Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators.

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

Human anti-C1q autoantibodies bind specifically to solid-phase C1q and enhance phagocytosis but not complement activation.

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology.

Mesenchymal stromal cells induce a permissive state in the bone marrow that enhances G-CSF-induced hematopoietic stem cell mobilization in mice.

Mesenchymal stromal cells (MSCs) support hematopoietic stem cells (HSCs) in vivo and enhance HSC engraftment and hematopoietic recovery upon cotransplantation with HSCs. These data have led to the hypothesis that MSCs may affect the HSC niche, leading to changes in HSC retention and trafficking. We studied the effect of MSC administration on the HSC compartment in the bone marrow (BM) in mice. After injection of MSCs, HSC numbers in the BM were decreased coinciding with an increased cell cycle activity compared with phosphate-buffered saline (PBS)-injected controls. Furthermore, the frequency of macrophages was significantly reduced and niche factors including Cxcl12, Scf, and Vcam were downregulated in endosteal cells. These BM changes are reminiscent of events associated with granulocyte colony-stimulating factor (G-CSF)-induced hematopoietic stem and progenitor cell (HSPC) mobilization. Interestingly, coadministration of MSCs and G-CSF resulted in a twofold increase in peripheral blood HSPC release compared with injection of G-CSF alone, whereas injection of MSCs alone did not induce HSPC mobilization. After intravenous administration, MSCs were only observed in the lungs, suggesting that they exert their effect on the HSC niche through a soluble mediator. Therefore, we tested the hypothesis that MSC-derived extracellular vesicles (EVs) are responsible for the observed changes in the HSC niche. Indeed, administration of EVs resulted in downregulation of Cxcl12, Scf, and Vcam and enhanced G-CSF-induced HSPC mobilization at similar levels as MSCs and G-CSF. Together, these data indicate that MSCs induce a permissive state in the BM, enhancing HSPC mobilization through the release of EVs.