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Activation of classical and lectin complement pathways contributes to several human diseases. Empasiprubart is a humanized recycling monoclonal antibody that inhibits both pathways by binding to the CCP2 domain of complement factor 2 (C2), an interaction that is dependent on both Ca 2+ and pH. Here, we resolve the crystal structure of empasiprubart complexed with C2, providing the molecular basis of its Ca 2+ dependency, and report a randomized, double-blind, placebo-controlled trial to assess the safety and tolerability (primary objectives) in addition to pharmacokinetics, pharmacodynamics, and immunogenicity (secondary objectives) of empasiprubart in 78 healthy participants (NCT04532125). A single intravenous (IV) dose of empasiprubart reduces circulating C2 levels by up to 99% and dose-dependently inhibits the classical and lectin pathways. Multiple IV empasiprubart doses reinforce reductions in free C2 levels, which persist until the endpoint of the study at 41 weeks. This prolonged reduction is in line with the empasiprubart elimination half-life (70–88 days). Single and multiple ascending doses of empasiprubart are generally safe and well tolerated. Overall, our results reveal in atomic detail the mechanism of empasiprubart and demonstrate that it is a first-in-class anti-C2 therapeutic antibody for use in complement-mediated diseases. Though the complement system is pivotal in the defence against infections, pathologic activation of the system contributes to disease. Here, authors show that their recently developed monoclonal antibody against complement factor 2, empasiprubart, inhibits the classical and lectin pathways in a clinical trial, and its crystal structure provides basis for its inhibitory properties, such as Ca 2+ binding.

The complement system which is part of the innate immune response against invading pathogens represents a powerful mechanism for killing of infected cells. Utilizing direct complement recruitment for complement‐mediated elimination of HIV‐1‐infected cells is underexplored. We developed a novel therapeutic modality to direct complement activity to the surface of HIV‐1‐infected cells. This bispecific complement engager (BiCE) is comprised of a nanobody recruiting the complement‐initiating protein C1q, and single‐chain variable fragments of broadly neutralizing antibodies (bNAbs) targeting the HIV‐1 envelope (Env) protein. Here, we show that two anti‐HIV BiCEs targeting the V3 loop and the CD4 binding site, respectively, increase C3 deposition and mediate complement‐dependent cytotoxicity (CDC) of HIV‐1 Env‐expressing Raji cells. Furthermore, anti‐HIV BiCEs trigger complement activation on primary CD4 T cells infected with laboratory‐adapted HIV‐1 strain and facilitates elimination of HIV‐1‐infected cells over time. In summary, we present a novel approach to direct complement deposition to the surface of HIV‐1‐infected cells leading to complement‐mediated killing of these cells. Synopsis This study reports the development of a novel therapeutic modality termed bispecific complement engager (BiCE) that directs complement activity to the surface of HIV‐1 envelope‐expressing cells, resulting in complement‐mediated elimination of these cells. (Created with BioRender.com). Two novel anti‐HIV BiCEs comprised of an anti‐C1q nanobody and a scFv of the bNAb 10‐1074 or 3BNC117, respectively, were constructed and characterized. Both anti‐HIV BiCEs showed complex formation with C1q and efficient neutralization of free virus in vitro . The two anti‐HIV BiCEs mediated complement activation and complement‐mediated lysis specific for HIV‐1 envelope‐expressing Raji cells in vitro . Ex vivo study showed an accelerated elimination over time of HIV‐1 infected primary CD4 T cells mediated by the anti‐HIV BiCEs. Graphical Abstract This study reports the development of a novel therapeutic modality termed bispecific complement engager (BiCE) that directs complement activity to the surface of HIV‐1 envelope‐expressing cells, resulting in complement‐mediated elimination of these cells.

The complement system which is part of the innate immune response against invading pathogens, represents a powerful mechanism for killing of infected cells. Utilizing direct complement recruitment for complement-mediated elimination of HIV-1-infected cells is underexplored. We developed a novel therapeutic modality to direct complement activity to the surface of HIV-1-infected cells. This bispecific complement engager (BiCE) is comprised of a nanobody recruiting the complement-initiating protein C1q, and single-chain variable fragments of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope (Env) protein. Here, we show that two anti-HIV BiCEs targeting the V3 loop and the CD4 binding site, respectively, increase C3 deposition and mediate complement-dependent cytotoxicity (CDC) of HIV-1 Env expressing Raji cells. Furthermore, anti-HIV BiCEs trigger complement activation on primary CD4 T cells infected with laboratory-adapted HIV-1 strain and facilitates elimination of HIV-1-infected cells over time. In summary, we present a novel approach to direct complement deposition to the surface of HIV-1-infected cells leading to complement-mediated killing of these cells.

The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of the C3(H2O) analog, C3MA, in which the C3 thioester is broken by aminolysis is indistinguishable from C3b except for the 77-residue anaphylatoxin (ANA) domain. In contrast, the reaction intermediate C3* formed during C3 adopts a dynamic conformation dramatically different from both C3 and C3MA/C3b. In C3*, unlocking of the macroglobulin (MG) 3 domain creates a large opening in the MG-ring through which the ANA domain translocates. In support of this mechanism, C3MA formation is inhibited by an MG3/MG4-interface-specific nanobody and prevented by linking the ANA domain to the C3 β-chain. Our study reveals an unexpected dynamic behavior of C3 where an exceptional conformational change allows the translocation of an entire domain through a large dynamic opening. These results form the basis for elucidation of the in vivo contribution of C3 hydrolysis to complement activation and offer a rational approach for modulation of C3(H2O) with the potential for preventing complement activation caused by intravascular hemolysis and surface contacts.

The CRISPR/Cas9 system has shown great potential for precisely editing genomic DNA sequences by introducing site-specific DNA cuts that are subsequently repaired by the cell. However, delivery of the CRISPR ribonucleoprotein remains an understudied area and hinders realizing the full potential of the system. We prepared Cas9 ribonucleoprotein complexes chemically conjugated to the 7D12 nanobody and demonstrate receptor-mediated transfection of Cas9 into A549 non-small-cell lung cancer cells via binding to the epithelial growth factor receptor for subsequent cell internalization. We further show that transfection with a Cas9 ribonucleoprotein targeting the BRCA2 gene results in an enhanced sensitivity to the chemotherapeutic drug Cisplatin, and thereby induces a synthetic dose lethality in A549 cells.