Targeting CD38 with isatuximab and a CD38/CD3 × CD28 trispecific T-cell engager (CD38-TCE). The KG-1 as well as the MOLM-13 and OCI-AML3 AML cell lines were selected because of their respective low and bright density of CD38. (A) Triplicates of antibody-dependent cellular cytotoxicity (ADCC) were performed after 24 hours of treatment with isatuximab (70 nM) in coculture with NK lymphocytes from healthy adults (1:1 effector to target cell [E:T] ratio). ADCC was measured as the percentage of annexin-V⁺ tumor cells. (B) Triplicates of antibody-dependent cellular phagocytosis (ADCP) in coculture with monocyte-derived macrophages obtained from healthy adults (1:2 Φ:AML cell ratio) were performed after labeling with violet proliferation dye (VPD) and pretreatment with isatuximab (70 nM) for 30 minutes. ADCP was measured as the percentage of CD14/CD300e/VPD triple-positive macrophages after 2 hours of coculture. (C) Tumor depletion in BM aspirates from older patients with AML (n = 17) after treatment with isatuximab (70 nM) during 24 hours. Results are represented as the relative increment observed in treated vs untreated samples. (D) Using the Infinicyt software (Cytognos, Salamanca, Spain), we merged the fetal calf serum files of blasts from older patients with AML (n = 241) with those from KG-1, MOLM-13 and OCI-AML3 cell lines. The mean fluorescence intensity (MFI) of CD38 (measured with the clone HB7 conjugated with APCH7; BD Biosciences, San Jose, CA) is represented by population-band histograms, and ordered from the lowest into the highest expression detected among AML cell lines. The MFI of CD38 required to trigger ADCC and ADCP induced by isatuximab is represented by a hypothetical vertical dash line for a schematic representation of the results described in panels A-B. (E) MOLM-13 cells were labeled with VPD, pretreated with isatuximab or the CD38-TCE and their respective isotypes for 30 minutes, and cocultured with PBMC from healthy adults (n = 6) in a 10:1 E:T ratio. After 48 hours, the activation of effector cells was measured according to the percentage of CD69⁺ T and NK lymphocytes. For statistical significance, the highest dose was compared with the isotype-treated condition: green and blue symbols represent T and NK cells, respectively; ∗ and # indicated treatment with isatuximab and the CD38-TCE, respectively. (F) Tumor cell death of MOLM-13 induced by pretreatment with isatuximab and the CD38-TCE was determined using annexin V staining after 24 and 48 hours of coculture in the presence of PBMC from healthy adults. (G) Tumor cell death in primary samples from patients with AML (n = 8) after treatment with isatuximab (700 pM) and the CD38-TCE (700 pM) for 48 hours. The percentage of tumor cell death was calculated as follows: [(% leukemic cells in untreated – % of leukemic cells in treated sample)/% of leukemic cells in untreated] × 100. Each colored line is a patient sample, and the 2 cases having tumor cells resistant to isatuximab while being sensitive to the CD38-TCE are represented by the pink and gray lines. (H) After 48 hours of treatment with the CD38-TCE (700 pM), the percentages of neutrophils, monocytes, NK, B, as well as CD4 and CD8 T lymphocytes among CD45⁺ nucleated cells in BM were normalized to those observed upon treatment with the isotype, which was set up to 100%. In all panels, 1 symbol (∗ or #), P < .05; 2 symbols, P < .01; 3 symbols, P < .001.