Anti-C4B monoclonal antibody, clone 21-18

Binding of Clq to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher Clq binding affinity compared with human Ig Gl, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of Ig Gl- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti EGFR-IgG1 did not promote CML of investigated target cells, anti EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti EGFR-IgG3 promoted strong Clq and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti EGFR-IgG3 triggered CML and to force a switch from classical complement pathway activation to Clq-dependent alternative pathway amplification. Together, these data suggest human anti EGFR-IgG3, although highly reactive with Clq, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway.

Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections.