Chemical crosslinkers
View Patent ↗Disclosed herein are methods of chemical conjugation comprising contacting a lysosomal enzyme with a first crosslinking agent to introduce aldehyde groups; contacting a lysosomal targeting peptide with a second crosslinking agent to introduce a hydrazide group at the N-terminal residue; contacting the lysosomal enzyme with aldehyde groups of step a. with the lysosomal targeting peptide with a hydrazide group at the N-terminal residue of step b; and forming a lysosomal enzyme-lysosomal targeting peptide conjugate.
1. A method of chemical conjugation comprising:
a. contacting a lysosomal enzyme with a first crosslinking agent to introduce aldehyde groups;
b. contacting a lysosomal targeting peptide with a second crosslinking agent to introduce a hydrazide group at the N-terminal residue;
c. contacting the lysosomal enzyme with aldehyde groups of a with the lysosomal targeting peptide with a hydrazide group at the N-terminal residue of b; and
d. forming a lysosomal enzyme-lysosomal targeting peptide conjugate.
2. The method of claim 1 wherein the lysosomal targeting peptide comprises variant insulin-like growth factor 2 (vIGF2) having the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
3. The method of claim 1 , wherein the first crosslinking agent comprises N-succinimidyl-4-formylbenzamide (S-4FB).
4. The method of claim 1 , wherein the second crosslinking agent comprises N-tert-butoxycarbonyl (tBoc)-protected hydrazide crosslinker (NHS-PEG4-tBoc-hydrazide).
5. The method of claim 1 , wherein the lysosomal enzyme comprises acid α-glucosidase (rhGAA), acid α-galactosidase A (GLA), acid β-glucuronidase (GUS), acid α-iduronidase A (IduA), acid iduronidate 2-sulfatase (I2S), β-hexosaminidase A (HexA), β-hexosaminidase B (HexB), acid α-mannosidase A, β-glucocerebrosidase (GlcCerase), acid lipase (LPA), or any combination thereof.
6. The method of claim 1 , wherein the lysosomal enzyme is human.
7. The method of claim 1 , wherein the lysosomal targeting peptide with a hydrazide group at the N-terminal residue is added in c at about four-fold molar excess.
8. The method of claim 1 , wherein aniline is added in c.