Véronique Blanc

The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents.

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.

SAR650984, A Novel Humanized CD38-Targeting Antibody, Demonstrates Potent Antitumor Activity in Models of Multiple Myeloma and Other CD38+ Hematologic Malignancies

Purpose: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984. Experimental Design: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice. Results: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells. Conclusion: These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies. Clin Cancer Res; 20(17); 4574–83. ©2014 AACR.

Abstract 4735: SAR650984: Characterization of a potent phase I humanized anti-CD38 antibody for the treatment of multiple myeloma and other hematologic malignancies.

SAR650984, a humanized antibody targeting the type II transmembrane glycoprotein CD38 is currently in phase I dose escalation in patients with selected CD38+ hematological malignancies. CD38 is an ectoenzyme which catalyzes the formation of nucleotide metabolites involved in calcium signalling. Nearly 90% of MM patients express CD38 and this expression correlates with poor disease prognosis. The robust multiple mechanisms of action of SAR650984 include antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis induction, leading to in vivo efficacy in different tumor models, as reported previously (AACR 2010 #714; AACR 2009 #859, #2048, #2797; ASH 2008 #2756). Here, we report additional studies aimed to further elucidate the mechanism of action of SAR650984. These studies include solving the crystal structure of the SAR650984/CD38 complex, measuring enzymatic inhibition, testing in vitro activity against human primary MM cells, studying the mechanism of apoptosis induction in vivo and lastly assessing in vivo activity in combination with bortezomib (Velcade®). Fab fragments derived from SAR650984 were co-crystallized with soluble human CD38 to a 1.5 A resolution. Loop H3 of the paratope (residues Asp100, Tyr101, Tyr102, Gly103, Ser104 and Asn105) protrudes out of the SAR650984 Fab structure and is particularly critical for the complex formation. Significant conformational changes are observed in CD38 upon binding of SAR650984 while the configuration of the key residues that are involved in the ADP-ribosyl cyclase activity of CD38 is conserved as well as the access to the catalytic site. SAR650984 shows potent inhibition of the catalytic activity of CD38 (86 % at 10 nM), Taken together this would suggest that SAR650984 is not a direct orthosteric antagonist but more likely an allosteric antagonist of CD38. Consistent with the in vitro activity observed against cell lines, SAR650984 also demonstrated potent pro-apoptotic activity against a panel of CD38+ human primary MM cells. In SCID mice bearing SU-DHL-8 lymphoma, SAR650984 induced apoptosis of tumor cells, evidenced by a robust and sustained induction of cleaved caspase 7. In combination with bortezomib, a standard of care for MM treatment, SAR650984 demonstrated synergy in NCI-H929 MM xenografts in SCID mice. Together with previous reports the humanized anti-CD38 antibody, SAR650984, has demonstrated potent anti-tumor activities including ADCC, CDC and induction of apoptosis which translated into potent in vivo anti-tumor efficacy. These data support the ongoing clinical development of SAR650984 as a promising therapeutic antibody for various CD38+ hematologic malignancies. Citation Format: Marie-Cecile Wetzel, Celine Nicolazzi, Francois Vallee, Jutta Deckert, Charles Dumontet, Adriana Plesa, Aimo Kannt, Vincent Mikol, Marielle Chiron, Loic Vincent, Veronique Blanc. SAR650984: Characterization of a potent phase I humanized anti-CD38 antibody for the treatment of multiple myeloma and other hematologic malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4735. doi:10.1158/1538-7445.AM2013-4735

Structural characterization of a potent humanized anti-CD38 antibody in phase I

CD38 is a type II transmembrane glycoprotein with both ADP-rybosyl cyclase and glycohydrolase activities. CD38 is highly expressed at the surface of malignant plasma cells of multiple myeloma. SAR650984 is a humanized IgG1 antibody targeting CD38 in early clinical developement that is acting through several potential mechanisms including ADCC, CDC and pro-apoptotic activity. Here we report further preclinical characterization of SAR650984 with a high resolution structure of Fab-SAR650984 in complex with CD38 allowing an epitope mapping. The crystal structure of SAR650984-Fab/huCD38 complex shows that SAR650984 neither blocks the access nor alters the configuration of the ADPRC catalytic site of CD38 although in vitro assays have demonstrated that SAR650984 behaves as a strong inhibitor of the ADPRC activity of CD38. These results suggest that SAR650984 is likely an allosteric antagonist of CD38 that alters the dynamics of enzyme upon binding.