Antibody-based targeting of CD44 induces apoptosis in CLL. (A) The anti-CD44 mAb IM7 shows a specific reduction of viability by inducing cell death only in Eµ-TCL1:CD44wt splenocytes vs those from Eµ-TCL1:CD44ko mice (n = 10; mean ± SEM, 0.61 ± 0.06 vs 0.94 ± 0.03; P = .002). Cell death induction is increased in combination with fludarabine at 50 µM (Eµ-TCL1:CD44wt 0.35 ± 0.04, Eµ-TCL1:CD44ko 0.67 ± 0.06; P = .0012). (B) The anti-CD44 mAb A3D8 significantly reduces cell viability (ATP activity at 5 µg/mL: ratio-to-control mean ± SEM, 0.65 ± 0.08; at 10 µg/mL: 0.66 ± 0.01) and (C) induces cell death (5 µg/mL: mean ± SEM, 1.42 ± 0.10; at 10µg/mL: 1.54 ± 0.23) as compared with isotype controls in short-term (24 hours) cultures of 19 human primary CLL samples, even in the presence of prosurvival IL-4/CD40L. (D) A3D8 shows significant reduction of CLL viability in suspension cultures as well as when added for 24 hours to CLL cocultures with prosurvival NKtert stromal cell support (n = 6; mean ± SEM, isotype control 1.70 ± 0.21 vs A3D8 at 5 µg/mL 1.04 ± 0.16; P = .026). (E) Incubation of CLL cells in vitro with anti-CD44 mAbs (left: IM7 on murine Eµ-TCL1 tumor cells; right: A3D3 on human CLL cells) leads to characteristic apoptotic figures (arrows: vacuolization, blebbing, karyorrhexis). Shown are representative Wright-Giemsa–stained cytospins of a total of 3 murine and 7 human CLL after 36 hours of incubation with 5 μg/mL isotype (no signs of antibody internalization) vs 5 μg/mL CD44 mAbs. Asterisk indicates spin artifact. (F) Eight-week-old C57BL6/C3H F1 female hosts sufficiently engrafted by Eµ-TCL1 tg cells from founder #586³¹  were intravenously injected with endotoxin-free IM7 or isotype-control mAbs, both at 50 μg/animal, for 1 or 2 (shown here) consecutive day(s). Leukemic burden in the PB was determined before as well as 24 hours (shown here) and 48 hours after treatment. The continued leukemic growth in the isotype-treated animals (P = .0056; paired Student’s t test) was strongly inhibited by IM7 treatment (n = 4/cohort). Mean ± SEM CD19⁺ leukocyte counts in PB at 24 hours postinjection (“after”) were 124 ± 13 for isotype control vs 57 ± 15 for IM7 (P = .0142, unpaired Student’s t test). Postmortem (48 hours after first injection) leukemic burden in bone marrow, liver, or spleen remained unaffected (not shown).