In Vitro Evaluation of Radioprotective and Radiosensitizing Effects of Rituximab

¹ School of Medicine, The Johns Hopkins University, Baltimore, Maryland
² Division of Nuclear Medicine, Russell H. Morgan Department of Radiological Sciences, School of Medicine, The Johns Hopkins University, Baltimore, Maryland

^* To whom correspondence should be addressed. E-mail: rwahl{at}jhmi.edu.

	Abstract

Clinical radioimmunotherapies with anti-CD20 monoclonal antibodies involve administering a predose of unlabeled anti-CD20 antibodies to favorably alter the biodistribution profile of the subsequently administered radiolabeled antibodies and mediate antitumor effects. Prior in vitro data suggested that unlabeled anti-CD20 monoclonal antibodies radiosensitize lymphoma cells as well. We assessed the antiproliferative and possible radiosensitizing capabilities of an anti-CD20 monoclonal antibody, rituximab. Methods: Luciferase-transfected (via a lentivirus vector) CD20⁺ human Raji lymphoma cells in log-phase growth were incubated with or without rituximab (20 µg/mL) for either 1 or 24 h before external-beam radiation exposure. Cell counts were measured with a luciferase assay at 24-h intervals. Subsets of these cells were also analyzed for cell cycle status by flow cytometry. Results: Rituximab pretreatment and irradiation were found to significantly inhibit tumor cell growth compared with irradiation alone (by a factor of 0.40 at 1 Gy [P < 0.01]). One hour of rituximab pretreatment modestly radiosensitized tumor cells at a radiation dose of 1 Gy (by a factor of 1.03 compared with the results for nonirradiated cells). At higher radiation doses (2 and 12 Gy), 1 h of rituximab pretreatment paradoxically radioprotected tumor cells by factors of 0.25 (P < 0.01) and 0.54 (P < 0.05), respectively. Rituximab predosing for 24 h was found to be radiosensitizing at a radiation dose of 4 Gy (by a factor of 2.84 [P < 0.01]) but radioprotective at radiation doses of 1, 8, and 12 Gy (by factors of 0.10 [P < 0.01], 2.50 [P < 0.01], and 2.07 [P < 0.05], respectively). These results correlated with retardation of the cell cycle at 6 d after rituximab administration, as determined by flow cytometry. Conclusion: Rituximab demonstrated a direct tumor antiproliferative effect in the absence of radiation. At lower levels of radiation exposure, rituximab radiosensitized Raji lymphoma cells. At higher doses of radiation, rituximab paradoxically protected tumor cells against ionizing radiation, possibly through effects on the cell cycle. These radiobiologic effects of rituximab should be carefully considered in the design of radioimmunotherapeutic trials.

Key Words: rituximab, radiosensitization, radioprotection, radioimmunotherapy