Abstract # 3935 , a novel CD38 for the treatment of B‐cell Non– Alba Matas‐Céspedes1, Anna Vidal‐Crespo1, Vanina Rodriguez1, Gaël Roué1, Elías Campo2 ,Dolors Colomer2 Jeroen J. Lammerts van Bueren3, Joost M. Bakker3, Adrian Wiestner4, Paul W.H.I. Parren3 , Patricia Pérez‐Galán1 1Department of Hemato‐Oncology, IDIBAPS. Barcelona, Spain 2Hematopathology Unit, Hospital Clinic. Barcelona, Spain 3Genmab. Utrecht, The Netherlands 4Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health. Bethesda, MD (USA)

Background 2. Daratumumab induces ADCC in the presence of external effectors in MCL, FL and CLL 5. Daratumumab significantly reduces SDF‐1/CXCL12 induced migration in CLL cells

•Daratumumab (DARA) is a human CD38 antibody with broad‐spectrum killing activity / A. PBMCs from healthy donors (n=2 in each experiment) were used as A NK cells •DARA induces killing of tumor cells mainly via complement‐dependent cytotoxicity (CDC) and antibody‐ effectors in a 1:50 ratio (T:E). Percentages of the effectors, NKs A C ns dependent cell‐mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011). (CD56+ and CD3‐) and monocytes/macrophages (CD14+), were •DARA is currently being evaluated in phase I/II in patients with , showing always quantified. A representative sample of a normal PBMC donor is shown. manageable adverse events and marked reductions in paraprotein and bone marrow plasma cells. B. Cell lines and primary cells from MCL, FL and CLL were treated with •Here, we have analyzed the potential of targeting of CD38 using DARA in three types of B‐cell non‐ Daratumumab (DARA) (0.001‐1 µg/ml) for 4h in the presence of Hodgkin lymphoma (B‐NHL): (MCL), (FL)) and chronic PBMCs. Cell death was assessed by Calcein release and fluorescence lymphocytic (CLL). B measured in a fluorimeter (Synergy HT, Biotek). B •MCL and CLL tumor cells show heterogeneous expression of CD38 and FL cells showed invariable high 6000 5000 CLL1 It has been described that CD38 cooperates with CXCL12‐

CD38 levels. MCL 4000 induced migration in CLL (Vaisitti et al, Leukemia 2010). count

•CD38 is specially attractive in CLL where high CD38 expression is a marker of bad prognosis and is 3000

cell For this reason we assessed if daratumumab could

cell 2000 expressed preferentially in the proliferating fraction of the tumor (Hamblin et al. Blood 1999 and 2002, FL interfere with this process. 1000 total A. Schematic view of migration assay. Damle RN. Blood 2007). 0 IgG1 Dara 30ug/mL Anti‐CXCR4 IgG1 Dara 30ug/mL Anti‐CXCR4 B. CLL cells were preincubated with the antibodies for CLL 30 min at 4ºC (IgG1 30µg/mL, Dara 30µg/mL or BL 9000 anti‐CXCR4 25µg/mL) in RPMI +0.5% BSA. Then cells 8000 Aims (positive control) CLL3 5 7000 were added to the upper chamber (5x10 cells in

count 6000

5000 100µl) and put in contact with the lower chamber cell 4000 cell

 1. Mechanisms of daratumumab citotoxicty in MCL, FL and CLL cells 3000 containing 200 ng/ml SDF‐1 /CXCL12 or not. After 2000 total 4h, CLL cells (CD19+ CD5+) in the lower chamber 2. Comparision of daratumumab activity with anti‐CD20 antibodies 1000 3. Daratumumab‐induced ADCC is comparable to anti‐CD20 antibodies 0 were counted in triplicates in a flow cytometer at fix IgG1 Dara 30ug/mL Anti‐CXCR4 IgG1 Dara 30ug/mL Anti‐CXCR4 3. Effect of daratumumab in combination with in MCL, FL and CLL cells flow rate. Two representative patients are shown. +SDF1/CXCL12 -SDF1/CXCL12 C. Summary of CLL patients analyzed. The percentage 4. Effect of daratumumab in SDF1/CXCL12‐induced migration in CLL primary cells of inhibition is referred to cells pretreated with IgG1 ns ns ns control. ns ns ns Results Lysis

Lysis Lysis

% % % Disclosure statement: J. Lammerts van Bueren , J. M. Bakker and P. W.H.I. Parren 1. Daratumumab does not induce CDC in MCL, FL or CLL cells are employees. P. Pérez‐Galán received research funding from Genmab.

A 120 HBL2 100 Cell lines and primary cells from MCL, FL and CLL were treated with 0.1µg/ml of either Daratumumab (DARA), JEKO 100 MCL (OFA) or (RITUX) for 4h in the presence of PBMCs. (ratio T: E= 1:50). Cell death was assessed by Calcein release REC 80 Conclusions MINO and fluorescence measured in a fluorimeter (Synergy HT,Biotek). 80 MCL1 WSU-FSCCL 60 FL1 60 RL FL CLL2 SC-1 40 CLL3 DOHH2 % Lysis % 40 Lysis % CLL7 • Daratumumab induces marginal CDC in MCL, FL or CLL cells. This limited CDC induction MEC2 20 20 JVM13 CLL CLL9 4. Lenalidomidepretreatment of PBMCs significantly increases Daratumumab DAUDI CLL10 BL 0 0 by daratumumab may be explained by the high expression of the complement inhibitors (positive control) ADCC activity -20 -20 IgG1 10 Dara 0.01 Dara 0.1 Dara 1 Dara 10 IgG1 10 Dara 0.01 Dara 0.1 Dara 1 Dara 10 CD46, CD55 and CD59 and by insufficient number of CD38 molecules/cell B A B Sample CD38 CD46 CD55 CD59 C CLL primary cells MCL and FL cell lines • Daratumumab induces ADCC in MCL, FL and CLL primary cells and cell lines in the 100 ** CLL9 100 DAUDI 98.9 88.02 17.19 9.09 DAUDI WSU-FSCCL RL CLL3 80 80 ** ** HBL2 41.3 99 97.4 97.35 600000 60 presence of PBMC effector cells. 60 JEKO 52.8 96.22 91.71 89.76 500000 40 400000 40* REC 99.4 93.34 87.43 86.74 MFI 58.097 MFI 15.139 MFI 15.342 300000 20 * * ** MINO 98.8 95.51 95.12 96.5 200000 Lysis % 20 • DaratumumabADCCinductioninMCL,FLandCLLiscomparabletoanti‐CD20 antibodies 0 Lysis % WSU-FSCCL 98.5 MINO REC 100000 88.65 93.31 94.69 0 0,01 0,1 1 0 0 RL 95 71.99 84.8 96.51 -20 0 0,01 0,1 1

CD38 molecules cell per -20 -40 SC-1 98.9 91.76 87.61 94.3 Antibody concentration (µg/ml) Antibody concentration (µg/ml) ofatutumab and rituximab -40 DOHH2 ND 96.84 73.06 98.13 MFI 17.249 MFI 13.120 MEC2 52.92 97.78 97.04 99.63 JVM13 58.31 89.84 85.54 97.22 • Lenalidomide pretreatment of effector PBMCs significantly enhances ADCC induced by CLL2 3 81.5 94.87 63.08 CLL5 44 82.88 74.92 72.9 100 WSU-FSCCL 100 REC Daratumumab in MCL, FL cell lines and CLL primary cells 80 * MCL, FL cell lines or CLL primary cells were treated with 80 Daratumumab (DARA) for 4h (A. 0.01‐1µg/ml,B.CLL A. Cell lines and primary cells from MCL, FL and CLL were treated with Daratumumab (DARA) (0.01‐10 µg/ml) for 45min in the 60 presence of 10% NHS (Normal Human Serum). Cell death was assessed by Calcein release and fluorescence measured in a 60 0.1µg/ml, MCL and FL 1µg/mL) in the presence of PBMCs. • Daratumumab significantly reduces SDF1/CXCL12‐induced migration in most CLL cells 40 fluorimeter (Synergy HT,Biotek). % Lysis % Lysis 40 that were pretreated or not with lenalidomide (LENA 3µM B. Expression of CD38 and the complement inhibitors CD46, CD55 and CD59. The percentage of positive cells above isotype is 20 20 72h). Cell death was assessed by Calcein release and 0 0 shown. 0 0,01 0,1 1 0 0,01 0,1 1 fluorescence measured in a fluorimeter (Synergy HT,

Antibody concentration (µg/ml) Biotek). C. Quantification of CD38 molecules per cell using calibration beads (Qifikit, Dako) in MCL and FL cell lines showing high CD38 Antibody concentration (µg/ml) Contact: [email protected] expression. Daudi cells were used as a reference of a cell line where DARA induces CDC. 2x105 CD38 molecules/cell seems the threshold for CDC induction on multiple myeloma cells Groen RW, Blood 2010, 116:3058). [email protected]