Blog #1: Every sdAb of the (path-)way: targeting factors and complexes across the complement system.

The complement system is a crucial part of our innate immune defence, activated by the binding and cleavage of a series of specialized proteins. When the activation or regulation of the complement system is out of balance, it can lead to various diseases. Furthermore, the exact working of the complement system is not fully understood yet. To better understand, measure, and treat the complement system and/or defects thereof, effective detection and modulatory agents are necessary. These tools can be used for both research, diagnostics, and therapeutics.

Single domain antibodies (sdAbs) are promising candidates for this role. They are small, stable, easy to develop, manufacture, and can be conjugated to other molecules. Moreover, they are compatible with hidden (cryptic) and structurally specific (conformational) epitopes. These features make sdAbs ideal for targeting the highly homologous components and complexes within the complement system.

At QVQ we are specialized in the development of sdAbs. In collaboration with the Medical Microbiology research group of Professor Suzan Rooijakkers at the University Medical Center Utrecht we aimed to develop sdAbs against multiple components of the complement system. The project was initiated by PhD candidate Eva Struijf and continued within QVQ with the aim to further develop the sdAb as portfolio products or acquisition by interested clients.

To obtain the complement specific sdAb llamas were immunized with complement proteins and complexes or bacteria covered (opsonized) with complement proteins. After immunization, sdAb-phage-libraries were created from each of the llama’s immune response. Next, we looked for the specific complement sdAb in these libraries using a process called biopanning (better known as selections). We created a broad range of sdAbs against several individual complement targets and complexes, which exhibit various functional activities. Below some of the key findings are highlighted. More information on the specific sdAb can be found in the thesis of dr. Eva Struijf1 and are summarized on a recent poster which was presented by our scientist dr. Gillian Dekkers at the Antibody Engineering and Therapeutics conference in London in June 2024.

Key findings

Firstly, by screening for both binding to C5 and for functional blocking of the complement pathways in complement-mediated red blood cell lysis assays, we found several anti-C5 sdAb. Two lead candidates were then further characterized and developed as QVQ portfolio products Q101 and Q102. In collaboration with the group of Professor Piet Gros, we showed that these blocking sdAbs bind with high affinity to two distinct epitopes on the C5 molecule.2 Interestingly, both Q101 and Q102 also bound to and blocked a natural occurring C5 variant R885H. This C5 variant is present in a subgroup of patients and is not targeted by the clinical C5 blocker Eculizumab®. These sdAb have to potential to improve therapeutic applications for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and other diseases, as it improves knowledge about C5 and C5 blockers such as Eculizumab®.

Secondly, selections and screening on the C5b6 complex revealed multiple sdAb candidates with C5b6, but not C5 or C6 specificity. The top candidates bind selectively to C5b6 and do not block the terminal pathway of complement. These characteristics make these molecules interesting reagents for diagnostic studies, which can tell us something about the degree of complement reaction that has taken place within patients.

Lastly, classical selections of two bio-panning rounds on complement components C1, C2, C4, C6, C7, and C9 revealed several binders per target with distinct sdAb sequences. Currently, these molecules are further developed and characterized.

Overall, we developed sdAbs targeting proteins and complexes across the complement system pathways. These agents are suitable for functional studies and have potential for research, diagnostic and therapeutic application. Furthermore, the presence of the immune libraries and panning outputs allows the development of many more sdAbs through additional panning campaigns followed by screening and Sanger or next generation sequencing.

References

1: Struijf, E. M. (2023). Nanobodies targeting complement Detecting and blocking complement activation. Utrecht University.

2: Struijf, E. M., et al (2023). Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies. Journal of Biological Chemistry, 299(8). https://doi.org/10.1016/j.jbc.2023.104956