Methods for coupling targeting peptides onto recombinant lysosomal enzymes for improved treatments of lysosomal storage diseases
Described herein are methods of making targeting peptides conjugated to a recombinant lysosomal enzyme by modifying the amino (N)-terminus and one or more lysine residues on a recombinant human lysosomal enzyme using a first crosslinking agent to give rise to a first crosslinking agent modified recombinant human lysosomal enzyme, modifying a lysine or cysteine within a short extension linker at the carboxyl (C)-terminus on a variant IGF-2 peptide having a short extension linker using a second crosslinking agent to give rise to a second crosslinking agent modified variant IGF-2 peptide, and then conjugating the first crosslinking agent modified recombinant human lysosomal enzyme to the second crosslinking agent modified variant IGF-2 peptide containing a short extension linker. Also described herein are conjugates synthesized using the methods disclosed herein. Also described herein are treatment methods using the disclosed conjugates.
1. A conjugate, comprising:
a variant IGF-2 peptide chemically conjugated to a recombinant human lysosomal the variant IGF-2 peptide comprises SEQ ID NO:2 or SEQ ID NO:6 or, comprises one or more of the following modifications with respect to SEQ ID NO: 8:
substitution of arginine for glutamic acid at position 6;
deletion of amino acids 1-4 and 6;
deletion of amino acids 1-4, 6 and 7;
deletion of amino acids 1-4 and 6 and substitution of lysine for threonine at position 7;
deletion of amino acids 1-4 and substitution of glycine for glutamic acid at position 6 and substitution of lysine for threonine at position 7;
substitution of leucine for tyrosine at position 27;
substitution of leucine for valine at position 43;
substitution of arginine for lysine at position 65; and
the IGF-2 peptide further comprises an affinity tag and/or a linker extension region.
2. The conjugate of claim 1 , wherein the recombinant human lysosomal enzyme comprises one or more modified lysine residues, a chemically modified N-terminus, or a combination thereof.
3. The conjugate of claim 1 , wherein the recombinant human lysosomal enzyme is human acid a-glucosidase (rhGAA).
4. The conjugate of claim 1 , wherein the chemical conjugation is via a cross linking agent comprises an aminoreactive bifunctional cross linker.
5. The conjugate of claim 1 , wherein the chemical conjugation is via a cross linking agent comprises N-succinimidyl 6-hydrazinonicotinate acetone (S-Hynic).
6. The conjugate of claim 1 , wherein the chemical conjugation is via a cross linking agent comprises sulfo-Nhydroxysuccinimide ester-phosphine (sulfo-NHS-phosphine).
7. The conjugate of claim 1 , wherein the-chemical conjugation is via a cross linking agent comprises N-hydroxysuccinimide ester-tetraoxapentadecane acetylene (NHS-PEG4-acetylene).
8. The conjugate of claim 1 , wherein the chemical conjugation is via a crosslinking agent comprises heterobifunctional cross linkers selected from difluorocyclooctyne (DIFO) and dibenzocyclooctyne (DIBO).
9. A method for treating a subject suffering from a lysosomal storage disease, the method comprising administering to the subject the conjugate of claim 1 in an amount sufficient to treat the lysosomal storage disease.
10. The method of claim 9 , wherein the lysosomal storage disease is at least one of the following: Pompe Disease, Fabry Disease, and Gaucher Disease, MPS I, MPS II, MPS VII, Tay Sachs, Sandhoff, a-mannosidosis, and Wohlman disease.
11. The method of claim 10 , wherein the lysosomal storage disease is Pompe Disease.
12. The method of claim 10 , wherein the lysosomal storage disease is Fabry Disease.
13. The method of claim 10 , wherein the lysosomal storage disease is Gaucher Disease.
14. The conjugate of claim 1 , wherein the IGF-2 peptide comprises SEQ ID NO:2.
15. The conjugate of claim 1 , wherein the IGF-2 peptide comprises SEQ ID NO:6.
16. The conjugate of claim 1 , wherein the IGF-2 peptide further comprises a linker, wherein said linker is 5 to 20 amino acid residues in length.
17. The conjugate of claim 16 , wherein said linker is about 10 amino acid residues in length.
18. The conjugate of claim 16 , wherein said linker comprises SEQ ID NO:3.
19. The conjugate of claim 16 , wherein said linker comprises SEQ ID NO:7.