusively [exclusive] in liver cells (hepatocytes). It is expressed in the plasma membrane and endoscopy. As schematically shown in Figure 20 (left), ASGPR is expressed in the plasma membrane and endoscopy. It is constitutively recycled between the lysosome and the cell. ASGPR is for degradation in lysosomes. Transports extracellular glycoproteins into the cell. Binds to membrane proteins or extracellular proteins. A bifunctional molecule containing part 1 and part 2 containing the ASGPR ligand is used to treat hepatocytes The use of this receptor for degrading the extracellular or membrane proteins described above is described in this specification. This is proven by the book.

[0221] In the ASGPR-related examples described herein, N-acetylgalactosamine (GalNAc ) polymers, in particular, poly(GalNAc-co-Ala) shown in Figure 20 (bottom left) The second portion, which includes the remer, is conjugated and binds to the target molecule to be degraded. We tested bifunctional molecules containing antibodies.

[0222] A difunctional molecule containing ASGPR ligand actually sends cargo into lysosomes in hepatocytes. To evaluate whether it can be captured, we use the assay schematically shown in Figure 21. Yes. Anti-mouse I was conjugated with poly(GalNAc-co-Ala) polymer. The difunctional molecule containing IgG antibody was tested, and the difunctional molecule was found to be extracellular fluorescently labeled mouse IgG ( We determined whether IgG-AF647 could be shuttled into the intracellular compartment of hepatocytes. In the example, a bifunctional antibody containing an anti-mouse IgG antibody conjugated to an M6PR ligand is used. Sex molecules were also tested. HEPG2 cells (hepatocellular carcinoma cell line) were treated with 50 nM IgG-AF 6 Cells were incubated with 47 and 25 nM anti-mouse conjugates for 1 hour. The contents were analyzed by flow cytometry. As shown by the bar graph, Ga When using an lNAc-containing conjugate, an approximately 8-fold increase in cell fluorescence was observed. The M6Pn-containing conjugate induced a 2x increase compared to the background. In hepatocytes where the expression level of ASGPR is higher than that of M6PR, cellular uptake occurs. GalNAc-containing conjugates are more effective in inducing mitigation. This demonstrates that.

[0223] Figure 22 shows the assay described in Figure 21, however, HUH7 cells (another liver cell) Results have been shown for HIV-AF647 in HUH7 cells (a type of splenic carcinoma cell line). Efficient uptake was observed. Furthermore, cells were subjected to M6PR or ASGPR inhibitors. These are monomers M6P (mM6P) and GalNAc (mGalNAc), respectively. This included incubated controls. When cells were incubated with inhibitors, Uptake is reduced, and uptake by GalNAc or M6Pn-containing conjugates is actually This indicates that the connections are mediated by ASGPR or M6PR, respectively.

[0224] [Example 4 - Using cetuximab-ASGPR ligand conjugate, hepatocytes Efficient degradation of EGFR in [location] Epidermal growth factor receptor (EGFR) is involved in proliferation and angiogenesis in hepatocellular carcinoma (HCC). It is known to induce EGFR. 68% of HCC patients express EGFR on HCC cells. Transplantation remains the best treatment option for HCC patients at present, and a good quality death The supply of organs from deceased donors is limited. Receptor tyrosine kinase (RTK) inhibitors or Antibodies are often used in treatment, but HCC cells utilize receptor tyrosine kinase (EGFR Heterodimerization of HER2, HER3, c-Met, IGF1R (same downstream effect) This leads to phosphorylation of Kutar, restoring oncogenic signaling via RTK crosstalk. This causes resistance to these treatments to develop. EGFR blocking antibodies are used to treat cephalomyelitis. Tuximab failed in a Phase II clinical trial in HCC patients.

[0225] In this embodiment, as schematically shown in Figure 23 (top), the ASGPR ligand (in this example) This is an anti-EGFR antibody conjugated to poly(GalNAc-co-Ala) ( In this example, a bifunctional molecule containing cetuximab (or similar) is involved in the uptake of EGFR in HCC cells. And whether it could induce degradation was evaluated. Cetuximab-M6PR-Rigan De Conjugate was also tested.

[0226] HEP3B cells were used in the first experiment. The cells were treated with 10 nM cetuximab conjugate. Incubate with the blot for 48 hours, then dissolve for Western blot analysis. The Western blot (shown below Figure 23) has 5 lanes: 1) No treatment, 2) EGF (a known downregulator of EGFR), 3) cetuximab, 4) cetuximab-Ga 5) Having an INAc conjugate and a cetuximab-M6Pn conjugate As shown, the cetuximab-GalNAc conjugate is efficient in reducing EGRFR Degradation, and greater EGFR degradation compared to cetuximab-M6Pn conjugate. This was demonstrated. Vinculin was used as a loading control.

[0227] HEPG2 cells were used in the second experiment. The results are shown in Figure 24. In HEPG2 cells Efficient degradation of EGFR was observed. Here, the degradation efficiency was probably similar to that of HEP3B. Due to the relative levels of EGFR in HEPG2 cell lines, cetuximab-GalN Similarities were observed between the Ac conjugate and the cetuximab-M6Pn conjugate. On the side, the relative mRNA levels of these receptors and EGFR in the two cell lines are shown. (Available in public databases). HEPG2 cells compared to HEP3B cells. Therefore, since the EGFR level is relatively low, these data suggest that the degradation agent is less effective ( Even in the case of tuximab-M6Pn conjugate, most membrane EGFRs are degraded. It has been done, and the residual EGFR seen in Western blot is internal EGFR. This suggests that it is possible. ASGPR levels are also monitored across different processes. This showed that the ASGPR level remained constant.

[0228] Next, EGFR degradation in HEP3B cells treated with cetuximab conjugate. A time series study was conducted to evaluate the results over time. The results are shown in Figure 25. As shown, 1 Within 2 hours, the cetuximab-GalNAc conjugate increased EGFR levels by 50%. Reduces to less than . Degradation increases over 48 hours. Cetuximab-M6Pn conjugate It did not reduce EGFR levels to below 50% at any of the time points in the study.

[0229] Immunofluorescence experiments are performed to determine whether the residual EGFR is membrane EGFR or intracellular EGFR. The results were evaluated. The cell contours for HEP3B and cetuximab are shown in Figure 26. Shows EGFR localization on the membrane. HEP3B treated with cetuximab-M6Pn conjugate. In this case, some of the EGFR localizes internally, while some remains on the membrane. HEP3B is cetuximab When treated with bu-GalNAc conjugate, EGFR was hardly observed on the membrane. The majority of EGFR is intracellular. Therefore, cetuximab-GalNAc conjugate The gate degrades most membrane-bound EGFR, leaving behind residues on the Western blot. Approximately 30% of EGFR is thought to be intracellular EGFR.

[0230] [Example 5 - HER via trastuzumab alone and trastuzumab conjugate] 2 decomposition] In this example, trastuzumab alone or the GalNAc-containing polymer shown in Figure 20 was used. In the presence of conjugated trastuzumab ("trastuzumab-GalNAc") The degree of HER2 degradation in HUH 7 and HEPG2 cells was evaluated. 7 and HEPG2 cells with 10 nM trastuzumab or trastuzumab-GalNA Incubate with C conjugate for 48 hours, then for Western blot analysis. Dissolved. The Western blot in Figure 27 shows three lanes for each cell line: 1) Treatment 1) None, 2) Trastuzumab, 3) Trastuzumab-GalNAc. (See bar graph in Figure 27) The rough graph shows the mean percentage of HER2 compared to the control in each cell line. To that end, in any cell line, trastuzumab alone or GalNAc-containing polyphosphate There is a statistical difference in HER2 degradation in the presence of trastuzumab conjugated to a remer. It was not. Therefore, trastuzumab-GalNAc conjugate is trastuzumab It did not enhance the degradation of HER2 compared to the degradation of HER2 in the presence of blu alone.

[0231] Thus, the above merely explains the principles of this disclosure. Those skilled in the art will understand that Although not explicitly described or illustrated herein, the principles of the present invention are embodied and their spirit and It will be understood that various configurations can be devised within the range. Furthermore, All examples and conditional statements listed in the details are in accordance with the principles of the present invention and the inventors' agreement. The primary intention is to help readers understand the concepts that have contributed to the advancement of the relevant technical field. It should be interpreted that this is not limited to such specifically listed examples and conditions. The principle, aspects, and embodiments of the present invention, as well as specific examples thereof, are listed below. All descriptions herein encompass both their structural and functional equivalents. This is intended to be the case. In addition, such equivalents are currently known equivalents and future developments. Both of the equivalents, that is, any development element that performs the same function regardless of its structure, It is intended to include. Therefore, the scope of the present invention is limited to the examples shown and described herein. It is not intended to be limited to specific embodiments.

Claims

1. It is a difunctional molecule, A first portion that specifically binds to cell surface molecules or extracellular molecules, A bifunctional molecule comprising a second portion that specifically binds to lysosome-targeting molecules.

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

3. The first portion specifically binds to a cell surface molecule, as described in claim 1 or claim 2. A bifunctional molecule.

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

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

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

7. The cell surface molecule is a tumor-associated antigen or a tumor-specific antigen, as described in claim 6. functional molecule.

8. The cell surface molecule is present on immune cells, as described in any one of claims 1 to 7. functional molecule.

9. The aforementioned immune cells include natural killer (NK) cells, macrophages, monocytes, neutrophils, and tree cells. Selected from the group consisting of rhizocytes, T cells, B cells, mast cells, basophils, and eosinophils, The bifunctional molecule according to claim 8.

10. The bifunctional molecule according to claim 8, wherein the cell surface molecule is an inhibitory immune receptor.

11. The bifunctional relationship according to claim 10, wherein the cell surface molecule is a ligand for an inhibitory immune receptor. molecule.

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

13. The aforementioned immune checkpoint molecules are PD-1, PD-L1, CTLA4, TIM3, L A bill is selected from a group consisting of members of the AG3, TIGIT, and B7 families. The bifunctional molecule described in item 12.

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

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

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

17. The bifunctionality according to claim 15, wherein the extracellular molecule is a cytokine or chemokine. molecule.

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

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

20. Claim 18 or 19, wherein the antibody specifically binds to cell surface molecules or extracellular molecules. The bifunctional molecule described above.

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

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

23. The bifunctional molecule according to claim 22, wherein the first portion is an antibody.

24. The aforementioned antibody is IgG, single-chain Fv (scFv), Fab, (Fab) ２ , (scFv') ２ The bifunctional molecule according to claim 23, which is a nanobody.

25. The second part is derived from polypeptides, ligands, aptamers, nanoparticles, and small molecules. A bifunctional molecule according to any one of claims 1 to 24, selected from the group.

26. The lysosome-targeting molecule is the mannose-6-phosphate receptor (M6PR). A bifunctional molecule described in any one of the requirements 1 to 25.

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

28. The one or more M6PR ligands, one or more mannose-6-phosphate A bifunctional molecule according to claim 27, comprising (M6P).

29. The one or more M6PR ligands include one or more M6P analogs. A bifunctional molecule according to claim 27 or claim 28.

30. The one or more M6P analogs are one or more mannose-6-phosphonates. The bifunctional molecule according to claim 29, comprising (M6Pn).

31. The second portion comprises 1 to 500 M6PR ligands, according to any of claims 27 to 30. A bifunctional molecule as described in item one.

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

33. The polymer scaffold is a glycopolymer containing one or more M6PR ligands. A bifunctional molecule according to claim 32.

34. The glycopolymer is functionalized with one or more M6PR ligands. The bifunctional molecule according to claim 33, which is a glycoprotein containing multiple amino acids.

35. The claim states that the glycoprotein is a glycoprotein derived from N-carboxyanhydride (NCA). The bifunctional molecule described in item 34.

36. Any one of claims 1 to 25, wherein the lysosome-targeting molecule is expressed on the surface of liver cells. The bifunctional molecule described in item 1.

37. The bifunctional relationship according to claim 36, wherein the lysosome-targeting molecule is expressed on the surface of hepatocytes. molecule.

38. The lysosome-targeting molecule is used in hepatocellular carcinoma (HCC) cells, fibrous liver cells, or A bifunctional molecule according to claim 36 or 37, expressed on both surfaces.

39. The claim states that the lysosome-targeting molecule is the asialoglycoprotein receptor (ASGPR). A bifunctional molecule as described in any one of items 36 to 38.

40. The second portion comprises the one or more ASGPR ligands as described in claim 39. A difunctional molecule.

41. The one or more ASGPR ligands, one or more N-acetylgalactoglutions The bifunctional molecule according to claim 40, comprising samine (GalNAc).

42. The one or more ASGPR ligands include one or more galactoses. The bifunctional molecule according to claim 40 or 41.

43. The one or more ASGPR ligands include one or more glucose molecules. A bifunctional molecule as described in any one of the requests 40 to 42.

44. Claims 40 to 43, wherein the second portion comprises 1 to 500 ASGPR ligands. A bifunctional molecule as described in either item.

45. The second portion comprises a polymer containing one or more ASGPR ligands, A bifunctional molecule according to any one of claims 40 to 44.

46. The second portion of claim 45 comprises poly(GalNAc-co-Ala). Functional molecules.

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

48. Claim 47, wherein the second part comprises a trivalent GalNAc-containing dendrimer scaffold. A difunctional molecule.

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

50. The second portion comprises a trivalent galactose-containing dendrimer scaffold, as described in claim 49. Bifunctional molecules.

51. Claim 3, wherein the first portion specifically binds to cell surface molecules expressed on hepatocytes. A bifunctional molecule as described in any one of items 6 to 50.

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

53. The aforementioned growth factor receptors include epidermal growth factor receptor (EGFR), C-Met, and insulin-like receptors. Growth factor 1 receptor (IGF1R), fibroblast growth factor 4 receptor (FGFR4), and Selected from the group consisting of platelet-derived growth factor receptors (PDGFRs), as described in claim 52. A bifunctional molecule.

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

55. The bifunctionality according to claim 54, wherein the first part is directly fused with the second part. molecule.

56. Claim 54 includes a spacer domain between the first part and the second part. A difunctional molecule.

57. The aforementioned bifunctional molecule The cell surface molecule or extracellular molecule and The lysosome targeting molecule and A bispecific antibody that specifically binds to the target, as described in any one of claims 1 to 56. functional molecule.

58. The bifunctional molecule includes the first portion conjugated to the second portion. A bifunctional molecule according to any one of claims 1 to 53, which is a conjugate.

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

60. Claim 58 or the claim including a second portion defined in any one of claims 27 to 35. A bifunctional molecule as described in item 59.

61. Claim 58 or the claim including a second part defined in any one of claims 40 to 50. A bifunctional molecule as described in item 59.

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

63. An expression vector comprising the nucleic acid described in claim 62.

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

65. Introducing the nucleic acid described in claim 62 or the expression vector described in claim 63 into cells. A method for producing the cells according to claim 64, including the method described in claim 64.

66. Claim 58, which includes conjugating the first part to the second part, or The method for producing a bifunctional molecule as described in claim 59.

67. The aforementioned conjugation process involves site-specifically conjugating the first portion to the second portion. The method according to claim 66, comprising djugate.

68. The first portion comprises a polypeptide, and the conjugation is the second portion The portion is site-specifically conjugated to a pre-selected amino acid of the first portion. The method according to claim 67, including the following.

69. Claim 6, wherein the pre-selected amino acid is located at the N-terminus or C-terminus of the first portion. The method described in 8.

70. The method according to claim 68, wherein the pre-selected amino acids are located inside the first portion. 。

71. The aforementioned pre-selected amino acids are non-natural amino acids, as per any one of claims 68 to 70. The method described in section [section number].

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

73. The second part described above is as defined in any one of claims 27 to 35, The method described in any one of paragraphs 66 to 72.

74. The second part described above is as defined in any one of claims 40 to 50, The method described in any one of paragraphs 66 to 72.

75. The claim states that the conjugation is performed by alkyne-azide cyclization addition. The method described in any one of items 66 to 74.

76. A method for degrading cell surface molecules or extracellular molecules, Lysosome-targeting molecules degrade the cell surface molecules or extracellular molecules in lysosomes. Under conditions of shuttle operation, the cell surface molecule or extracellular molecule according to any of claims 1 to 61 A method comprising contacting the difunctional molecule described in item 1.

77. The difunctional molecule is the cell surface molecule or extracellular molecule in the presence of only the first portion. Compared to the degradation of molecules, this enhances the degradation of the cell surface molecules or extracellular molecules. The method described in claim 76.

78. The method according to claim 76 or claim 77, wherein the method is carried out in vitro.

79. The method according to claim 76 or claim 77, wherein the method is carried out in vivo.

80. A pharmaceutical composition, A bifunctional molecule according to any one of claims 1 to 61, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

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

82. The pharmaceutical composition according to claim 80 or claim 81 is administered to an individual in need thereof. A method that includes the act of doing so.

83. An effective amount of the pharmaceutical composition according to claim 80 or claim 81 is administered to an individual having cancer. Methods of treating cancer, including [specific treatment / procedure].

84. The first part described above includes a cell surface molecule on a cancer cell, a ligand for a cell surface molecule on a cancer cell, and an immunoglobulin. Cell surface molecules on disease cells, ligands for cell surface molecules on immune cells, inhibitory immune receptors, It specifically binds to molecules selected from the group consisting of ligands for inhibitory immune receptors. The method described in item 83.

85. The individual has hepatocellular carcinoma (HCC), and the first portion is a part of the HCC cells of the individual. Claim 83 or Claim The method described in 84.

86. The first portion binds to growth factors on the HCC cells of the individual, as described in claim 85. The method.

87. The first part is a group consisting of EGFR, C-Met, IGF1R, and FGFR4. The method according to claim 90, wherein the growth factor is selected from the following.

88. The second part described above is as defined in any one of claims 36 to 50, The method described in any one of items 85 to 87.

89. A method for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC), wherein individuals requiring ADCC... A method comprising administering the pharmaceutical composition according to claim 80 or claim 81 to a person.

90. A method for enhancing the immunogenicity of cancer in an individual, as described in claim 80 or claim 81 A method comprising administering the listed pharmaceutical composition to an individual.

91. The first part is a group consisting of an inhibitory immune receptor and a ligand for an inhibitory immune receptor. The method according to claim 89 or claim 90, wherein the molecule is specifically bound to a selected molecule.

92. The aforementioned administration is by parenteral administration, according to any one of claims 82 to 91. Method of description.

93. The aforementioned bifunctional molecule is a cell surface molecule or extracellular molecule in the presence of only the first portion. The claim states that the degradation of the cell surface molecules or extracellular molecules is enhanced relatively compared to the degradation of the cell surface molecules or extracellular molecules. The method described in any one of items 82 to 91.

94. It's a kit, A bifunctional molecule according to any one of claims 1 to 61, Fingers for degrading cell surface molecules or extracellular molecules to which the first part specifically binds. A kit including instructions.

95. The aforementioned instructions are for the in vitro degradation of the cell surface molecules or extracellular molecules. The kit according to claim 94.

96. The aforementioned instructions are for the in vivo degradation of the cell surface molecules or extracellular molecules. A kit according to claim 94.

97. It's a kit, A bifunctional molecule according to any one of claims 1 to 61 or claim 80 or claim The pharmaceutical composition described in 81, Instructions for administering the aforementioned bifunctional molecule or pharmaceutical composition to an individual requiring it. A kit that includes [the above].

98. The claim states that the bifunctional molecule or pharmaceutical composition is present in one or more unit doses. The kit described in 97.

99. The bifunctional molecule or pharmaceutical composition is present in two or more unit doses as described in claim 97. A kit containing [something].

100. Polymer scaffolding and, One or more mannose-6-phosphate receptors (M6P) bound to the polymer scaffold A glycopolymer containing R) ligand.

101. The glycopolymer is functionalized with one or more M6PR ligands. The glycopolymer according to claim 100, which is a glycoprotein containing multiple amino acids.

102. The glycoprotein is a glycoprotein derived from N-carboxyanhydride (NCA). The glycopolymer described in item 101.

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

104. The one or more M6PR ligands include one or more M6P analogs. A glycopolymer as described in any one of the requests 100 to 103.

105. The one or more M6P analogs are one or more mannose-6-phosphonates. The glycopolymer according to claim 104, comprising (M6Pn).

106. Claims 100 to 105, wherein the polymer scaffold contains 1 to 500 M6PR ligands. The glycopolymer described in any one of the items.

107. A method for producing a glycopolymer according to any one of claims 100 to 106, 、107 Attaching one or more M6PR ligands to the polymer scaffold, or The polymer scaffold is formed from monomers functionalized with one or more M6PR ligands. A method that includes achieving something.

108. The scaffold is functionalized with one or more M6PR ligands. The synthesis is carried out by solid-phase synthesis, as described in claim 107, which is polymerized from monomers. Method of description.

109. The glycopolymer is a glycoprotein polymer, and the synthesis is a solid-phase peptide. The method according to claim 108, wherein the method is by synthesis.

110. A glycopolymer according to any one of claims 100 to 106, Includes instructions for conjugating the glycopolymer to the target molecule, kit.

111. The reagent further comprises a reagent for conjugating the glycopolymer into a target molecule. The kit according to claim 110.

112. The kit according to claim 110 or 111, wherein the target molecule is a polypeptide.

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

114. Claim 110, wherein the target molecule specifically binds to a cell surface molecule or an extracellular molecule. A kit as described in any one of items ~113.

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

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

117. The monomer according to claim 115, wherein the monomer is a non-natural amino acid.

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

119. The one or more M6PR ligands include one or more M6P analogs. A monomer described in any one of the requests 115 to 118.

120. The one or more M6P analogs are one or more mannose-6-phosphonates. The monomer according to claim 119, comprising (M6Pn).

Images