SAR650984 (SAR) Directly Promotes Homotypic Adhesion-Related Multiple Myeloma (MM) Cell Death and SAR-Induced Anti-MM Activities Are Enhanced By Pomalidomide, More Potently Than Lenalidomide
Authors: Jiang Hua , An Gang , Acharya Chirag , Zhong Mike Y , Cai Ti , Song Zhili , Yang Guang , Adrian Francisco , Munshi Nikhil C. , Tai Yu-Tzu , Anderson Kenneth C. 
Published: October 2019

Abstract
SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among > 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express > 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients.

Disclosures
Cai: Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.