Evaluation of FcR-directed immunotherapeutic strategies to manipulate macrophages in in vitro and ex vivo models of chronic inflammation

Macrophages are a fundamental part of the immune system and are responsible for diverse physiological processes including inflammation, tissue repair and homeostasis. Displaying extraordinary diversity and plasticity, macrophages are clustered in an intricate model, with two extremes – pro-inflammatory (M1) versus anti-inflammatory (M2) phenotypes. Mirroring the Th1/Th2 dichotomy, M1/M2 participate in initiation of the inflammation process and guide it until final resolution. While the M1 phenotype dominates in the early inflammation phase by initiating and orchestrating the immune response, a transition towards predominance of the M2 phenotype occurs at the final stage, which is important for resolution, tissue repair, and final healing. However, in a variety of immune-mediated inflammatory diseases, like rheumatoid arthritis, chronic cutaneous inflammation, this M1/M2 balance is disturbed and a persistency of M1 arrests the pro-inflammatory phase, contributing directly to the chronicity. To address the lack of available strategies for a targeted treatment of the mentioned diseases, in this thesis an emphasis will be put on the development of novel immunotherapeutical approaches. Recently, our group showed in a series of experiments that CD64-directed depletion of M1 using recombinant ETA'-based immunotoxin (IT) was able to reverse this increased M1/M2 ratio. This approach could be utilized in order to influence pathogenic changes of the M1/M2 ratio in a therapeutic setting.In this thesis, first “proof-of-concept” in the human system is demonstrated by testing the IT-treatment ex vivo using biopsies from patients with chronic skin inflammation (atopic dermatitis or chronic diabetic wounds). Next to normalizing the M1/M2 ratio as reported in the previous in vivo animal study, also the cytokine levels (e.g. IL-6 and IL-10) could successfully be normalized towards the anti-inflammatory profile. Furthermore, the collected data in this work provides additional evidence for the large macrophage heterogeneity as well as plasticity demonstrated by the ability of M1 to be functionally repolarized into the M2 direction and vice versa. This key observation points towards a potential novel therapeutic approach with regard to macrophages-targeted therapy: the modulation, rather than the elimination of M1 macrophages. Unlike the CD64-targeting IT therapy, the immunomodulation aims to intervene in the inflammation process and restores the cytokine balance without affecting the macrophage viability. Therefore, two immunomodulatory fusion proteins were designed to target CD64, the distinctive marker for M1, and deliver two different human effector proteins - thioredoxin 1 (Trx-1) and suppressor of cytokine signaling 2 (SOCS-2). The constructs were genetically engineered as C-terminal fusion partners to H22(scFv), expressed in HEK293T c ells and purified using affinity chromatography. The identity and binding specificity of the macrophage immunomodulatory proteins (MIMPs) were successfully confirmed using SDS-PAGE followed by Coomassie staining, western blotting, and flow cytometry, respectively. Primary macrophages were derived from human PBMCs and used to investigate the immunomodulatory activity of both MIMPs. Two different experimental approaches termed “priming” and “treatment” were used in vitro –– which represent both application paths – systemic and local, respectively. While H22(scFv)-Trx-1 failed to induce substantial repolarization, H22(scFv)-SOCS-2 successfully attenuated the expression of pro-inflammatory cytokines in M1 (e.g. IL-6, IL-12, TNF-α and IL-1β). In addition, it also induced secretion of anti-inflammatory IL-10. However, H22(scFv)-Trx-1 was able to attenuate the oxidative stress in M1. Furthermore, priming of non-polarized macrophages with both MIMPs proved successful in prevention of the later polarization into the M1-like phenotype upon stimulation with wound exudate. Herein the superior effect of H22(scFv)-SOCS-2 was again demonstrated by the substantial induction of IL-10 production. In the future, the clinical relevance of these findings should be confirmed in ex vivo experiments using human biopsies or in an appropriate preclinical animal model. In a parallel line of exploration, the suitability of the Fc-alpha receptor (FcαR, CD89) for immunotherapy-targeting macrophages was investigated in vitro. To that aim, the receptor density of CD89 and CD64 was determined by flow cytometry. Although CD89 was expressed at lower levels than CD64 on both M1 and M2, it was preferentially expressed by M1. To develop CD89-targeted immunotherapy, an IT comprising a CD89-binding scFv and ETA' was recombinantly expressed and subsequently affinity purified. Successful binding, internalization and receptor-dependent cytotoxicity of this IT was confirmed on CD89+ pro-monocytic cell lines (HL-60, THP-1, MonoMac-1 and U937), whereby IC50 values tended to be dependent on the nature of the stimulus (e.g. IFN-γ or TNF-α). Nevertheless, the CD89(scFv)-ETA' failed to induce apoptosis in primary human PBMC-derived macrophages regardless of their phenotype, but these findings revealed possible utilization of the CD89-directed IT in the context of myeloid leukemia, neutrophilia and similar diseases.In conclusion, results of this thesis provide new insights into the immunomodulation of macrophages using a well validated CD64-targeted delivery system as well as the function of SOCS-2 and Trx-1 in the context of polarized macrophages during chronic inflammation. The CD64 but not the CD89 path in combination with a suitable effector payload comprises a promising therapeutic strategy for inflammatory diseases, strengthening the immunotherapeutic armamentarium