Poster CC16 - Localized versus Systemically Delivered Immunomodulation for Treatment of Psoriasis

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 disease state. Our group has taken two different approaches for addressing clearance; (1) the design of a localized IDO therapeutic through fusion with Galectin3 (Gal3) as an anchoring moiety, and (2) conjugation of IDO with poly(ethylene glycol) (PEG) to increase its circulating persistence for systemic delivery. 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. The research presented here compares the localized versus systemically delivered approach for immunomodulation, using the mouse model of psoriasis.

Materials and

Methods: The design of the IDO-Gal3 fusion protein has been previously described, and was recombinantly expressed in E. coli and validated for purity through SDS-PAGE. To conjugate the PEG polymer to IDO, 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. PEG-IDO’s therapeutic efficacy 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 at the clinical onset of disease, day 3, with a subsequent dose delivered on day 8 as needed. The immune cell population was analyzed at the midpoint of disease, day 7, and cells were isolated from both the dermis and the epidermis layers before stained for both myeloid and lymphoid markers and analyzed via flow cytometry.

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 2 hours post injection. IMQ-induced psoriasis mice treated with IDO-Gal3 showed significantly lower cumulative clinical scores from day 6 onwards, as well as significantly lower area under the curve as compared to saline control mice. Mice treated with 10kDa PEG-IDO required two doses, one at day 3 and a follow up at day 8, to maintain a comparable decrease in scores as IDO-Gal3 treatment. 30kDa PEG-IDO, which persisted longer in circulation, only required one treatment on day 3 to reduce clinical scores for the entirety of the experiment, suggesting that circulation time directly impacts the length of therapeutic efficacy of PEG-IDO. For IDO-Gal3 treated mice, there was a reduction in neutrophils in both the dermis and epidermis at day 7; as neutrophils are pathogenic in psoriasis, this reduction in the neutrophil population in our IDO-Gal3 treated mice may be due to the decrease in disease severity seen by the clinical scores. Additionally, there were fewer activated TCRbeta+ and CD4+ T cells of the dermis in the IDO-Gal3 treated mice, indicating that IDO-Gal3 treatment reduces inflammatory markers in the lymphoid population, which also may correlate to the reduction in clinical scores. These data demonstrate that IDO can be engineered for either localized or systemic delivery, and maintains its immunomodulatory effect in both formulations, as seen by the positive effect on clinical scores in IMQ-induced psoriasis mice. Future work will be done to determine the immune cell population of PEG-IDO treated mice, as well as determine the mechanism of action behind IDO’s immunomodulatory effect.

Acknowledgements (Optional):