Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor
Henrik Pedersen1, Rasmus Kjeldsen Jensen1, Annette Gudmann Hansen2
1Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
Insights
Researchers developed novel nanobodies targeting complement component C3b, a key driver of complement system pathology. The fusion protein EWEnH shows promise for treating complement-driven diseases in vivo.
Area of Science:
- Immunology
- Biochemistry
Background:
- The complement system is crucial for innate immunity but can cause disease when dysregulated.
- Complement component C3b is central to complement activation and a therapeutic target.
- Previous work identified nanobody hC3Nb1 binding to C3 and its products.
Purpose of the Study:
- To develop novel nanobodies targeting complement C3b for therapeutic applications.
- To engineer fusion proteins with enhanced specificity and functionality for complement inhibition.
Main Methods:
- Modification of nanobody hC3Nb1 with a Glu-Trp-Glu motif to create EWE nanobody.
- Fusion of EWE nanobody with complement Factor H (FH) CCP domains to generate EWEnH and EWEµH.
- Assays to evaluate binding affinity, Factor I (FI)-mediated cleavage support, and inhibition of complement deposition.
Main Results:
- EWE nanobody demonstrated specificity for C3 degradation products.
- EWEnH and EWEµH supported FI-mediated cleavage of human and rat C3b.
- All tested proteins (EWE, EWEµH, EWEnH) bound C3b and iC3b with high affinity and inhibited alternative pathway complement deposition.
- EWEnH exhibited excellent solubility and cross-reactivity with human and rat C3b.
Conclusions:
- Engineered nanobodies, particularly EWEnH, effectively inhibit complement activation.
- EWEnH demonstrates potential as a therapeutic candidate for in vivo studies in rodent models of complement-mediated diseases.
Abstract:
The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.
