Figure 4.

CT-011 enhances immune complex formation between effector NK cells and target MM cells. (A) A representative example of the flow cytometric technique to evaluate immune complex formation is shown, where primary, human NK cells, cultured as before for 72 hours in IL-2 and control mAb or CT-011, were stained with CFSE. Primary MM tumor cell targets were isolated from MM patient marrow aspirates and stained with PKH. Flow gates were created with PKH⁺ events representing MM target cells on the y-axis and CFSE⁺ effector NK cells on the x-axis. Double-positive events (ie, PKH⁺CFSE⁺) were interpreted as immune complex formations (shaded right top panel of flow diagrams). The left panel is representative of findings in control conditions; the right panel is representative of findings where NK cells were pretreated with CT-011. (B) Immune complex formation between NK cells and the K562, U266, RPMI8226 cell lines as well as with primary MM tumor cells was evaluated. At baseline, less than 1% of gated events in all conditions were PKH⁺CFSE⁺. However, NK cells pretreated with CT-011 led to statistically significant increases in immune complex formation with K562 (*P = .02), U266 (**P = .007), RPMI8226 (***P = .01), and primary MM tumor cell targets (****P = .009). All results shown are from at least 3 independent experiments with each target. (C) In this figure, total PBMCs obtained from patients with MM (rather than purified NK cells as in panels A-B) were treated for 48 hours in IL-2 with control or CT-011 mAb and immune complex formation in response to CT-011 against autologous CD138⁺PD-L1⁺ MM tumor cells or CD138⁻PD-L1⁻ cellular marrow elements was compared. Compared with control-treated effector cells, CT-011 increased immune complex formation with MM tumor cells (*P < .001) but not against normal marrow cells. Data shown are from 3 independent experiments in n = 3 patients.