Francois Vallee

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.

Selective blockade of the hydrolysis of the endocannabinoid 2-arachidonoylglycerol impairs learning and memory performance while producing antinociceptive activity in rodents

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery

Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

Discovery and Pharmacokinetic and Pharmacological Properties of the Potent and Selective MET Kinase Inhibitor 1-{6-[6-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl]benzothiazol-2-yl}-3-(2-morpholin-4-ylethyl)urea (SAR125844).

The HGF/MET pathway is frequently activated in a variety of cancer types. Several selective small molecule inhibitors of the MET kinase are currently in clinical evaluation, in particular for NSCLC, liver, and gastric cancer patients. We report herein the discovery of a series of triazolopyridazines that are selective inhibitors of wild-type (WT) MET kinase and several clinically relevant mutants. We provide insight into their mode of binding and report unprecedented crystal structures of the Y1230H variant. A multiparametric chemical optimization approach allowed the identification of compound 12 (SAR125844) as a development candidate. In this chemical series, absence of CYP3A4 inhibition was obtained at the expense of satisfactory oral absorption. Compound 12, a promising parenteral agent for the treatment of MET-dependent cancers, promoted sustained target engagement at tolerated doses in a human xenograft tumor model. Preclinical pharmacokinetics conducted in several species were predictive for the observed pharmacokinetic behavior of 12 in cancer patients.

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

Abstract 2911: SAR125844: a potent and selective ATP-competitive inhibitor of MET kinase

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The tyrosine kinase MET is a membrane receptor that is essential for embryonic development and wound healing in normal cells. Stimulation of MET by its natural ligand, the hepatocyte growth factor (HGF), induces cell proliferation, migration, and invasion. Abnormal MET activation (over-expression of MET protein, amplification or mutations of the MET gene) has been observed in multiple human cancer types. We report herein the discovery of a potent and selective small molecule inhibitor (SAR125844) with potential therapeutic application in cancer patients with deregulated MET-dependent malignancies. Our initial hit identification approach was based on the biochemical screen of an in-house kinase inhibitor biased library where a series of benzimidazole sulfonate derivatives were identified with sub-micromolar MET inhibition. In particular, the initial hit exhibited an IC50 of 140 nM vs MET but it also had strong affinity for CDK9 (IC50= 6 nM), a CDK isoform involved in gene transcription. Chemical modifications of the series to dial out CDK9 affinity and remove potential normal cell cytotoxicity led to a more selective derivative with IC50s of 80nM and 1355nM vs MET and CDK9 respectively. Further sub-structural exploration allowed us to identify a heteorocyclic moiety which was shown by X-ray data to specifically interact with Tyr1230 in a non active conformation of the protein. The resulting highly favourable U-shape mode of binding in MET of representative examples from these series (e.g. IC50= 1nM) was not tolerated in CDK9 (IC50 > 10µM). Final multi-parametric medicinal chemistry optimisation led to SAR125844 with single digit nanomolar antiproliferative activity on MET-amplified cell lines. SAR125844 is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. This compound exhibits also satisfactory eADMET in vitro properties and has shown moderate total plasma clearance, large volume of distribution and moderate to long terminal elimination half-life in rats. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2911. doi:1538-7445.AM2012-2911

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.