Engineering an indoleamine 2,3-dioxygenase immunotherapy via selective cysteine-to-serine mutations.

Indoleamine 2,3-dioxygenase is an immunomodulatory enzyme that shows great promise when delivered exogenously as a protein therapeutic. However, IDO activity is under complex redox control, mediated in part by multiple cysteine residues within its primary sequence. We have characterized three IDO mutants in which solvent-accessible cysteine residues were mutated to chemically-similar serine residues, "IDOC4S4" with C112S, C159S, C206S, and C308S mutations and "IDOC5S3" with C112S, C159S, and C308S mutations based on prior reports that C206 is necessary for catalytic function, and IDOC0S8, in which all cysteine residues were mutated to serines. IDOC0S8 was expressed in poor yield and demonstrated less than 1% activity when compared to wild-type IDO. In contrast, IDOC4S4 and IDOC5S3 demonstrated robust enzymatic activity, though IDOC5S3 had a slower V max than wild-type and IDOC4S4. Computational predictions and experimental measurements suggested a high degree of structural similarity between the wild-type IDO and IDOC4S4, with subtle perturbation of α-helical content for IDOC5S3. The structure of IDOC0S8 was predicted to be significantly different than that of wild-type IDO. IDOC4S4 and IDOC5S3 were more stable than wild-type IDO over time at physiological, ambient, and reduced temperatures, likely due to diminished oxidation of the mutant IDO forms. Based on the increased V max and robust thermal stability of IDOC4S4, we fused it to the anchoring moiety galectin 3, to evaluate its effectiveness in a mouse model of psoriasis. The IDOC4S4-galectin-3 fusion blunted the rate and severity of disease as compared to wild-type IDO-galectin-3 fusion. When compared to historical data with Cys-Ala IDO mutants, this study highlights the importance of employing amino acid substitution according to similarity in isosteric and isostructural shape to advance IDO as an immunomodulatory therapeutic.