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Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4–4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.

Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research.

Biosensors based on the combination of semiconductor quantum dots (QDs) and Förster resonance energy transfer (FRET) have demonstrated many advantages for simple, fast, sensitive, and multiplexed diagnostics. However, the implementation of QDs as functional standard materials into homogeneous (single-step) FRET immunoassays has not yet been accomplished, because profound investigations of antibody-conjugation strategies concerning their influence on diagnostic performance for quantifying clinical biomarkers are lacking. Here, we report about a systematic study of size, type, orientation, specificity, nonspecific binding, and cross-reactivity of antibodies conjugated to QDs for single and duplexed EGFR and HER2 immunoassays. Time-gated terbium-to-quantum dot FRET detection on a clinical immunoassay fluorescence plate reader (KRYPTOR) enabled a direct comparison of matuzumab, cetuximab, trastuzumab, and pertuzumab monoclonal antibodies and EgA1, EgB4, 11A4, and 18A12 VHH nanobodies conjugated to 605 and 650 nm emitting QDs. Detection limits of 2.9 ng/mL EGFR, using cetuximab and matuzumab conjugates, and 8.0 ng/mL HER2, using oriented 11A4 and 18A12 conjugates, demonstrated the capability of detecting concentrations well below the clinical cutoff values. Multiplexed assays could quantify EGFR and HER2 at low nanomolar concentrations from the same sample. Our results show that careful optimization of QD-antibody conjugation is a prerequisite to implementing QDs into applied clinical diagnostics.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.