The complement system, which comprises around 40 soluble blood proteins, is an important part of the body’s innate immune system. It initiates a cascading immune response when activated by various triggers, which can be recognized by the classical pathway (typically triggered by antibody complexes), the lectin pathway (triggered by mannose) or the alternative pathway (triggered by LPS). Various reactions occur at different levels in the cascade, ranging from opsonization (phagocyte action) to inflammation and finally to cell lysis (membrane attack complex). A wide range of danger signals can activate the complement system – not only bacteria and viruses, but also foreign surfaces and misfolded proteins. So assessing its activity is an important part of drug toxicity studies. However, there’s also a growing interest in inhibiting the complement system as a mode of action in drug development.
Complement inhibition is especially promising for conditions where the innate immune system does damage to the body. Soliris and Ultomiris, the first complement-inhibiting drugs to reach the market, treat rare complement-driven diseases, including paroxysmal nocturnal hemoglobinuria (PNH), a condition where the complement system attacks the red blood cells. Today there are many complement inhibitors in development, targeting everything from rheumatic arthritis and sepsis to diseases of the eye.Most of these novel drugs block complement activity at the level of the C5 protein, just like Soliris and Ultomiris do. The C5 target is now “proven in combat”, and there are at least ten C5 inhibitors already in or soon moving into clinical trials. However, the complement cascade can potentially be blocked at any level. In nephrology, for example, there are at least nine different complement targets being investigated. Each complement target, of course, comes with its own advantages and concerns. As an example, the classical, lectin and alternative pathways all converge at the C3 protein level. Inhibiting at or above the C3 cascade would therefore prevent phagocyte activity in addition to inflammation and membrane attack complex formation. Yet phagocyte activity is also one of the body’s vital cleaning mechanisms, as it removes cellular debris and antibody complexes. So blocking at such a high level could increase the risk of bacterial infections and certain autoimmune diseases, as seen in patients with upstream deficiencies in the complement system. Blocking at the C5 protein level, in turn, is associated with an increased risk of meningitis.
Given the complexities of complement activity, what factors should be considered when planning a mode of action study for a new complement inhibitor? The most obvious is the level at which the drug blocks the complement cascade, which will influence the types of readouts that are relevant. Does it target the opsonization of cells and tissues? Does it target chemoattraction mechanisms and the bridges between innate and adaptive immunity? Does it target the final stages of the cascade and membrane attack complex? In addition, it’s important to know if both complement inhibition and complement activation should be evaluated. If the molecule is an antibody, for example, it has the potential to activate the classical pathway through its Fc region. This is a common mode of action for many therapeutic antibodies, especially in oncology, but it constitutes a risk factor for other antibodies. (For more on this, be sure to (read our earlier blog post on CDC). Knowing that the Fc region is fully deactivated avoids the risk of simultaneously braking and accelerating complement activity.
Yet perhaps the biggest question is whether to evaluate complement inhibition in serum or whole blood. Both options reflect the entire complement cascade since all the cascade proteins are present in the fluid phase. However, serum lacks key regulatory elements with high relevance to complement activity. One element is the cellular phase, which has important complement-influencing functions. By leaving out the blood cells, serum ignores the regulation of complement mechanisms by various components in the cell membranes. Likewise, serum misses some of the naturally occurring complement variations between donors. Complement responses differ significantly between individuals, in part due to differences in their complement protein levels. Such variations can have a significant impact on the outcome of clinical studies.
In short, testing complement activity in whole blood can be as valuable in mode of action studies as it is in toxicity studies. A whole blood model can capture the full interaction of a complement-inhibiting drug with the liquid and cellular phases, including the significant crosstalk between the complement and coagulation cascades. Moreover, flow cytometry can be used with whole blood to quantify and categorize cell losses due to phagocyte action or membrane attack complex.
Simply put, the more accurately a preclinical model mimics the human body, the better it can predict the body’s immune response. Curious to know more about complement inhibition and how it can be evaluated effectively? We’d be happy to share experience from our own testing platform, which uses fresh, circulating whole blood. To discuss its possibilities or to request data samples, simply get in touch with us here.