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

A method of making a targeting peptide conjugated to a recombinant lysosomal enzyme, the method comprising:

• conjugating a first crosslinking agent modified recombinant human lysosomal enzyme to one or more second crosslinking agent modified variant IGF-2 peptides, wherein the first crosslinking agent modified recombinant lysosomal enzyme comprises a recombinant lysosomal enzyme characterized as having a chemically modified N-terminus and one or more modified lysine residues;
• and
• the one or more second cross linking agent modified variant IGF-2 peptides comprise one or more variant IGF-2 peptides comprising a modified amino acid within a short extension linker at the amino (N)-terminus,
• wherein
• the lysosomal enzyme is selected from the group consisting of human acid α-glucosidase, human acid αgalactosidase A, human acid β-glucuronidase, human acid α-iduronidase A, human acid iduronidate 2-sulfatase, human β-hexosaminidase A, human β-hexosaminidase B, human acid α-mannosidase A, human β-glucocerebrosidase, human acid lipase, and any combinations thereof, and
• the one or more variant IGF-2 peptides comprising SEQ ID NO:
• 2 with one or more of the following modifications with respect to the amino acid sequence of SEQ ID NO:2:
• 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 variant IGF-2 peptide comprises an affinity tag and/or a linker extension region of at least 5 amino acids preceding IGF-2.