Engineering Reactive Oxygen Species-Resistant Galectin-1 Dimers with Enhanced Lectin Activity

Galectin-1 is an immunomodulatory carbohydrate-binding protein with demonstrated efficacy in various preclinical models. However, its potential for clinical use is challenged by two features of the protein. First, galectin-1 (Gal-1) can be inactivated in oxidative environments, such as sites of inflammation, via covalent cross-linking of surface-exposed cysteine residues. Second, the active conformation of galectin-1 is a noncovalent homodimer with a micromolar dissociation constant. Together, these features necessitate frequent administration of high doses of galectin-1 for therapeutic efficacy. To address this challenge, we report an engineered dimeric variant of Gal-1 that is resistant to oxidative inactivation. Specifically, to prevent oxidative inactivation we mutated 3 of 4 surface cysteine residues to serine residues on Gal-1 ("Tri Gal-1"), and then cross-linked two Tri Gal-1 molecules with poly(ethylene glycol) diacrylate to create a stable homodimer ("Tri-PEG-Tri"). Our data demonstrate that cysteine-to-serine galectin-1 mutants retain the carbohydrate-binding properties and pro-apoptotic activity of wild-type Gal-1. Mutants lacking all surface cysteine residues are completely resistant to covalent cross-linking in oxidative environments. At sufficient polymer:protein ratios, poly(ethylene glycol) diacrylate reacts with the surface cysteine on two Tri Gal-1 molecules to form Tri-PEG-Tri. The effective dose of Tri-PEG-Tri is more than an order of magnitude lower than that of non-PEGylated Gal-1. Together, these data demonstrate reactive oxygen species (ROS)-resistant Tri-PEG-Tri dimers with enhanced lectin activity that may be broadly useful for improving the therapeutic efficacy of Gal-1 in immune modulation, transplant tolerance, and treatment of chronic inflammation.