US2017042979A1PendingUtilityA1

Targeting of glycoprotein therapeutics

61
Assignee: GENZYME CORPPriority: Apr 6, 2005Filed: Oct 18, 2016Published: Feb 16, 2017
Est. expiryApr 6, 2025(expired)· nominal 20-yr term from priority
Inventors:James Stefano
A61P 43/00A61P 3/00A61K 47/64A61K 38/47A61K 47/61C12Y 302/01022C12N 9/2465C12N 9/96A61K 47/60A61K 47/48215
61
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Claims

Abstract

Methods of making ligand-decorated polymer conjugates of therapeutic glycoproteins are described. Improved targeting of glycoproteins to specific tissues is achieved by masking the natural carbohydrate and other surface determinants with high molecular weight polymers, such as, e.g., PEG, polysialic acid, etc., which in turn are decorated with target-specific ligands. In some embodiments, acid-labile linkages in such conjugates or rapidly degradable masking groups allow for the intracellular release of the polymer from the glycoprotein, for example, under conditions found in lysosomes.

Claims

exact text as granted — not AI-modified
1 . A conjugate comprising:
 (1) a therapeutic glycoprotein (G),   (2) a masking moiety (M) covalently linked to an oligosaccharide side chain of the glycoprotein through a first linker (L 1 ), and   (3) a targeting moiety (T) covalently linked to the masking moiety through a second linker (L 2 ),   wherein the glycoprotein is released from the conjugate under lysosomal conditions.   
     
     
         2 . The conjugate of  claim 1  comprising G(L 1 -M(L 2 -T) n ) m , wherein 1≦n≦20 and 1≦m≦20, independently of each other. 
     
     
         3 . The conjugate of  claim 1 , wherein the glycoprotein is a non-viral protein chosen from an enzyme and an antibody. 
     
     
         4 . The conjugate of  claim 3 , wherein the enzyme is a lysosomal enzyme. 
     
     
         5 . The conjugate of  claim 4 , wherein the lysosomal enzyme is selected from the group consisting of α-galactosidase A, acid ceramidase, acid α-L-fucosidase, glucocerebrosidase, acid β-galactosidase, iduronate-2-sulfatase, α-L-iduronidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, arylsulfatase B, arylsulfatase A, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase, acid α-glucosidase, β-hexosaminidase B, heparan N-sulfatase, α-N-acetylglucosaminidase,
 acetyl-CoA:α-glucosaminiden-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, α-N-acetylgalactosaminidase, sialidase, β-glucuronidase, and β-hexosaminidase A. 
 
     
     
         6 . The conjugate of  claim 5 , wherein the lysosomal enzyme is chosen from acid α-glucosidase and acid sphingomyelinase. 
     
     
         7 . The conjugate of  claim 5 , wherein the lysosomal enzyme is chosen from α-galactosidase A and glucocerebrosidase. 
     
     
         8 . The conjugate of  claim 1 , wherein the masking moiety is a polymer selected the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polymethacrylate (PMA), polysialic acid (PSA), hyaluronic acid (HA), albumin, immunoglobulin (IgG), dextran sulfate, polyethyleneimine (PEI), polyacrylamide, α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA), poly(vinylpyrrolidone-co-dimethyl maleic anhydride (poly(VP-co-DMMAn), N-(2-hydroxypropyl)methacrylimide) (HMPA) and a hydroxy alkyl starch (HAS). 
     
     
         9 . The conjugate of  claim 8 , wherein the PEG is a star-PEG or a pendant-PEG. 
     
     
         10 . The conjugate of  claim 1 , wherein the masking moiety is degradable under the lysosomal conditions thereby allowing the release of the glycoprotein. 
     
     
         11 . The conjugate of  claim 1 , wherein the targeting moiety is a receptor-binding peptide, an antibody to a receptor, or a natural receptor ligand. 
     
     
         12 . The conjugate of  claim 1 , wherein at least one of the linkers L 1  and L 2  comprises a labile group which allows the glycoprotein to be released under the lysosomal conditions. 
     
     
         13 . The conjugate of  claim 12 , wherein the linker comprising the labile group is L 1 . 
     
     
         14 . The conjugate of  claim 12 , wherein the labile group is acid-labile. 
     
     
         15 . The conjugate of  claim 14 , wherein the acid-labile group is chosen from hydrazone, imino, ester, and amido. 
     
     
         16 . The conjugate of  claim 14 , wherein the acid-labile group is a hydrazone group. 
     
     
         17 . The conjugate of  claim 16 , wherein at least one of L 1  and L 2  comprises a hydrazone-containing group chosen from: 
       
         
           
           
               
               
           
         
         wherein Ar is aryl or heteroaryl. 
       
     
     
         18 . The conjugate of  claim 17 , wherein at least one of L 1  and L 2  comprises a hydrazone-containing group chosen from: 
       
         
           
           
               
               
           
         
       
     
     
         19 . The conjugate of  claim 1 , wherein at least one of L 1  and L 2  comprises an acid-labile group and a disulfide. 
     
     
         20 . The conjugate of  claim 19 , wherein acid-labile group is chosen from hydrazone, imino, ester, and amido. 
     
     
         21 . The conjugate of  claim 19 , wherein acid-labile group is a hydrazone group. 
     
     
         22 . The conjugate of  claim 19 , wherein at least one of L 1  and L 2  comprises a group chosen from: 
       
         
           
           
               
               
           
         
       
     
     
         23 . The conjugate of  claim 1  comprising the formula chosen from: 
       
         
           
           
               
               
           
         
       
     
     
         24 . The conjugate of  claim 23  comprising the formula chosen from: 
       
         
           
           
               
               
           
         
       
     
     
         25 . A conjugate comprising:
 (1) a therapeutic glycoprotein (G),   (2) a first means for masking an oligosaccharide side chain recognition by its cognate receptor, said first means being covalently linked to the glycoprotein through a first linker (L 1 ), and   (3) a second means for targeting the conjugate to a specific tissue or organ, said first means being covalently linked to the first means through a second linker (L 2 ),   wherein the glycoprotein is released from the conjugate under lysosomal conditions.   
     
     
         26 . A pharmaceutical composition, comprising the conjugate of  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         27 . A method of making the conjugate of  claim 1 , comprising:
 (i) providing a masking moiety comprising a first functional group and a second functional group,   (ii) reacting the first functional group with a carbohydrate side chain of a glycoprotein, and   (iii) reacting the second functional group with a targeting moiety.   
     
     
         28 . A method of treating a lysosomal storage disorder in a mammal, comprising administering to the mammal the conjugate of  claim 1 . 
     
     
         29 . A conjugate comprising:
 (1) a therapeutic glycoprotein (G),   (2) a masking moiety (M) covalently linked to the glycoprotein through a first linker (L 1 ), and   (3) a targeting moiety (T) covalently linked to the masking moiety through a second linker (L 2 ),   wherein the glycoprotein is released from the conjugate under lysosomal conditions.   
     
     
         30 . The conjugate of  claim 29 , comprising G(L 1 -M(L 2 -T) n ) m , wherein 1≦n≦20 and 1≦m≦20, independently of each other. 
     
     
         31 . The conjugate of  claim 29 , wherein the glycoprotein is a non-viral protein chosen from an enzyme and an antibody. 
     
     
         32 . The conjugate of  claim 31 , wherein the enzyme is a lysosomal enzyme. 
     
     
         33 . The conjugate of  claim 32 , wherein the lysosomal enzyme is selected from the group consisting of α-galactosidase A, acid ceramidase, acid α-L-fucosidase, glucocerebrosidase, acid β-galactosidase, iduronate-2-sulfatase, α-L-iduronidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, arylsulfatase B, arylsulfatase A, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase, acid α-glucosidase, β-hexosaminidase B, heparan N-sulfatase, α-N-acetylglucosaminidase,
 acetyl-CoA:α-glucosaminiden-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, α-N-acetylgalactosaminidase, sialidase, β-glucuronidase, and β-hexosaminidase A. 
 
     
     
         34 . The conjugate of  claim 33 , wherein the lysosomal enzyme is chosen from α-galactosidase A and glucocerebrosidase. 
     
     
         35 . The conjugate of  claim 29 , wherein the masking moiety is a polymer selected the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polymethacrylate (PMA), polysialic acid (PSA), hyaluronic acid (HA), albumin, immunoglobulin (IgG), dextran sulfate, polyethyleneimine (PEI), polyacrylamide, α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA), poly(vinylpyrrolidone-co-dimethyl maleic anhydride (poly(VP-co-DMMAn), N-(2-hydroxypropyl)methacrylimide) (HMPA) and a hydroxy alkyl starch (HAS). 
     
     
         36 . The conjugate of  claim 35 , wherein the PEG is a star-PEG or a pendant-PEG. 
     
     
         37 . The conjugate of  claim 29 , wherein the masking moiety is degradable under the lysosomal conditions thereby allowing the release of the glycoprotein. 
     
     
         38 . The conjugate of  claim 29 , wherein the targeting moiety is a non-endogenous protein, a receptor-binding peptide, an antibody to a receptor, or a natural receptor ligand. 
     
     
         39 . The conjugate of  claim 29 , wherein at least one of the linkers L 1  and L 2  comprises a labile group which allows the glycoprotein to be released under the lysosomal conditions. 
     
     
         40 . The conjugate of  claim 39 , wherein the labile group is acid-labile. 
     
     
         41 . The conjugate of  claim 40 , wherein the acid-labile group is chosen from hydrazone, imino, ester, and amido. 
     
     
         42 . The conjugate of  claim 41 , wherein the acid-labile group is a hydrazone group. 
     
     
         43 . The conjugate of  claim 39 , wherein the labile group is a disulfide. 
     
     
         44 . A conjugate comprising:
 (1) a therapeutic glycoprotein (G),   (2) a masking moiety (M) covalently linked to an amino acid residue of the glycoprotein through a first linker (L 1 ), and   (3) a targeting moiety (T) covalently linked to the masking moiety through a second linker (L 2 ),   wherein the glycoprotein is released from the conjugate under lysosomal conditions.   
     
     
         45 . The conjugate of  claim 44 , comprising G(L 1 -M(L 2 -T) n ) m , wherein 1≦n≦20 and 1≦m≦20, independently of each other. 
     
     
         46 . The conjugate of  claim 44 , wherein the glycoprotein is a non-viral protein chosen from an enzyme and an antibody. 
     
     
         47 . The conjugate of  claim 46 , wherein the enzyme is a lysosomal enzyme. 
     
     
         48 . The conjugate of  claim 47 , wherein the lysosomal enzyme is selected from the group consisting of α-galactosidase A, acid ceramidase, acid α-L-fucosidase, glucocerebrosidase, acid β-galactosidase, iduronate-2-sulfatase, α-L-iduronidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, arylsulfatase B, arylsulfatase A, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase, acid α-glucosidase, β-hexosaminidase B, heparan N-sulfatase, α-N-acetylglucosaminidase,
 acetyl-CoA:α-glucosaminiden-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, α-N-acetylgalactosaminidase, sialidase, β-glucuronidase, and β-hexosaminidase A. 
 
     
     
         49 . The conjugate of  claim 48 , wherein the lysosomal enzyme is chosen from α-galactosidase A and glucocerebrosidase. 
     
     
         50 . The conjugate of  claim 44 , wherein the amino acid residue is a lysine residue. 
     
     
         51 . The conjugate of  claim 44 , wherein the masking moiety is a polymer selected the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polymethacrylate (PMA), polysialic acid (PSA), hyaluronic acid (HA), albumin, immunoglobulin (IgG), dextran sulfate, polyethyleneimine (PEI), polyacrylamide, α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA), poly(vinylpyrrolidone-co-dimethyl maleic anhydride (poly(VP-co-DMMAn), N-(2-hydroxypropyl)methacrylimide) (HMPA) and a hydroxy alkyl starch (HAS). 
     
     
         52 . The conjugate of  claim 51 , wherein the PEG is a star-PEG or a pendant-PEG. 
     
     
         53 . The conjugate of  claim 44 , wherein the masking moiety is degradable under the lysosomal conditions thereby allowing the release of the glycoprotein. 
     
     
         54 . The conjugate of  claim 44 , wherein the targeting moiety is a non-endogenous protein, a receptor-binding peptide, an antibody to a receptor, or a natural receptor ligand. 
     
     
         55 . The conjugate of  claim 44 , wherein at least one of the linkers L 1  and L 2  comprises a labile group which allows the glycoprotein to be released under the lysosomal conditions. 
     
     
         56 . The conjugate of  claim 54 , wherein the labile group is a disulfide. 
     
     
         57 . The conjugate of  claim 56 , wherein at least one of the linkers L 1  and L 2  comprises a disulfide selected from: 
       
         
           
           
               
               
           
         
         wherein 2≦p≦12. 
       
     
     
         58 . The conjugate of  claim 55 , wherein the labile group is acid-labile. 
     
     
         59 . The conjugate of  claim 58 , wherein the acid-labile group is chosen from hydrazone, imino, ester, and amido. 
     
     
         60 . The conjugate of  claim 59 , wherein the acid-labile group is a hydrazone group. 
     
     
         61 . The conjugate of  claim 60 , wherein at least one of L 1  and L 2  comprises a group selected from: 
       
         
           
           
               
               
           
         
         wherein Ar is aryl or heteroaryl. 
       
     
     
         62 . The conjugate of  claim 56 , comprising the formula: 
       
         
           
           
               
               
           
         
         wherein 2≦p≦12 and 2≦q≦12, independently of each other. 
       
     
     
         63 . The conjugate of  claim 12 , wherein the labile group comprises a disulfide. 
     
     
         64 . The conjugate of  claim 63 , wherein at least one of the linkers L 1  and L 2  comprises a disulfide selected from: 
       
         
           
           
               
               
           
         
         wherein 2≦p≦12. 
       
     
     
         65 . A pharmaceutical composition, comprising the conjugate of  claim 44  and a pharmaceutically acceptable excipient. 
     
     
         66 . A method of making the conjugate of  claim 44 , comprising:
 (i) providing a masking moiety comprising a first functional group and a second functional group,   (ii) reacting the first functional group with an amino acid residue of a glycoprotein, and   (iii) reacting the second functional group with a targeting moiety.   
     
     
         67 . A method of treating a lysosomal storage disorder in a mammal, comprising administering to the mammal the conjugate of  claim 44 .

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