Determinants of IgG-mediated complement activation

Antibodies (immunoglobulins; IgG) are some of the most important molecules of our immune defense against pathogens and tumor cells. These Y-shaped proteins recognize and tightly bind specific structures on the surface of viruses, bacteria and tumor cells via their two "arms", the so- called Fab regions, and thus mark them for destruction by the immune system. This is possible because the stem of the Y-shaped antibody, the so-called Fc region, then protrudes from the surface of the target cell, and other molecules of the immune system, in particular components of the classical complement pathway, can recognize it. If everything goes right, these molecules bind to the Fc region and to each other, ultimately causing the perforation of the cell membrane thus the elimination of the antibody-tagged target cell. There are four different subclasses of IgG antibodies (IgG1 - IgG4) in our blood that differ mainly in the length and flexibility of the so-called `hinge` region (the joint that connects the Fab and Fc regions in the center of the Y). In this project, we will investigate how exactly these IgG subclasses induce the elimination of target cells. Of particular interest is whether a recently discovered mechanism that allows IgG1 molecules to arrange themselves into antibody-hexamers (snowflake-like structures of six Y-shaped antibodies each) after binding to a target cell also applies to the other subclasses (IgG2, IgG3 and IgG4). Hexamerization represents a decisive step in the activation of the classical complement pathway for IgG1, and we will study whether this is the case for the other subclasses as well. In addition, we will elucidate further potential influences on the successful activation of the complement system, such as the binding strength between antibody and target cell and differences in antibody glycosylation (the type of attached sugar molecules). We will employ a combination of several high-end microscopy techniques (high-speed atomic force microscopy, single-molecule fluorescence microscopy) as well as techniques for quantifying the interactions between antibodies, cell membranes and complement proteins (single-molecule force spectroscopy and quartz-crystal microbalance), which together will allow us to decipher these processes both structurally and dynamically. The project is carried out by Dr. Johannes Preiner (project leader) in cooperation with Dr. Jaroslaw Jacak at the University of Applied Sciences Upper Austria, Department of Medical Technology, Campus Linz.

The classical complement pathway is an important branch of the human immune system that plays an increasingly important role in the elimination of infections, but also in targeted immunotherapy for cancer. This mechanism is initiated by the binding of IgG antibodies to certain surface structures (antigens) of infected cells, bacteria or cancer cells, and ends in their elimination from the organism. The capacity for activation differs greatly between the four IgG antibody subclasses found in humans, but a general mechanism that can explain these differences has not yet been identified. In the project, these differences were experimentally characterized at the molecular level using several biophysical techniques, resulting in a mechanistic model that can now be used to make mathematical predictions about the activation of the classical complement pathway. Using high-speed atomic force microscopy, the involved molecular processes could be directly "filmed", and the underlying interactions were precisely quantified by employing single-molecule force spectroscopy, quartz crystal microbalance and grating-coupled interferometry. Complementary to the biophysical methods, cell and lipid vesicle-based assays were also carried out together with cooperation partners (Genmab, Netherlands). It was shown that complement activation depends on the respective ability of the IgG antibodies to form sufficiently large oligomers - i.e. complexes of four, five, or six IgG antibodies - on antigenic surfaces, to which another component of the classical complement pathway (the complement protein C1) then may bind with at least four of its total of six possible binding sites, ultimately leading to its activation. In addition to the antibody subclasses, the influence of other variables such as antibody concentration, antigen density, exposure times, point mutations, or the presence of proteins that bacteria use to defend themselves against the complement system were also investigated in detail and combined into a coherent mechanistic model. In future, this can serve as a basis for the optimization of antibodies by means of protein engineering and the design of improved immunotherapies.