The complement system is a tightly controlled proteolytic cascade in the innate immune system, which tags intruding pathogens and dying host cells for clearance. An essential protein in this process is complement component C3. Uncontrolled complement activation has been implicated in several human diseases and disorders and has spurred the development of therapeutic approaches that modulate the complement system. Here, using purified proteins and several biochemical assays and surface plasmon resonance, we report that our nanobody, hC3Nb2, inhibits C3 deposition by all complement pathways. We observe that the hC3Nb2 nanobody binds human native C3 and its degradation products with low nanomolar affinity and does not interfere with the endogenous regulation of C3b deposition mediated by Factors H and I. Using negative-stain EM analysis and functional assays, we demonstrate that hC3Nb2 inhibits the substrate–convertase interaction by binding to the MG3 and MG4 domains of C3 and C3b. Furthermore, we notice that hC3Nb2 is cross-reactive and inhibits the lectin and alternative pathway in murine serum. We conclude that hC3Nb2 is a potent, general, and versatile inhibitor of the human and murine complement cascades. Its cross-reactivity suggests that this nanobody may be valuable for analysis of complement activation within animal models of both acute and chronic diseases.

The complement system is an intricate cascade of the innate immune system and plays a key role in microbial defense, inflammation, organ development, and tissue regeneration. There is increasing interest in developing complement regulatory and inhibitory agents to treat complement dysfunction. In this study, we describe the nanobody hC3Nb3, which is specific for the C-terminal C345c domain of human and mouse complement component C3/C3b/C3c and potently inhibits C3 cleavage by the alternative pathway. A high-resolution structure of the hC3Nb3–C345c complex explains how the nanobody blocks proconvertase assembly. Surprisingly, although the nanobody does not affect classical pathway–mediated C3 cleavage, hC3Nb3 inhibits classical pathway–driven hemolysis, suggesting that the C-terminal domain of C3b has an important function in classical pathway C5 convertase activity. The hC3Nb3 nanobody binds C3 with low nanomolar affinity in an SDS-resistant complex, and the nanobody is demonstrated to be a powerful reagent for C3 detection in immunohistochemistry and flow cytometry. Overall, the hC3Nb3 nanobody represents a potent inhibitor of both the alternative pathway and the terminal pathway, with possible applications in complement research, diagnostics, and therapeutics.

The complement system is part of the innate immune response, where it provides immediate protection from infectious agents and plays a fundamental role in homeostasis. Complement dysregulation occurs in several diseases, where the tightly regulated proteolytic cascade turns offensive. Prominent examples are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and Alzheimer’s disease. Therapeutic intervention targeting complement activation may allow treatment of such debilitating diseases. In this review, we describe a panel of complement targeting nanobodies that allow modulation at different steps of the proteolytic cascade, from the activation of the C1 complex in the classical pathway to formation of the C5 convertase in the terminal pathway. Thorough structural and functional characterization has provided a deep mechanistic understanding of the mode of inhibition for each of the nanobodies. These complement specific nanobodies are novel powerful probes for basic research and offer new opportunities for in vivo complement modulation.

Methyl pyrazines, particularly 2,5-dimethylpyrazine (DMP) and 2,3,5-trimethylpyrazine (3MP) are among the major flavor compounds associated with various seed and grain food processing operations, including roasting and drying. Synthetic polymers selective for DMP and 3MP were prepared by a molecular imprinting technique, and the binding characteristics of the imprinted polymers were examined by chromatographic methods. Despite the low molecular weight and lack of strongly interacting pendant functional groups in the pyrazine targets, the polymers showed selectivity for their respective templates over other pyrazines. Such polymers are envisioned to be of potential use in the analysis of flavor compounds in the food industry and in the selective isolation or enrichment of flavor profiles.

The integrin receptor α_Mβ₂ mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of α_Mβ₂ signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human α_Mβ₂ headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H α_M variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the α_M subunit in an Mg²⁺-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the α_M subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound α_Mβ₂ with iC3b, suggesting that it may represent a novel high-affinity proteinaceous α_Mβ₂-specific agonist. Taken together, our data suggest that the iC3b–α_Mβ₂ complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to α_Mβ₂.