Complement Regulation

The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last two decades had led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. Therapeutic complement inhibition therefore means deliberately blocking one or more steps of that cascade to reduce harmful inflammation, opsonization, or cell lysis, while balancing preservation of host defense and clearance functions. A central question is to choose between two opposing approaches: “proximal vs terminal” blockade. In a proximal strategy (e.g., C3 or pathway initiators) you reduce upstream amplification and all downstream effectors, but impairs opsonization and some host-defense functions. In a terminal strategy (e.g. C5/C5a or MAC) initial upstream sensing/opsonization is kept intact, but it primarily prevents MAC formation and/or (C5a) anaphylatoxin signaling and therefore is sometimes more selective for tissue-damage control. It is likely that a variety of inhibitors will become available in the near term.

Which targets to choose is depending on disease biology and severity of expected side-effects.

Pathway initiators. This are clear examples of proximal targets of inhibition and mostly associated with the CP&LP (eg, C1s, C2 and MASP-2). A clear choice when the onset of disease is restricted to one specific pathway, leaving the function of the other pathways intact.

1. Terminal pathway (TP). This is the choice if you want to block the complement systems as a whole or to prevent the specific damage of unwanted MAC incorporation. A strong way to do this is via the C5, C5a, C5aR axis. C5 activation is the gatekeeper for the TP and MAC generation. In disease where MAC-mediated damage or C5a signaling is key ((e.g., PNH, aHUS), blocking this axis is very effective and through the use of eculizumab demonstrated proven to be safe and effective. Blockage upstream C6-C9 could be an alternative if you only want to prevent MAC formation (eg some cases in neurology and prevention of transplantation damage).
2. Convertases and related. When the amplification of the cascade is the main target of complement inhibition, mainly via the malfunction of the regulation of the AP, the central C3 convertase or the individual AP regulators (eg fB,FH, properdin) could be the target of interest. This kind of inhibition can be very powerful with the disadvantage of introducing some side effects, like the risk of infection.
3. Pathway initiators. This are clear examples of proximal targets of inhibition and mostly associated with the CP&LP (eg, C1s, C2 and MASP-2). A clear choice when the onset of disease is restricted to one specific pathway, leaving the function of the other pathways intact.

The last and upcoming decade a plethora of inhibitors are discovered, currently in trials and making the way into the clinic in the coming period. A diverse range of molecules have shown to be effective.

1. Antibody (fragments). An antibody is the most well-known category and they have the advantage of a long half-life, high specificity and safety profile. Nowadays all kind of (Fc) modifications even improve its function. The use of eculizumab/ravulizumab (αC5) in the field of complement paved the way for complement inhibition as a whole and many more antibodies are to come (eg , pozelimab (C5); sutimlimab (C1s), narsoblimab (MASP2), crovalimab (C5)).
2. Small molecules. While biologics have traditionally been used for complement inhibition, small molecules are emerging as attractive alternatives due to their oral bioavailability, rapid pharmacokinetics, and potential for precise, pathway-selective modulation (peptides (zilucoplan (C5), AMY101 (C3), small molecules (compstatin (C3), danicopan (fD)), aptamers (zimura (C5), avacopan (C5aR1), etc)
3. Recombinant/engineered protein based regulators. By leveraging natural regulatory mechanisms with enhanced specificity and optimized pharmacological properties, these agents enable precise control of complement activation while preserving essential immune functions (eg mini-FH, berinert/Cinryze (c1s/r;MASP)).
4. Combinations of the above mentioned classes.

With the rapid expansion of complement-targeted therapeutics, there is a growing need for robust diagnostics and biomarkers to support patient selection, therapeutic monitoring, and clinical decision-making. Beyond use in clinical trials, these tools are essential for accurate disease diagnosis and longitudinal follow-up in routine care. The heterogeneity of complement-mediated diseases, is driven by patient genetics, the presence and functional relevance of individual biomarkers, and the involvement of protein complexes. This necessitates a diverse portfolio of assays and technologies to adequately address clinical needs.

1. Biomarker quantification. Besides the specific target of the applied complement inhibitor, a few basic complement biomarkers are generally quantified: C3&C4 in plasma, which gives an indication for complement activation/consumption. Clinically quantified with nephelometry and when a more accurate method is required ELISA. This is also the platform of choice for activation products like C3a, C3d, C4d, C5a and TP soluble TCC (sC5b-9). These markers could monitor effectiveness of treatment, therapy breakthrough, inflammatory potential and overall complement mediated immune activation.
2. Functional assay (hemolytic, convertase, pathway activity). Functional assays are essential to early decipher assay specificity or to demonstrate pharmacodynamic effects. Classical CH50 and AP50 serum-based hemolytic assays are used to assess functional activity of the CP or AP. These hard to standardize assays are in many places replaced with so-called pathway-ELISAs. They measure CP/LP/AP functional activation using specifically coated surfaces plus in vitro TCC complex formation and its detection. This offers higher throughput and standardization.
3. Cell based assays. These not commonly used assays often rely on (red) blood cell flowcytometry. It can detect opsonization, complement receptors and intracellular processes. Is for example critical in PNH to detect opsonization/extravascular hemolysis risk under anti-C5 therapy, thereby demonstrating whether proximal C3 deposition continues.
4. Drug related & pharmacokinetics (auto, idiotypic, MS, IA). To measure the actual drug concentration for antibodies, peptides and small molecules. It can be applied for dosing and therapy monitoring.
5. Disease markers (non-complement). Complement inhibition and therapy also ask for disease specific context. Such assays should be selected to match target biology and clinical questions. For PNH this could be e.g. hemoglobin and reticulocyte count and in kidney diseases creatin, electrolytes and proteinuria/albuminuria are of interest.

As the field of complement inhibition is evolving, new targets and therapeutics will arise and thereby the need for new or more sensitive or simultaneous (quantitative) detection of related (complement) biomarkers. Furthermore, it is becoming more apparent we do not only have to look into the fluid phase (serum/plasma, CSF, urine), but also more locally at the site of activation/inhibition and even intracellularly. Other challenges are training of new complement specialists and (clinical) laboratory specialists to deal with the specific characteristics and interpretation of complement results and proper sample handling. With the rise of complement (inhibition) in the clinic also the need for further standardization and specific guidelines for assay design and interpretation are of importance as well.