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. 2021 Jul 14;9(7):817.
doi: 10.3390/biomedicines9070817.

The Dual Role of a Polyvalent IgM/IgA-Enriched Immunoglobulin Preparation in Activating and Inhibiting the Complement System

Carolin Schmidt  1 , Sabrina Weißmüller  1 , Fabian Bohländer  2 , Matthias Germer  3 , Martin König  1 , Alexander Staus  4 , Andrea Wartenberg-Demand  5 , Corina C Heinz  6 , Jörg Schüttrumpf  7
Affiliations

The Dual Role of a Polyvalent IgM/IgA-Enriched Immunoglobulin Preparation in Activating and Inhibiting the Complement System

Carolin Schmidt et al. Biomedicines. .

Abstract

Activation of the complement system is important for efficient clearance of a wide variety of pathogens via opsonophagocytosis, or by direct lysis via complement-dependent cytotoxicity (CDC). However, in severe infections dysregulation of the complement system contributes to hyperinflammation. The influence of the novel IgM/IgA-enriched immunoglobulin preparation trimodulin on the complement pathway was investigated in in vitro opsonophagocytosis, binding and CDC assays. Immunoglobulin levels before and after trimodulin treatment were placed in relation to complement assessments in humans. In vitro, trimodulin activates complement and induces opsonophagocytosis, but also interacts with opsonins C3b, C4b and anaphylatoxin C5a in a concentration-dependent manner. This was not observed for standard intravenous IgG preparation (IVIg). Accordingly, trimodulin, but not IVIg, inhibited the downstream CDC pathway and target cell lysis. If applied at a similar concentration range in healthy subjects, trimodulin treatment resulted in C3 and C4 consumption in a concentration-dependent manner, which was extended in patients with severe community-acquired pneumonia. Complement consumption is found to be dependent on underlying immunoglobulin levels, particularly IgM, pinpointing their regulative function in humans. IgM/IgA provide a balancing effect on the complement system. Trimodulin may enhance phagocytosis and opsonophagocytosis in patients with severe infections and prevent excessive pathogen lysis and release of harmful anaphylatoxins.

Keywords: C4; CDC; IgA; IgG; IgM; anaphylatoxins; complement factors C3; immunomodulation; opsonophagocytosis; polyvalent immunoglobulin.

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Conflict of interest statement

Authors are employees of Biotest AG, Dreieich, Germany. The authors declare that the research was conducted in the absence of any other commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Trimodulin induces concentration-dependent phagocytosis and opsonophagocytosis of S. aureus bioparticles. (A) Phagocytosis and (B) (opsono)phagocytosis of S. aureus bioparticles by differentiated HL-60 cells in the absence of complement (−, with hiNHSmin), or in the presence of active complement (+, with NHSmin), respectively. Additionally, assays included no (0, white bars, with FB only) or different trimodulin concentrations as indicated below the figures, representing final trimodulin concentrations in the assays. Bars represent mean + standard deviation (SD, n = 6 independent experiments). Reactions without active complement (−) display phagocytosis activity. Reactions with active complement (+) display phagocytosis plus opsonophagocytosis. Statistical analyses were performed using an unpaired Wilcoxon–Mann–Whitney test. Significance is shown with asterisk indicating a p-value of: * p < 0.05; ** p < 0.01; *** p < 0.0005; **** p < 0.0001; ns: non-significant. FB: formulation buffer; NHSmin: normal human serum depleted for IgM and IgG; hiNHSmin: heat-inactivated NHSmin; SD: standard deviation.
Figure 2
Figure 2
Trimodulin inhibits the detection of the opsonins C3b and C4b concentration-dependently. Opsonin binding assay (ELISA) showing different dilutions of three batches trimodulin (green) and a single intravenous IgG preparation (IVIg) batch (blue). Deposition of (A) C3b and (B) C4b was detected with an anti-human C3b or C4b antibody, respectively. Data points represent the mean from six independent experiments performed in duplicates. Error bars indicate the standard deviation (±SD). (C) Western blot analysis of the supernatant of the opsonin ELISA. Loading of the different controls and trimodulin samples on the gel is indicated. C3 fragments (C3b, iC3b, C3c) were detected with a goat anti-human C3c antibody (Dako Agilent, Santa Clara, USA) and a secondary CF488A-labeled donkey anti-goat antibody (blue; Biotium, San Francisco, CA, USA). IgM was detected with a mouse anti-human IgM antibody and a CF588-labeled donkey anti-mouse antibody (green). The plasma control contains EDTA-plasma only, while the trimodulin control contains trimodulin without EDTA-plasma. OD: optical density; ELISA: enzyme-linked immunosorbent assay.
Figure 3
Figure 3
Trimodulin reduces complement-dependent cytotoxicity (CDC) lysis activity in a concentration-dependent manner. Percentage of viable Ramos (RA1) cells after pre-incubation in normal human serum (NHS) with Rituximab (20 µg/mL) plus increasing concentrations of (A) trimodulin or (B) IVIg inducing CDC lysis via the classical pathway (CP). Cell viability of Ramos cells was analyzed by staining the membrane attack complex (MAC)-perforated cells with propidium iodide and measurement in a flow cytometer. As control, the FB plus Rituximab (0 mg/mL Ig) was used to determine the base level of CDC (white bars). Bars and error bars indicate mean + SD from six independent experiments performed in duplicates. Statistical analyses were performed using an unpaired Wilcoxon–Mann–Whitney test. Significance is shown ns: non-significant or with asterisks indicating a p-value: * p < 0.05; ** p < 0.01. FB: formulation buffer; CP: classical pathway; SD: standard deviation.
Figure 4
Figure 4
Trimodulin reduces C5a detection in a concentration-dependent manner. Anaphylatoxin binding assay showing different dilutions of trimodulin (green) and IVIg (blue) compared to the formulation buffer control (0, white bars). C5a was generated with zymosan via the AP and its binding to Igs was analyzed. Free C5a was detected by using a commercial C5a ELISA Kit. The amount of unbound C5a was set to 100% in the FB control. Data represent mean + SD from three independent experiments performed in duplicates. AP: alternative complement pathway; FB: formulation buffer; SD: standard deviation; ELISA: enzyme-linked immunosorbent assay.
Figure 5
Figure 5
Pharmacodynamics of complement factor C3 and C4 upon trimodulin treatment in healthy subjects and sCAP patients. (A–D) Results from the phase I clinical trial with trimodulin (Study 970) showing the change in C3 and C4 serum concentrations before (Day 0), during and after single (1x 182.6 mg/kg BW, (A,B), n = 4) or multiple (5x 182.6 mg/kg BW, (C,D), n = 6) doses of trimodulin (arrows) in healthy subjects. Multiple doses were given on five consecutive days 1 through 5. (E,F) Results from the phase II clinical trial with trimodulin (Study 982, CIGMA). Multiple (5x 182.6 mg/kg BW) doses of trimodulin (arrows) were given to three patients with sCAP. Complement was assed at the end of infusion. Change was calculated as percentage change (% change) from baseline per subject and the mean was calculated per treatment group. ((A,B): mean values, (C,D): mean values ± SD with n = 6; (E,F): single patient values). BW: body weight; SD: standard deviation; C3: complement factor 3.
Figure 6
Figure 6
Interactions of trimodulin and IVIg with different complement factors in the classical complement pathway. Due to IgM content trimodulin activates the classical complement pathway effectively (bold green arrows) compared to IVIg (thin green arrows). This leads to effective C3b/C4b opsonization of pathogens, opsonophagocytosis and to pathogen clearance. Furthermore, trimodulin inhibits the downstream CDC pathway by reducing the formation of the MAC complex (bold red blocking line) more effectively as compared to IVIg (thin red blocking line) and consequently the release of toxic mediators released by pathogens, or in case of autoimmune diseases by the host cells. The release of toxins results in inflammation, as well as the recruitment of neutrophils, which secrete more inflammatory cytokines. Both inflammation and C5a release induce coagulation processes.
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