Methods for coupling targeting peptides onto recombinant lysosomal enzymes for improved treatments of lysosomal storage diseases

Patent metadata

Field	Value
Publication number	US-10660972-B2
Application number	US-201615347006-A
Country	US
Kind code	B2
Filing date	Nov 9, 2016
Priority date	May 27, 2011
Publication date	May 26, 2020
Grant date	May 26, 2020

Abstract

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.

First claim

What is claimed:

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.

Assignees

• Amicus Therapeutics Inc

Inventors

• Do Hung

Classifications

• C07K1/18

  Ion-exchange chromatography · CPC title

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• A61K38/47

  acting on glycosyl compounds (3.2), e.g. cellulases, lactases · CPC title

• C07K1/306

  by crystallization · CPC title

• C07K14/575

  Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695) · CPC title