A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy - PubMed (original) (raw)

A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy

Hongjie Wang et al. Blood. 2010.

Abstract

Many tumors, including lymphomas, up-regulate expression of CD46 to escape destruction by complement. Tumor cells are therefore relatively resistant to therapy by monoclonal antibodies, which act through complement-dependent cytotoxicity (CDC). From an Escherichia coli expression library of adenovirus type 35 fiber knob mutants, we selected a variant (Ad35K(++)) that had a higher affinity to CD46 than did the natural Ad35 fiber knob. We demonstrated that incubation of lymphoma cells with recombinant Ad35K(++) protein resulted in transient removal of CD46 from the cell surface. Preincubation of lymphoma cells with Ad35K(++) sensitized cells to CDC, triggered by the CD20-specific monoclonal antibody rituximab. In xenograft models with human lymphoma cells, preinjection of Ad35K(++) dramatically increased the therapeutic effect of rituximab. Blood cell counts and organ histology were normal after intravenous injection of Ad35K(++) into mice that express human CD46. The presence of polyclonal anti-Ad35K(++) antibodies did not affect the ability of Ad35K(++) to enhance rituximab-mediated CDC in in vitro assays. The Ad35K(++)-based approach has potential implications in monoclonal antibody therapy of malignancies beyond the combination with rituximab.

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Figures

Figure 1

Ad35 knob interaction with CD46. (A) Partial amino acid sequence of wild-type fiber knob (Ad35K) and the Ad35 knob mutants Ad35K-279 (ablated for CD46 binding) and Ad35K++. The corresponding affinities are indicated. The localization of the critical amino acid residues within a 3-dimensional model of the Ad35 fiber knob domain has been reported recently., (B) CD46 surface levels. Raji cells were incubated with 20 μg/mL Ad35K-279, Ad35K, Ad35++, or with anti-CD46 mAb. At the indicated time points, surface CD46 levels of cells were analyzed by flow cytometry. Shown is the percentage of CD46 levels (mean values and standard deviations), compared with CD46 mean fluorescence intensity of PBS-treated cells; n > 6; P = .021 for Ad35K versus Ad35K++ samples (15 minutes, 6 hours, 24 hours, and 48 hours combined). (C) Ad35 knob surface levels. Raji cells were incubated with Ad35K or Ad35K++ at room temperature for 1 hour. After washing with PBS, cells were incubated with fresh medium at 37°C for 15 minutes (■) or 6 hours (□). The levels of cell-bound Ad35 knob were analyzed by flow cytometry with the use of a mouse anti-His tag antibody followed by an anti–mouse antibody conjugated with Alexa Fluor 488. Shown is the fluorescence intensity (mean values and SDs); n > 6. (D-I) Immunofluorescence microscopy analysis of CD46, Cy3-Ad35K++, cathepsin B, and caveolin. For better clarity of cytoplasmic signals, the microphotographs shown are from HeLa cells. Similar results were obtained with Raji cells. (J) Inhibition of Ad35-GFP infection. Raji cells were treated with 20 μg/mL Ad35K-279, Ad35K, or Ad35K++ for 24 hours and, after washing, infected with Ad35-GFP at the indicated MOIs (multiplicities of infection). GFP expression was analyzed 24 hours later. Shown are the mean values and SDs; n = 3.

Figure 2

Ad35K++-sensitized cytolysis of lymphoma cells in vitro. (A top) Scheme of the experiment with Raji cells. Raji cells were incubated with anti-CD46 mAb, Ad35K-279, Ad35K, or Ad35K++. Eight hours later, daclizumab or rituximab were added to cells and incubated at room temperature. After 30 minutes, NHS was added, and viable cells were counted 3 hours later based on trypan blue exclusion. (Bottom) Percentage of viable cells compared with PBS-treated cells. Shown are the mean values and standard deviations; n > 6. (B-C) Studies with other lymphoma cell lines (B) or primary cells from patients with B-CLL (C). The experimental conditions were as described in panel A. Significant differences (P < .05) are marked by an asterisk. (D) Studies with normal human PBMCs. Human PBMCs (pooled from 3 healthy donors) were sorted for CD20+ cells with the use of fluorescence-activated cell sorting. CD20+ cells were cultured for 3 days. A total of 105 cells were treated with Ad35K++ (25 μg/mL), followed by rituximab (15 μg/mL) and NHS (25% final concentration) 8 hours later. Four hours after adding NHS, viable cells were counted based on trypan blue exclusion. Cell viability of PBS-treated cells was taken as 100%. There was no change in cell viability for cells incubated with Ad35K++, rituximab, or NHS alone. (E) Human PBMCs were cultured for 3 days and treated as described in panel D. Shown are the mean values and standard deviations; n = 5.

Figure 3

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.

Figure 4

Effect of Ad35K++ on rituximab therapy in Farage xenograft model. (A) Distribution of human CD20+ Farage cells. Note the follicular tumor growth, particularly in the spleen. (B) Treatment scheme. Treatment was started at day 21 after intravenous injection of Farage cells. (C) Flow cytometry for huCD20 cells; n = 7. The differences between rituximab and Ad35K++/rituximab are significant for all 3 tissues (P < .03). (D) Kaplan-Meier survival study after 1 round of treatment; n = 7.

Figure 5

Immunogenicity of Ad35K++. (A) Analysis of Ad knob-specific antibodies in serum from patients with cancer. Recombinant Ad5 hexon, Ad5 knob, Ad35K, and Ad35K++ as well as denaturated Ad5 particles were separated by polyacrylamide gel electrophoresis and stained with Coomassie blue (left). The proteins were plotted to nitrocellulose filter, which were incubated with human serum and subsequently with mouse anti–human immunoglobulin G–HRP conjugates (right). Human serum reacted with multimeric Ad5 hexon, trimeric Ad5 knobs, and virion pentons (∼ 70- and 80-kDa bands) but not with Ad35K or Ad35K++. Shown is a representative sample. (B) Analysis of knob antibodies in serum from mice that were injected subcutaneously with Ad35K++ (Ad35K++ vacc) or injected intravenously with Ad35K++ (Ad35K++ i.v.). Shown are representative samples. Photographs were taken with a digital Panasonic Lumix TZ-3 camera and processed in Adobe Photoshop. (C) Effect of anti-Ad35K++ serum on rituximab-mediated CDC. Raji cells were preincubated with mouse serum at a final dilution of 1:20 for 10 minutes at room temperature. Control serum was from normal mice. Test serum was from vaccinated mice that received subcutaneous Ad35K++ vaccinations. Then, 25 ng/mL Ad35K or Ad35K++ was added for 10 hours, followed by incubation with 15 μg/mL rituximab. CDC was induced 30 minutes later by adding NHS. Viable cells were counted 3 hours later. Shown are the mean values and SDs; n = 5.

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A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy - PubMed (original) (raw)