Pretargeted radioimmunotherapy (PRIT) has the potential to increase the dose of radionuclide delivered to tumors while limiting radiation to normal tissues. The purpose of this phase 1 trial is to assess safety of this multistep approach using a novel tetrameric single-chain anti-CD20-streptavidin fusion protein (B9E9FP) as the targeting moiety in patients with B-cell non-Hodgkin lymphoma (NHL), and to characterize its pharmacokinetics and immunogenicity. All patients received B9E9FP (160 mg/m2 or 320 mg/m2); either 48 or 72 hours later, a synthetic clearing agent (sCA) was administered (45 mg/m2) to remove circulating unbound B9E9FP. 9OYttrium (90Y; 15 mCi/m2)/111In (5 mCi)-DOTA-biotin was injected 24 hours later. There were 15 patients enrolled in the study. B9E9FP had a mean plasma half-life (T1/2) of 25 ± 6 hours with a reduction in plasma level of more than 95% within 6 hours of sCA administration. 90Y/111In-DOTA-biotin infusion resulted in rapid tumor localization and urinary excretion. The ratio of average tumor to whole-body radiation dose was 49:1. No significant hematologic toxicities were noted in 12 patients. There were 2 patients who had hematologic toxicity related to progressive disease. There were 2 complete remissions (90 and 325 days) and one partial response (297 days). B9E9FP performs well as the targeting component of PRIT with encouraging dosimetry, safety, and efficacy. A dose escalation trial of 90Y-DOTA-biotin in this format is warranted. © 2004 by The American Society of Hematology.