US2022023434A1PendingUtilityA1
Bifunctional Molecules for Lysosomal Targeting and Related Compositions and Methods
Assignee: UNIV LELAND STANFORD JUNIORPriority: Dec 19, 2018Filed: Dec 18, 2019Published: Jan 27, 2022
Est. expiryDec 19, 2038(~12.4 yrs left)· nominal 20-yr term from priority
A61K 47/6849C07K 16/42C07K 16/18C07K 2317/55C07K 16/2863C07K 2317/732A61K 47/61A61K 47/6897C07K 16/2881C07K 16/32C07K 2317/77A61K 47/6855A61K 47/6873C07K 16/28A61P 35/00A61K 47/6425C07K 16/2827
60
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Claims
Abstract
Provided are bifunctional molecules that include a first moiety that specifically binds a cell surface molecule or extracellular molecule, and a second moiety that specifically binds a lysosomal targeting molecule. The bifunctional molecules find use, e.g., for targeted degradation of cell surface and extracellular molecules (e.g., proteins) via the endosomal/lysosomal pathway. Also provided are compositions and kits that include the bifunctional molecules, as well as methods of using the bifunctional molecules. Methods of making bifunctional molecules are also provided.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A bifunctional molecule comprising:
a first moiety that specifically binds a cell surface molecule or extracellular molecule; and a second moiety that specifically binds a lysosomal targeting molecule.
2 . The bifunctional molecule of claim 1 , wherein the bifunctional molecule enhances degradation of the cell surface molecule or extracellular molecule relative to degradation of the cell surface molecule or extracellular molecule in the presence of the first moiety alone.
3 . The bifunctional molecule of claim 1 or claim 2 , wherein the first moiety specifically binds a cell surface molecule.
4 . The bifunctional molecule of claim 3 , wherein the cell surface molecule is a cell surface receptor.
5 . The bifunctional molecule of claim 4 , wherein the cell surface receptor is a growth factor receptor.
6 . The bifunctional molecule of any one of claims 1 to 5 , wherein the cell surface molecule is present on a cancer cell.
7 . The bifunctional molecule of claim 6 , wherein the cell surface molecule is a tumor-associated antigen or a tumor-specific antigen.
8 . The bifunctional molecule of any one of claims 1 to 7 , wherein the cell surface molecule is present on an immune cell.
9 . The bifunctional molecule of claim 8 , wherein the immune cell is selected from the group consisting of: a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a basophil, and an eosinophil.
10 . The bifunctional molecule of claim 8 , wherein the cell surface molecule is an inhibitory immune receptor.
11 . The bifunctional molecule of claim 10 , wherein the cell surface molecule is a ligand of an inhibitory immune receptor.
12 . The bifunctional molecule of claim 8 , wherein the cell surface molecule is an immune checkpoint molecule.
13 . The bifunctional molecule of claim 12 , wherein the immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and a member of the B7 family.
14 . The bifunctional molecule of claim 1 , wherein the first moiety specifically binds an extracellular molecule.
15 . The bifunctional molecule of claim 14 , wherein the extracellular molecule is a ligand for a cell surface receptor.
16 . The bifunctional molecule of claim 15 , wherein the extracellular molecule is a growth factor.
17 . The bifunctional molecule of claim 15 , wherein the extracellular molecule is a cytokine or a chemokine.
18 . The bifunctional molecule of claim 14 , wherein the extracellular molecule is an antibody.
19 . The bifunctional molecule of claim 18 , wherein the antibody is an autoantibody.
20 . The bifunctional molecule of claim 18 or claim 19 , wherein the antibody specifically binds to a cell surface molecule or an extracellular molecule.
21 . The bifunctional molecule of any one of claims 1 to 20 , wherein the first moiety is selected from the group consisting of: a polypeptide, a ligand, an aptamer, a nanoparticle, and a small molecule.
22 . The bifunctional molecule of claim 21 , wherein the first moiety is a polypeptide.
23 . The bifunctional molecule of claim 22 , wherein the first moiety is an antibody.
24 . The bifunctional molecule of claim 23 , wherein the antibody is an IgG, a single chain Fv (scFv), Fab, (Fab) 2 , (scFv′) 2 , or a nanobody.
25 . The bifunctional molecule of any one of claims 1 to 24 , wherein the second moiety is selected from the group consisting of: a polypeptide, a ligand, an aptamer, a nanoparticle, and a small molecule.
26 . The bifunctional molecule of any one of claims 1 to 25 , wherein the lysosomal targeting molecule is a mannose-6-phosphate receptor (M6PR).
27 . The bifunctional molecule of claim 26 , wherein the second moiety comprises one or more M6PR ligands.
28 . The bifunctional molecule of claim 27 , wherein the one or more M6PR ligands comprise one or more mannose-6-phosphates (M6P).
29 . The bifunctional molecule of claim 27 or claim 28 , wherein the one or more M6PR ligands comprise one or more M6P analogs.
30 . The bifunctional molecule of claim 29 , wherein the one or more M6P analogs comprise one or more mannose-6-phosphonates (M6Pn).
31 . The bifunctional molecule of any one of claims 27 to 30 , wherein the second moiety comprises from 1 to 500 M6PR ligands.
32 . The bifunctional molecule of any one of claims 27 to 31 , wherein the second moiety comprises a polymer scaffold that displays the one or more M6PR ligands.
33 . The bifunctional molecule of claim 32 , wherein the polymer scaffold is a glycopolymer comprising the one or more M6PR ligands.
34 . The bifunctional molecule of claim 33 , wherein the glycopolymer is a glycoprotein comprising one or more amino acids functionalized with the one or more M6PR ligands.
35 . The bifunctional molecule of claim 34 , wherein the glycoprotein is a N-carboxyanhydride (NCA)-derived glycoprotein.
36 . The bifunctional molecule of any one of claims 1 to 25 , wherein the lysosomal targeting molecule is expressed on the surface of liver cells.
37 . The bifunctional molecule of claim 36 , wherein the lysosomal targeting molecule is expressed on the surface of hepatocytes.
38 . The bifunctional molecule of claim 36 or claim 37 , wherein the lysosomal targeting molecule is expressed on the surface of hepatocellular carcinoma (HCC) cells, fibrotic liver cells, or both.
39 . The bifunctional molecule of any one of claims 36 to 38 , wherein the lysosomal targeting molecule is asialoglycoprotein receptor (ASGPR).
40 . The bifunctional molecule of claim 39 , wherein the second moiety comprises one or more ASGPR ligands.
41 . The bifunctional molecule of claim 40 , wherein the one or more ASGPR ligands comprises one or more N-acetylgalactosamines (GalNAc).
42 . The bifunctional molecule of claim 40 or claim 41 , wherein the one or more ASGPR ligands comprises one or more galactoses.
43 . The bifunctional molecule of any one of claims 40 to 42 , wherein the one or more ASGPR ligands comprises one or more glucoses.
44 . The bifunctional molecule of any one of claims 40 to 43 , wherein the second moiety comprises from 1 to 500 ASGPR ligands.
45 . The bifunctional molecule of any one of claims 40 to 44 , wherein the second moiety comprises a polymer comprising the one or more ASGPR ligands.
46 . The bifunctional molecule of claim 45 , wherein the second moiety comprises poly(GalNAc-co-Ala).
47 . The bifunctional molecule of claim 41 , wherein the second moiety comprises a monovalent, bivalent, or trivalent GalNAc-containing dendrimer scaffold.
48 . The bifunctional molecule of claim 47 , wherein the second moiety comprises a trivalent GalNAc-containing dendrimer scaffold.
49 . The bifunctional molecule of claim 42 , wherein the second moiety comprises a monovalent, bivalent, or trivalent galactose-containing dendrimer scaffold.
50 . The bifunctional molecule of claim 49 , wherein the second moiety comprises a trivalent galactose-containing dendrimer scaffold.
51 . The bifunctional molecule of any one of claims 36 to 50 , wherein the first moiety specifically binds a cell surface molecule expressed on hepatocytes.
52 . The bifunctional molecule of claim 51 , wherein the cell surface molecule is a growth factor receptor.
53 . The bifunctional molecule of claim 52 , wherein the growth factor receptor is selected from the group consisting of epidermal growth factor receptor (EGFR), C-Met, insulin like growth factor 1 receptor (IGF1R), fibroblast growth factor receptor 4 (FGFR4), and platelet-derived growth factor receptor (PDGFR).
54 . The bifunctional molecule of any one of claims 1 to 53 , wherein the first moiety is a polypeptide and the second moiety is a polypeptide, and wherein the bifunctional molecule is a fusion protein comprising the first moiety fused to the second moiety.
55 . The bifunctional molecule of claim 54 , wherein the first moiety is fused directly to the second moiety.
56 . The bifunctional molecule of claim 54 , comprising a spacer domain between the first moiety and the second moiety.
57 . The bifunctional molecule of any one of claims 1 to 56 , wherein the bifunctional molecule is a bispecific antibody that specifically binds:
a cell surface molecule or extracellular molecule; and
a lysosomal targeting molecule.
58 . The bifunctional molecule of any one of claims 1 to 53 , wherein the bifunctional molecule is a conjugate comprising the first moiety conjugated to the second moiety.
59 . The bifunctional molecule of claim 58 , wherein the first moiety is an antibody.
60 . The bifunctional molecule of claim 58 or claim 59 , comprising a second moiety as defined in any one of claims 27 to 35 .
61 . The bifunctional molecule of claim 58 or claim 59 , comprising a second moiety as defined in any one of claims 40 to 50 .
62 . A nucleic acid encoding the bifunctional molecule of any one of claims 54 to 57 .
63 . An expression vector comprising the nucleic acid of claim 62 .
64 . A cell comprising the nucleic acid of claim 62 or the expression vector of claim 63 .
65 . A method of producing the cell of claim 64 , comprising introducing into a cell the nucleic acid of claim 62 or the expression vector of claim 63 .
66 . A method of making the bifunctional molecule of claim 58 or claim 59 , comprising conjugating the first moiety to the second moiety.
67 . The method according to claim 66 , wherein the conjugating comprises site-specifically conjugating the first moiety to the second moiety.
68 . The method according to claim 67 , wherein the first moiety comprises a polypeptide, and wherein the conjugating comprises site-specifically conjugating the second moiety to a pre-selected amino acid of the first moiety.
69 . The method according to claim 68 , wherein the pre-selected amino acid is at the N-terminus or C-terminus of the first moiety.
70 . The method according to claim 68 , wherein the pre-selected amino acid is internal to the first moiety.
71 . The method according to any one of claims 68 to 70 , wherein the pre-selected amino acid is a non-natural amino acid.
72 . The method according to any one of claims 66 to 71 , wherein the first moiety is an antibody.
73 . The method according to any one of claims 66 to 72 , wherein the second moiety is as defined in any one of claims 27 to 35 .
74 . The method according to any one of claims 66 to 72 , wherein the second moiety is as defined in any one of claims 40 to 50 .
75 . The method according to any one of claims 66 to 74 , wherein the conjugating is by alkyne-azide cycloaddition.
76 . A method of degrading a cell surface molecule or extracellular molecule, comprising:
contacting the cell surface molecule or extracellular molecule with the bifunctional molecule of any one of claims 1 to 61 under conditions in which the lysosomal targeting molecule shuttles the cell surface molecule or extracellular molecule to the lysosome for degradation.
77 . The method according to claim 76 , wherein the bifunctional molecule enhances degradation of the cell surface molecule or extracellular molecule relative to degradation of the cell surface molecule or extracellular molecule in the presence of the first moiety alone.
78 . The method according to claim 76 or claim 77 , wherein the method is performed in vitro.
79 . The method according to claim 76 or claim 77 , wherein the method is performed in vivo.
80 . A pharmaceutical composition comprising:
the bifunctional molecule of any one of claims 1 to 61 ; and a pharmaceutically acceptable carrier.
81 . The pharmaceutical composition of claim 80 , wherein the composition is formulated for parenteral administration.
82 . A method comprising administering to an individual in need thereof the pharmaceutical composition of claim 80 or claim 81 .
83 . A method of treating cancer comprising administering to an individual having cancer an effective amount of the pharmaceutical composition of claim 80 or claim 81 .
84 . The method according to claim 83 , wherein the first moiety specifically binds to a molecule selected from the group consisting of: a cell surface molecule on a cancer cell, a ligand for a cell surface molecule on a cancer cell, a cell surface molecule on an immune cell, a ligand for a cell surface molecule on an immune cell, an inhibitory immune receptor, and a ligand of an inhibitory immune receptor.
85 . The method according to claim 83 or claim 84 , wherein the individual has hepatocellular carcinoma (HCC), the first moiety binds a cell surface molecule on HCC cells of the individual, and the second moiety binds ASGPR.
86 . The method according to claim 85 , wherein the first moiety binds a growth factor on HCC cells of the individual.
87 . The method according to claim 90 , wherein the first moiety binds a growth factor selected from the group consisting of: EGFR, C-Met, IGF1R, and FGFR4.
88 . The method according to any one of claims 85 to 87 , wherein the second moiety is as defined in any one of claims 36 to 50 .
89 . A method of enhancing antibody-dependent cellular cytotoxicity (ADCC) comprising administering to an individual in need of ADCC the pharmaceutical composition of claim 80 or claim 81 .
90 . A method of enhancing immunogenicity of a cancer in an individual, comprising administering to the individual the pharmaceutical composition of claim 80 or claim 81 .
91 . The method according to claim 89 or claim 90 , wherein the first moiety specifically binds to a molecule selected from the group consisting of: an inhibitory immune receptor, and a ligand of an inhibitory immune receptor.
92 . The method according to any one of claims 82 to 91 , wherein the administering is by parenteral administration.
93 . The method according to any one of claims 82 to 91 , wherein the bifunctional molecule enhances degradation of the cell surface molecule or extracellular molecule relative to degradation of the cell surface molecule or extracellular molecule in the presence of the first moiety alone.
94 . A kit comprising:
the bifunctional molecule of any one of claims 1 to 61 ; and instructions for degrading the cell surface molecule or extracellular molecule to which the first moiety specifically binds.
95 . The kit of claim 94 , wherein the instructions are for degrading the cell surface molecule or extracellular molecule in vitro.
96 . The kit of claim 94 , wherein the instructions are for degrading the cell surface molecule or extracellular molecule in vivo.
97 . A kit comprising:
the bifunctional molecule of any one of claims 1 to 61 or the pharmaceutical composition of claim 80 or claim 81 ; and instructions for administering the bifunctional molecule or pharmaceutical composition to an individual in need thereof.
98 . The kit of claim 97 , wherein the bifunctional molecule or pharmaceutical composition is present in one or more unit dosages.
99 . The kit of claim 97 , wherein the bifunctional molecule or pharmaceutical composition is present in two or more unit dosages.
100 . A glycopolymer comprising:
a polymer scaffold; and one or more mannose-6-phosphate receptor (M6PR) ligands attached to the polymer scaffold.
101 . The glycopolymer of claim 100 , wherein the glycopolymer is a glycoprotein comprising one or more amino acids functionalized with the one or more M6PR ligands.
102 . The glycopolymer of claim 101 , wherein the glycoprotein is a N-carboxyanhydride (NCA)-derived glycoprotein.
103 . The glycopolymer of any one of claims 100 to 102 , wherein the one or more M6PR ligands comprise one or more mannose-6-phosphates (M6P).
104 . The glycopolymer of any one of claims 100 to 103 , wherein the one or more M6PR ligands comprise one or more M6P analogs.
105 . The glycopolymer of claim 104 , wherein the one or more M6P analogs comprise one or more mannose-6-phosphonates (M6Pn).
106 . The glycopolymer of any one of claims 100 to 105 , wherein the polymer scaffold comprises from 1 to 500 M6PR ligands.
107 . A method of making the glycopolymer of any one of claims 100 to 106 , comprising:
attaching the one or more M6PR ligands to the polymer scaffold; or
synthesizing the polymer scaffold from monomers functionalized with the one or more M6PR ligands.
108 . The method according to claim 107 , wherein the scaffold is polymerized from one or more monomers functionalized with the one or more M6PR ligands, and wherein the synthesizing is by solid-phase synthesis.
109 . The method according to claim 108 , wherein the glycopolymer is a glycoprotein polymer, and wherein the synthesizing is by solid-phase peptide synthesis.
110 . A kit comprising:
the glycopolymer of any one of claims 100 to 106 ; and instructions for conjugating the glycopolymer to a molecule of interest.
111 . The kit of claim 110 , further comprising reagents for conjugating the glycopolymer to a molecule of interest.
112 . The kit of claim 110 or claim 111 , wherein the molecule of interest is a polypeptide.
113 . The kit of claim 112 , wherein the polypeptide is an antibody.
114 . The kit of any one of claims 110 to 113 , wherein the molecule of interest specifically binds a cell surface molecule or extracellular molecule.
115 . A monomer functionalized with one or more mannose-6-phosphate receptor (M6PR) ligands.
116 . The monomer of claim 115 , wherein the monomer is an amino acid.
117 . The monomer of claim 115 , wherein the monomer is a non-natural amino acid.
118 . The monomer of any one of claims 115 to 117 , wherein the one or more M6PR ligands comprise one or more mannose-6-phosphates (M6P).
119 . The monomer of any one of claims 115 to 118 , wherein the one or more M6PR ligands comprise one or more M6P analogs.
120 . The monomer of claim 119 , wherein the one or more M6P analogs comprise one or more mannose-6-phosphonates (M6Pn).Join the waitlist — get patent alerts
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