CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia by Edwin Bremer, Bram ten Cate, Douwe F. Samplonius, Lou F. M. H. de Leij, and Wijnand Helfrich Blood Volume 107(7):2863-2870 April 1, 2006 ©2006 by American Society of Hematology

Solution behavior and stability of scFvCD7:sFasL. Solution behavior and stability of scFvCD7:sFasL. (A) Culture medium derived from CHO-K1 production cells containing scFvCD7:sFasL was subjected to SE-FPLC using a calibrated HiLoad 16/60 FPLC column. The apoptotic activity of each individual fraction was assessed using the FasL-sensitive CD7-positive MOLT16 cells in the presence or absence of CD7-blocking mAb TH69. The horizontal bar in the graph indicates the fractions pooled for further testing. (B) scFvCD7:sFasL was stored for various lengths of time, up to 9 days, at 37°C in the presence of 15% serum. At serial time points the remaining apoptotic activity of the stored scFvCD7:sFasL was assessed by treatment of FasL-sensitive CD7-positive CEM cells for 16 hours. Apoptotic activity was assessed by ΔΨ. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology

CD7-restricted binding and apoptosis induction by scFvCD7:sFasL. CD7-restricted binding and apoptosis induction by scFvCD7:sFasL. (A) CD7-restricted binding of scFvCD7:sFasL was assessed using CD7-positive CEM cells. Cells were incubated with scFvCD7:sFasL (solid line) or with unconditioned medium (filled area). Specific binding was demonstrated by preincubating CEM cells with mAb TH69 followed by incubation with scFvCD7:sFasL (dashed line). Binding of scFvCD7:sFasL was determined by flow cytometry using PE-conjugated anti-FasL mAb. (B) CD7-restricted induction of apoptosis by scFvCD7:sFasL was assessed using CD7-positive cells (MOLT-16, CEM, Jurkat, and HUT-78) and CD7-negative Raji cells. All cell types were treated for 16 hours with increasing concentrations of scFvCD7:sFasL, after which apoptosis was assessed by ΔΨ. Additionally, Raji cells were treated with increasing concentrations of cross-linked sFasL (cr-sFasL). Indicated values are mean + SEM of 3 independent experiments. (C) Jurkat cells were treated for 16 hours with scFvCD7:sFasL (20 ng/mL) in the presence or absence of MAb TH69 or FasL-neutralizing MAb Alf2.1. Apoptosis was assessed by staining for apoptotic DNA-fragmentation using mAb F7-26. Horizontal bars indicate the percentage of apoptosis. (D) To compare the specific apoptotic activity of scFvCD7:sFasL with that of the related TRAIL fusion protein scFvCD7: sTRAIL, Jurkat cells were treated with equimolar concentrations of scFvCD7:sFasL and scFvCD7:sTRAIL for 16h. After treatment, apoptosis was assessed by ΔΨ. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology

Potent induction of apoptosis in CD7-negative bystander tumor cells. Potent induction of apoptosis in CD7-negative bystander tumor cells. Mixed cultures of Ramos.CD7 target cells and Raji bystander cells (ratio 1:1) were treated for 16 hours with scFvCD7:sFasL (100 ng/mL) in the presence or absence of mAb TH69 or mAb Alf2.1. The differential fluorescent labeling of target and bystander cell populations was used to separately evaluate apoptosis induction by ΔΨ in Ramos.CD7 target cells (A) and Raji bystander cells (B). Indicated values are mean + SEM of 3 independent experiments. Statistical analysis was performed using 2-tailed Student t test. *P < .05; **P < .01. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology

Treatment of normal human leukocytes, activated T cells, and HUVECs with scFvCD7:sFasL. Treatment of normal human leukocytes, activated T cells, and HUVECs with scFvCD7:sFasL. (A) Resting PBLs were subjected to treatment with scFvCD7:sFasL (325 ng/mL), or cross-linked sFasL (100 ng/mL) for up to 7 days, after which experimental apoptosis was assessed by annexin V/PI staining. Indicated values are representatives of 3 independent experiments. (B) Isolated PBLs were mixed at a ratio of 1:10 with DiI-labeled Jurkat cells. Mixed cultures were treated for 24 hours with scFvCD7:sFasL (100 ng/mL) or secondarily cross-linked sFasL (100 ng/mL). Differential fluorescent labeling of Jurkat target cells and PBLs was used to separately evaluate apoptosis induction by annexinV staining. Indicated values are representatives of 3 independent experiments. (C) Activated T cells were subjected to treatment with scFvCD7:sFasL (325 ng/mL) for up to 7 days, after which apoptosis was assessed by annexin V/PI staining. Indicated values are representatives of 3 independent experiments. (D) Resting HUVECs were treated for 24 hours with scFvCD7:sFasL (100 ng/mL), secondarily cross-linked sFasL (100 ng/mL), or actinomycin D (2 μg/mL). Apoptosis was assessed by ΔΨ. (E) Resting HUVECs were mixed with fluorescently labeled Jurkat cells (ratio 1:1) and treated with scFvCD7:sFasL (100 ng/mL) or actinomycin D (2 μg/ml) for 24 hours. Differential fluorescent labeling of Jurkat target cells and HUVEC bystander cells was used to separately evaluate apoptosis by ΔΨ. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology

Additive tumoricidal effect of scFvCD7:sFasL with several classes of antileukemia agents. Additive tumoricidal effect of scFvCD7:sFasL with several classes of antileukemia agents. Jurkat cells were treated for 16 hours with scFvCD7:sFasL (5 ng/mL) alone or in combination with vincristin (0.1 ng/mL) (A), farnesyl transferase inhibitor L-744832 (25 μM) (B), and the proteasome inhibitor bortezomib (10 nM) (C). Apoptosis induction was assessed by ΔΨ. The CI was calculated as described in “Materials and methods” and used to determine the cooperative effect of combination treatment. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology

Cotreatment of PBLs, activated T cells, or HUVECs with scFvCD7:sFasL and several classes of antileukemia agents. Cotreatment of PBLs, activated T cells, or HUVECs with scFvCD7:sFasL and several classes of antileukemia agents. PBLs (A), activated T cells (B), and HUVECs (C) were treated with scFvCD7:sFasL (40 ng/mL) combined with vincristine (0.5 ng/mL), L-744832 (25 μM), or bortezomib (40 nM). Apoptosis was assessed by annexin V/PI staining. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology

Treatment of T-ALL, PTCL, and CD7-positive AML patient-derived blood samples in vitro. Treatment of T-ALL, PTCL, and CD7-positive AML patient-derived blood samples in vitro. (A) Blood cells derived from 4 patients with T-ALL, 1 patient with PTCL, and 1 patient with CD7-positive AML were subjected to treatment with scFvCD7:sFasL (150 ng/mL) and analyzed for apoptosis induction using annexin V/PI staining. (B) Primary CD7-positive AML cells were subjected to treatment with scFvCD7:sFasL alone or in the presence of mAb TH69 for 16 hours, after which apoptosis was assessed by annexin V/PI staining. Statistical analysis was performed using 2-tailed Student t test. *P < .05. (C) Primary T-ALL cells were subjected to scFvCD7:sFasL (150 ng/mL) and then assessed for the presence of active caspase-3 using fluorescent microscopy as described in “Fluorescence microscopy of activated caspase-3.” (D) Primary CD7-positive AML cells were subjected to single-agent treatment with scFvCD7:sFasL (100 ng/mL), vincristine (10 ng/mL), amsacrine (1 μM), or to combination treatment. Apoptosis was assessed by annexin V/PI staining. The CI was used to determine the cooperative effect of combination treatment. Edwin Bremer et al. Blood 2006;107:2863-2870 ©2006 by American Society of Hematology