C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr₂s₂) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r₂s₂). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r₂s₂ proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r₂s₂ contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r₂s₂. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

Antibodies are important mediators of the human complement response, which offers critical protection against microbial infections and damaged host cells (1). In order to initiate a complement response, an antibody molecule first needs to bind antigens on the target cell via its antigen-binding (Fab) domains (2–5). Subsequently, the antibody’s constant (Fc) domain recruits the first complement protein complex, C1, to the cell surface (SI Appendix, Fig. S1A). The large C1 complex (also denoted as C1qr₂s₂, 766 kDa) consists of the recognition protein C1q (410 kDa) and a heterotetramer of serine proteases C1r and C1s (denoted C1r₂s₂, 356 kDa) (SI Appendix, Fig. S1B). While C1q is responsible for antibody recognition, its attached proteases C1r₂s₂ induce the activation of downstream enzymatic complexes (i.e., C3 convertases [C4b2b (6)]) that catalyze the covalent deposition of C3-derived molecules (e.g., C3b and its degradation product iC3b) onto the target cell surface (SI Appendix, Fig. S1A) (7, 8). C3b opsonizes the target cell surface and can induce formation of lytic pores (membrane attack complex [MAC]) in the target cell membrane (9–11). In contrast to human cells and gram-negative bacteria, gram-positive bacteria are not susceptible to the MAC due to their thick cell wall (12). On these bacteria, efficient decoration with C3b and iC3b is essential for triggering effective phagocytic uptake of target cells via complement receptors (CR) expressed on phagocytes of which the integrin CR3 (also denoted CD11b/CD18) is considered most important (13, 14).In recent years, our insights into IgG-dependent complement activation have increased significantly. A combination of structural, biophysical, and functional studies revealed that surface-bound IgG molecules (after Fab-mediated antigen binding) require organization into higher-ordered structures, namely hexamers, to induce complement activation most effectively (15–19). Hexamerized IgGs are being held together by noncovalent Fc–Fc interactions and form an optimal platform for C1q docking (SI Appendix, Fig. S1A). C1q has a “bunch of tulips–” like structure, consisting of six collagen arms that each end in a globular (gC1q) domain (SI Appendix, Fig. S1B) that binds the Fc region of an IgG. As the affinity of C1q for a single IgG is very weak (20, 21), avidity achieved through simultaneous binding of its globular domains to six oligomerized IgG molecules is paramount for a strong response (15, 17–19). Furthermore, it was found that IgG hexamerization could be manipulated by specific point mutations in the Fc–Fc contact region that enhance such oligomerization (15, 18, 22). While these hexamer-enhancing mutations in IgG potentiate the efficacy of MAC-dependent cytotoxicity on tumor cells and gram-negative bacteria (15, 23), their effect on complement-dependent phagocytosis is not known.Because complement is an important effector mechanism to kill bacteria and tumor cells, development of complement-enhancing antibodies represents an attractive strategy for immune therapies (1, 24). Immunotherapy based on human monoclonal antibodies is not yet available for bacterial infections (25–28). Such developments are mainly hampered by the fact that little is known about the basic mechanisms of complement activation on bacterial cells. For instance, we do not understand why certain antibodies induce complement activation on bacteria and others do not. In this study, we set out to investigate how antibacterial IgGs induce an effective complement response. By surprise, we noticed that C1q–IgG stability differs between human IgG subclasses. More detailed molecular investigations revealed that C1r₂s₂ proteases are important for generating stable C1q–IgG complexes on various target surfaces. Furthermore, we demonstrate that C1q–IgG stability is influenced by antibody oligomerization. These molecular insights into C1q binding to surface-bound IgGs may pave the way for optimal design of antibody therapies.