Methods for coupling recombinant lysosomal enzyme targeting peptides to optimize the treatments of lysosomal deposition diseases

摘要

A method of preparing a recombinant lysosomal enzyme conjugated targeting peptide, method comprising the following: (a) i. modifying the amino (N) terminus and one or more lysine residues in a recombinant human lysosomal enzyme, using a first crosslinking agent to generate a recombinant human lysosomal enzyme modified by a first crosslinking agent; ii. modifying the first amino acid of a short linker at the amino (N) terminus in a peptide of variant IGF-2, using a second crosslinking agent to generate a peptide of variant IGF-2 modified by the second crosslinking agent and iii. conjugating the modified human recombinant lysosomal enzyme with the first cross-linking agent to the peptide of variant IGF-2 containing a short linker modified by the second cross-linking agent, or (b) conjugating a heterobifunctional cross-linking agent with a peptide of variant IGF-2 ; and conjugating the IGF-2 variant peptide modified by a heterobifunctional crosslinking agent with a recombinant human lysosomal enzyme, by reaction with the N-terminus and one or more lysine residues in the recombinant human lysosomal enzyme, or (c) conjugating a crosslinking agent. heterobifunctional with a recombinant human lysosomal enzyme by reaction with the N-terminus and one or more lysine residues in the recombinant human lysosomal enzyme; and conjugating the recombinant human lysosomal enzyme modified by the heterobifunctional crosslinking agent with a peptide of variant IGF-2; where the recombinant human lysosomal enzyme is selected from recombinant human α-glucosidase (rhGAA), recombinant human δ-galactosidase A (GLA), recombinant human β-glucuronidase (GUS), recombinant human δ-iduronidase A (IduA), isuronidate 2 Recombinant Human-Sulphatase (I2S), Recombinant Human ß-Hexosaminidase A (HexA), Recombinant Human ß-Hexosaminidase B (HexB), Recombinant Human δ-Mannosidase A, Recombinant Human ß-Glucocerebrosidase (GlcCerase), Recombinant Human Lipase Acid (Lipase) ) or any of its combinations and where the first crosslinking agent is selected from N-succinimidyl-6-hydrazinonicotinate acetone (S-Hynic), sulfo-succinimidyl-6-hydrazonicotinate acetone (sulfo-S-HyNic), C6-succinimidyl-6 -hydrazino-nicotinamide (C6-SHynic), succinimidyl-4-hydrazidoterephthalate hydrochloride (SHTH) succinimidyl-4-hydrazino nicotinate hydrochloride (SHNH) or N-hydroxysuccinimide-ester- (PEG) n-hydrazide, where n is represented by 3-24 u PEG units; and the second crosslinking agent is selected from PEG4-pentafluorobenzene-4-formylbenzoate (PEG4-PFB), succinimidyl-4-formylbenzoate (SFB), and C6-succinimidyl-4-formylbenzoate (C6-SFB) or the first crosslinking agent is selected from N-hydroxysuccinimide ester phosphine (NHS-phosphine), sulfo-Nhydroxysuccinimide ester-phosphine (sulfo-NHS-phosphine), N-hydroxysuccinimide ester-tetraoxapentadecane-acetylene (NHS35 PEG4-acetylene) or N-hydroxysuccinimide ester- (PEG) , where n represents 3-24 PEG units, cleavable heterobifunctional cross-linkers, such as NHS-PEG3-SS-acetylene or heterobifunctional cyclooctin-containing cross-linkers, such as difluorocyclooctin (DIFO) and dibenzocyclooctin (DIBO); and the second crosslinking agent is selected from N-hydroxysuccinimide ester-PEG4-azide (HS-PEG4-azide), N-hydroxysuccinimide ester azide (NHS-azide), N-hydroxysuccinimide ester- (PEG) n azide, where n represents 3-24 PEG units, or NHS-PEG3-SS-azide or the heterobifunctional crosslinking agent is selected from m-maleimidobenzyl-N-hydroxysuccinimide ester (MBS), sulfo-m-maleimidobenzyl-N-hydroxysuccinimide ester (sulfo-MBS) and sulfosuccinimidyl- 4- (Nmaleimidomethyl) cyclohexan-1-carboxylate (SMCC); and where the IGF-2 variant peptide comprises one or more of the following modifications with respect to the native human IGF-2 sequence: substitution of leucine for tyrosine at position 27; replacement of leucine by valine at position 43; substitution of arginine for lysine at position 65; and / or the IGF-2 variant peptide comprises an affinity tag and / or a linker extension region of at least 5 amino acids preceding IGF-2. substitution of arginine for glutamic acid at position 6; removal of amino acids 1-4 and 6; deletion of amino acids 1-4, 6 and 7; removal of amino acids 1-4 and 6 and substitution of lysine for threonine at position 7; removal of amino acids 1-4 and substitution of glycine for glutamic acid at position 6 and substitution of lysine for threonine at position 7;