Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical activity of allogeneic SLAMF7-specific CAR T-cells (UCARTCS1) in multiple myeloma

doi: 10.1136/jitc-2023-008769

Figure Lengend Snippet: UCARTCS1-mediated lysis of primary MM cells. (A) BM-MNC obtained from 29 patients were incubated with UCARTCS1 cells or control (non-transduced (NT), SLAMF7/TCRαβ double knock-out) T-cells at different E:T ratios for 24 hours, after which the surviving MM cells were enumerated using flow cytometric analysis. Data represent mean±SEM. Mann-Whitney U test was used to calculate significance between both groups. (B) Representative flow cytometry contour plots showing SLAMF7-positive MM cells from a newly diagnosed MM patient, untreated (left) or treated with UCARTCS1 cells (right; E:T ratio of 1.5:1). After exclusion of doublets and dead Fixable Near-IR positive cells, MM cells were identified as CD38 + CD138 + cells. (C) UCARTCS1-mediated lysis of MM cells was similar when using UCARTCS1 cells from two different healthy T-cell donors. UCARTCS1 cells (light blue) and control NT T-cells (black) from donor one were tested with 16 BM samples; UCARTCS1 cells (dark blue) and control NT T-cells (gray) from donor two were tested with 13 BM samples. Mann-Whitney test was used to evaluate significance between both groups. (D) UCARTCS1-mediated lysis of MM cells in patient subgroups according to prior treatment: 10 BM samples from newly diagnosed MM patients (NDMM; green), 10 from daratumumab-naïve RRMM patients (RRMM; blue), and nine from daratumumab-refractory MM patients (DRMM; red). Solid lines represent UCARTCS1 cells and dotted lines control (non-transduced (NT), SLAMF7/TCRαβ double knock-out) T-cells. Data represent mean±SEM. Patient subgroups were compared using the Kruskal-Wallis test. (E) IFN-y, TNF-α, IL-17a, IL-10, IL-6, IL-4, IL-2, and granzyme B were measured in the cell supernatants of BM-MNCs treated with UCARTCS1 (blue) or control (non-transduced (NT), SLAMF7/TCRαβ double knock-out) T-cells (gray) for 24 hours by using a flow cytometry based assay (cytokines) or an ELISA (granzyme B). BM-MNCs were obtained from six DRMM patients. Data represent mean±SEM. Mann-Whitney U test was used to calculate significance between both groups. *p<0.05, **p<0.01, ****p<0.0001. BM-MNCs, bone marrow mononuclear cells; E:T ratio, effector to target-ratio; MM, multiple myeloma; ns, not significant; TCR, T-cell receptor.

Article Snippet: Specifically, plasma cells and other immune cell subsets were identified and analyzed for cell surface marker expression levels by staining 0.5–1.0×10 6 cells with CD45-KO, CD56 PC7, CD138 PE (Beckman Coulter), CD38-multi-epitope-FITC (Cytognos), CD3 BUV395, CD4 BUV737, CD8 BUV496, CD14 BB700, CD19 PE-CF594, CD127 PE-C7, CD319 (SLAMF7) AF647 (all BD Biosciences) and CD25 PE (DAKO).

Techniques: Lysis, Incubation, Control, Knock-Out, MANN-WHITNEY, Flow Cytometry, Enzyme-linked Immunosorbent Assay