Effect of Ad35K⁺⁺ in Raji lymphoma xenograft model. (A) Distribution of human CD20⁺ Raji cells. At day 14 after intravenous Raji cell injection, femurs, spleens, and mesenteric lymph nodes were harvested. Lymph node and spleen sections were analyzed by immunofluorescence microscopy with FITC-labeled anti–human CD20 antibodies. For bone sections, the Klear Mouse DAB detection kit (Golden Bridge International Inc) was used. Positive staining appears in brown. Specificity of staining was confirmed by staining with corresponding isotype-matched antibodies (negative control) and antibodies specific to a human mitochondrial marker (positive control). Representative sections are shown. The scale bars represent 40 μm. (B) Scheme of experimental setting no. 1. CB17-SCID/beige mice received 3 × 10⁶ human lymphoma Raji cells by tail vein injection. Fourteen days later, when control mice developed the first clinical symptoms, animals were intravenously injected with Ad35K-279 or Ad35K⁺⁺. Rituximab or PBS was given intravenously 10 hours later. In the first experiment, mice were killed 12 hours later, and tissues were analyzed for human CD20⁺ cells. In a second experiment, animals were monitored every 12 hours for onset of paralysis or morbidity, which served as the end point for Kaplan-Meier survival studies. (C) Percentage of human CD20⁺ Raji cells in bone marrow and mesenteric lymph nodes of treated mice measured by flow cytometry (12 hours after treatment); n = 5. The differences between rituximab and Ad35K⁺⁺/rituximab are significant (P < .02) for both, bone marrow and spleen. The differences between Ad35K-279, Ad35K⁺⁺, and rituximab are not significant. (D) Kaplan-Meier survival study; n = 10. (E) Scheme of experimental setting no. 2. At day 13 after lymphoma cell injection, the first treatment cycle was started with 2 intravenous injections of 50 μg of Ad35K⁺⁺ 6 hours apart. Six hours after the second Ad35K⁺⁺ injection, mice received an intravenous injection of 50 μg of rituximab. A second treatment cycle was started 36 hours later. Onset of hind leg paralysis served as an end point in survival studies. (F) Kaplan-Meier survival study. Mice received either 1 treatment cycle [1 × (rituximab + Ad35K⁺⁺)] or 2 cycles [2 × (rituximab + Ad35K⁺⁺)], or the indicated control injections; n = 10.