PEGylation of Indoleamine 2,3-Dioxygenase for Systemic Immune Regulation

Introduction:: Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes the rate limiting step of tryptophan catabolism, and has been shown to play a critical role in the promotion of immune tolerance and restoration of immune homeostasis in many inflammatory and autoimmune diseases. Additionally, previous work has demonstrated IDO delivered exogenously demonstrates immunosuppressive programming of dendritic cells to maintain an immature state. These properties make IDO an attractive protein therapy candidate. However, it faces the challenge of quick clearance from the site of administration, and a shortened timeline for addressing systemic disease state. Many therapeutic molecules have addressed these challenges through the conjugation of poly(ethylene glycol) (PEG) chains to the molecule of interest, thus increasing their hydrodynamic radius and circulation time within the blood. The research presented here demonstrates coupling PEG with IDO and creating a modified protein-polymer complex that can persist in circulation while maintaining its active enzymatic function, resulting in a therapeutic suitable for systemic inflammation and immunosuppression. Our PEGylation process was optimized through testing of various reaction conditions and polymer chain length, with the final PEG-IDO product reliably still active despite the addition of the PEG and had improved circulation as compared to wild type (WT) IDO alone. Using PEG-IDO in the mouse model for multiple sclerosis and for psoriasis showed strong indication of its efficacy as a systemic immunotherapeutic.


Materials and Methods:: To conjugate the PEG polymer to the protein, PEG chains with a single maleimide reactive group were combined with IDO, in molar excess of PEG:IDO. Different molar excesses of PEG and polymer chain lengths were assessed, and successful conjugation was confirmed through SDS-PAGE. To determine IDO activity, PEG-IDO was incubated with tryptophan and the electron donors, methylene blue and ascorbic acid, and the presence of the tryptophan metabolite kynurenine was measured via kinetic absorbance at 321 nm. Circulation of PEG-IDO was determined via daily tail vein bleeds after a bolus injection of protein. IDO quantity in blood samples was analyzed via Western blot and quantified after comparison against a standard curve of IDO in serum. To assess therapeutic efficacy, two animal models were used. A mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), was used to determine systemic effects of circulating PEG-IDO. Disease state was assessed via scoring of clinical observations regarding tail and leg paralysis, with higher scores indicating greater degree of paralysis. Mice were treated at the clinical onset of disease. The effect of PEG-IDO on regional immune activation was assessed with a mouse model for psoriasis, imiquimod (IMQ)-induced psoriasis. IMQ cream was applied daily for 14 days, and progress of disease was measured via a modified version of the psoriasis area and severity index (PASI), to quantify the severity of erythema, scaling, and skin thickening. Mice were treated with two doses of PEG-IDO, at day 3 and day 8.


Results, Conclusions, and Discussions:: We were able to optimize PEG-IDO formulation through varying the reaction conditions. Increasing the molar ratio of PEG-IDO increased the number of PEG chains conjugated, as well as increasing the reaction efficiency. PEGylated IDO maintained its enzymatic activity after conjugation with the polymer, across all molar ratios. PEGylation had an increased effect on circulation half-life, where increasing the polymer chain length resulted in an increased circulation time. 10k PEG-IDO had a circulation half-life of 21 hours and 30k PEG-IDO persisted in circulation through all 7 days of sampling, while WT IDO was barely detectable even 2 hours post injection. EAE scores in PEG-IDO treated mice dropped compared to the saline control, and were significantly different by 22 days post induction, indicating that PEG-IDO not only reduced clinical scores, but persisted in such a manner that maintained this reduction. Additionally, cumulative scores were significantly lower in PEG-IDO treated mice. Flow cytometry analysis of the brain and spinal cord showed increased Foxp3+ T cells in PEG-IDO treated mice, indicating an increase in regulatory T cells and a return to an immune system homeostatic condition. IMQ-induced psoriasis mice treated with two doses of PEG-IDO showed significantly lower cumulative clinical scores from day 5 onwards until day 13, as well as had significantly lower area under the curve as compared to saline control mice.

These data demonstrate that IDO can be PEGylated while still maintaining enzymatic activity. PEG-IDO persists longer in vivo as compared to wild type IDO, and has a positive effect on clinical scores in EAE and IMQ-induced psoriasis mice. Future work will be done on how best to modulate PEG-IDO circulation time, as well as determine the mechanism of action behind its efficacy as an immunomodulatory treatment.


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