Anti-C1s humanized monoclonal antibody SAR445088: A classical pathway complement inhibitor specific for the active form of C1s.

The objective of this study was to characterize the complement-inhibiting activity of SAR445088, a novel monoclonal antibody specific for the active form of C1s. Wieslab® and hemolytic assays were used to demonstrate that SAR445088 is a potent, selective inhibitor of the classical pathway of complement. Specificity for the active form of C1s was confirmed in a ligand binding assay. Finally, TNT010 (a precursor to SAR445088) was assessed in vitro for its ability to inhibit complement activation associated with cold agglutinin disease (CAD). TNT010 inhibited C3b/iC3b deposition on human red blood cells incubated with CAD patient serum and decreased their subsequent phagocytosis by THP-1 cells. In summary, this study identifies SAR445088 as a potential therapeutic for the treatment of classical pathway-driven diseases and supports its continued assessment in clinical trials.

C1s Inhibition by BIVV009 (Sutimlimab) Prevents Complement-Enhanced Activation of Autoimmune Human B Cells In Vitro.

The classical pathway of complement (CP) can mediate C3 opsonization of Ags responsible for the costimulation and activation of cognate B lymphocytes. In this manner, the complement system acts as a bridge between the innate and adaptive immune systems critical for establishing a humoral response. However, aberrant complement activation is often observed in autoimmune diseases in which C3 deposition on self-antigens may serve to activate self-reactive B cell clones. In this study, we use BIVV009 (Sutimlimab), a clinical stage, humanized mAb that specifically inhibits the CP-specific serine protease C1s to evaluate the impact of upstream CP antagonism on activation and proliferation of normal and autoimmune human B cells. We report that BIVV009 significantly inhibited complement-mediated activation and proliferation of primary human B cells. Strikingly, CP antagonism suppressed human Ig-induced activation of B cells derived from patients with rheumatoid arthritis. These results suggest that clinical use of CP inhibitors in autoimmune patients may not only block complement-mediated tissue damage, but may also prevent the long-term activation of autoimmune B cells and the production of autoantibodies that contribute to the underlying pathologic condition of these diseases.

The structural determinants of checkpoint activation.

Here, we demonstrate that primed, single-stranded DNA (ssDNA) is sufficient for activation of the ATR-dependent checkpoint pathway in Xenopus egg extracts. Using this structure, we define the contribution of the 5'- and 3'-primer ends to Chk1 activation when replication is blocked and ongoing. In addition, we show that although ssDNA is not sufficient for checkpoint activation, the amount of ssDNA adjacent to the primer influences the level of Chk1 phosphorylation. These observations define the minimal DNA requirements for checkpoint activation and suggest that primed ssDNA represents a common checkpoint activating-structure formed following many types of damage.

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.