We chose nude mice, because they are less sensitive to radiation than SCID mice, and used the Ramos human B-cell lymphoma cell collection inoculated subcutaneously, since tumor growth was dependable and predictable. and discusses how some fundamental changes could potentially enhance the response and period from radionuclide-targeted therapy. Keywords: CD20, CD22, non-Hodgkin lymphoma, pretargeting, radioimmunotherapy Introduction Radioimmunotherapy (RAIT) is an approved approach for the treatment of follicular and transformed non-Hodgkin lymphoma (NHL) with two radiolabeled anti-CD20 IgG products.1C3 90Y-ibritumomab tiuxetan (Zevalin?; Cell Therapeutics, Seattle, Washington) and 131I-tositumomab (Bexxar?; GlaxoSmithKline, Philadelphia, PA) have shown improved objective responses over the corresponding anti-CD20 IgG used with each agent; namely, rituximab (Biogen Idec, Cambridge, MA) and tositumomab, respectively, which themselves have significant anti-tumor activity.4, 5 New clinical studies are lending increasing support for their use in frontline and consolidation settings,2, 6C22 but new laboratory investigations are providing insights into ways that could improve responses, in some cases RF9 with less toxicity. In order to improve these treatments, we need first to understand the current treatment regimens, and how each of these brokers was developed. Monoclonal antibody-based therapeutics in hematopoietic malignancies started with an examination of several naked murine monoclonal antibodies in lymphoma and leukemias. Some objective responses were observed, but overall the anti-tumor effects were minimal, in many respects limited by the amount of antibody and the number of treatments that could be given with the murine IgG.23C32 For example, in a small pilot study, Press et al. reported progressively better objective anti-tumor responses in patients given a continuous infusion of the murine anti-CD20 IgG 1F5, but it required nearly 2 grams of antibody to get a total response. Today, this amount of antibody is usually commonplace for antibody therapeutics, but at that time, this was beyond the capability of most facilities to produce, and development of an anti-mouse antibody response further restricted the treatment. However, preclinical models often found that immunoconjugates (antibodies conjugated with radionuclide, drugs, or toxins) were much more active RF9 than the corresponding unlabeled antibody and required less antibody. After DeNardo et al. reported encouraging responses with a radioiodinated anti-HLA-DR antibody in B-cell malignancies, a number of other studies using antibodies to CD37, CD20 and CD22 were pursued, first MTC1 in a myeloablative setting with bone marrow support, and then with non-myeloablative doses, to have more substantial anti-tumor activity than that reported with the naked antibodies.33C37 The initial studies performed with CD37 and CD20 antibodies found a more favorable biodistribution for the radioconjugates when additional unlabeled anti-CD20 IgG was mixed with the radioimmunoconjugate.34, 35 Imaging studies showed there was intense uptake of the radioimmunoconjugate in the spleen, accompanied by rapid blood clearance, when low protein doses were used.38 Since all of the above-mentioned antibodies bind to normal and malignant B-cells, the large repository B-cells in the spleen (normal and malignant B-cells, often resulting in splenomegaly) acted as a sink, taking up the small amount of radioconjugate from your blood before it had an opportunity to circulate and localize in all tumor sites. Administering additional antibody protein reduced splenic uptake and slowed the radioconjugates clearance from blood. Antibodies against different antigens require different amounts of antibody for this to occur.34, 35, 39C44 Adding additional antibody to an immunoconjugate raises concerns that this unlabeled antibody would compete for the binding sites in the tumor, reducing the amount of the radioimmunoconjugate localized in the tumor. However, in nude mice bearing Raji human B-cell lymphoma xenografts, Buchsbaum et al.45 confirmed that tumor localization of the radiolabeled anti-B1 antibody (murine anti-CD20 IgG2a; tositumomab) could be enhanced by first pre-dosing the animals with the unlabeled antibody before the radioimmunoconjugate. Although mouse B-cells do not have cross-reactivity with the anti-B1 antibody, nude mice have low levels of murine IgG2a.46, 47 As a RF9 consequence, when small amounts of exogenously added murine IgG2a are given to the mice, the antibody is rapidly bound by cells in the spleen having receptors for this IgG form, causing increased splenic uptake and rapid blood clearance, which subsequently reduced the amount of antibody available for tumor targeting. The pre-dose preoccupies these receptors, allowing the radioimmunoconjugate to circulate longer in the blood, thus giving it more opportunity to bind to the tumor. While this is not the same mechanism of antibody.