title: Functional characterization of a new IgM- and IgA-enriched immunoglobulin preparation
Creators: name: Schmidt, Carolin
Publisher: name: UNSPECIFIED
Year: 2020

description: The development and the functional characterization of human plasma-derived immunoglobulin (Ig) preparations are of high clinical relevance for the therapy of patients who suffer from antibody depletion, autoimmune or inflammatory disorders. Currently, mainly intravenous immunoglobulin G (IgG) preparations (IVIgs) are in use to treat a broad range of pathological conditions, like primary and secondary immunodeficiencies (PID; SID), but also for immunomodulation. Nevertheless, there is a need to improve the exploitation of the plasma as well as the efficacy of polyclonal Ig preparations without an increase in plasma demand. Especially fractions containing IgA and IgM molecules may be advantageous, because of their versatile pharmacological effects that could be exploited therapeutically. Trimodulin (name of the active substance, for differentiation from a standard IVIg preparation) is a new plasma-derived Ig preparation for intravenous application and is unique, as it contains besides IgG also defined amounts of IgM as well as IgA molecules compared to currently available standard IVIg preparations. It was developed for the treatment of patients with severe infections and was already tested in a phase II clinical trial (CIGMA study), where a promising potential in the therapy of patients with severe community-acquired pneumonia (sCAP) could be demonstrated. Based on the novelty and complexity of this Ig preparation that contains besides IgG also IgM as well as IgA molecules, there is still a need for a detailed understanding of the complex mode of action (MoA) of trimodulin regarding its antimicrobial as well as its immunomodulatory mechanisms. Particularly the complement system is an important tool of the humoral immune system, because its activation leads to the killing of invading pathogens via various effector functions. Nevertheless, it can also be harmful to the host when the immune system is over-activated and can lead to tissue damage as well as multiple organ failure. Therefore, it is important that the complement system is balanced between activation and inhibition. It was hypothesized that trimodulin exhibits an ambivalent effect regarding complement activation to ensure the neutralization of invading microorganisms but also to protect the host against inflammation caused by a hyper-activated immune system. In order to evaluate the ability to inhibit complement activation, the binding of trimodulin to activated complement factors (C3b, C4b, C5a and C3a) was analyzed in ELISA settings. It was demonstrated that trimodulin is able to interact with C3b as well as C4b molecules by binding and an overlay of these complement factors. The addition of an IVIg control (Intratect) showed only slight effects and the decrease in the detection of C3b as well as C4b was obviously stronger with the IgM- and IgA-enriched preparation. Moreover, a trend in reducing the detection of C5a with trimodulin was revealed, whereas no interaction of trimodulin with activated C3a molecules could be demonstrated. Furthermore, a complement-dependent cytotoxicity (CDC) assay was used to analyze if trimodulin can even functionally prevent complement activation. Therefore, the CDC of target cells (Ramos cell line) was measured after addition of trimodulin and an obvious reduction in the CDC of Ramos cells was detected. This demonstrated for the first time that trimodulin is able to inhibit activated complement factors, which could be used to prevent inflammation in patients with an over-stimulated immune response. Furthermore, the results clearly showed the benefit of using trimodulin to prevent the over-stimulation of the complement system, over a standard IVIg preparation that was not able to reduce the CDC in this assay. In order to analyze if trimodulin, which contains besides IgG and IgA also ⋲ 23% IgM, is able to activate the complement system and to induce the opsonophagocytosis of pathogens, two opsonophagocytosis assays (OPAs) were developed. In general, IgM molecules are able to activate the complement system via the classical pathway more effectively than IgG molecules. The OPA using living E. coli bacteria unfortunately did not depict the opsonophagocytic capacity of trimodulin, but showed only the IgG-mediated phagocytosis of the bacteria. This was assumed to be due to a high non-specific killing of the used complement source, which destroyed high amounts of E. coli bacteria even in the absence of trimodulin. For this reason, a second OPA was established by using S. aureus bioparticles and the opsonophagocytic capacity of trimodulin was evaluated in a fluorescence-activated cell sorter (FACS). The analysis showed that trimodulin was able to activate the complement system concentration-dependently and thereby to trigger the opsonophagocytosis of S. aureus bioparticles by effector cells (HL-60 cell line) in low concentrations. Additionally, it was detected that even in the OPA setting trimodulin was able to inhibit the complement system in high doses. The results of this PhD thesis obviously support the theory that trimodulin exhibits an ambivalent effect regarding complement activity and are in accordance with various studies that also show dual mechanisms of Ig preparations in activating and inhibiting the complement system. Moreover, it is assumed that the treatment with trimodulin could help to reduce inflammation caused by an over-stimulation of the complement system and simultaneously ensuring the neutralization of harmful pathogens. Furthermore, the PhD thesis gives new insights in the MoA by confirmation of the complement activating as well as inhibitory capacity of trimodulin and emphasizes the crucial advantages of using an IgM- and IgA-enriched preparation compared to a standard IVIg preparation.