Radioimmunotherapy with anti-CD20 monoclonal antibodies is a promising new treatment approach for patients with relapsed B-cell lymphomas. However, the majority of patients treated with conventional radiolabeled anti-CD20 antibodies eventually have a relapse because the low tumor-to-blood and tumor-to-normal organ ratios of absorbed radioactivity limit the dose that can be safely administered without hematopoietic stem cell support. This study assessed the ability of a streptavidin-biotin "pretargeting" approach to improve the biodistribution of radioactivity in mice bearing Ramos lymphoma xenografts. A pretargeted streptavidin-conjugated anti-CD20 1F5 antibody was infused, followed 24 hours later by a biotinylated N-acetylgalactosamine-containing "clearing agent" and finally 3 hours later by 111 In-labeled DOTA-biotin. Tumor-to-blood ratios were 3:1 or more with pretargeting, compared with 0.5:1 or less with conventional 111 In-1F5. Tumorto-normal organ ratios of absorbed radioactivity up to 56:1 were observed with pretargeting, but were 6:1 or less with conventional 111 In-1F5. Therapy experiments demonstrated that 400 Ci (14.8 MBq) or more of conventional 90 Y-1F5 was required to obtain major tumor responses, but this dose was associated with lethal toxicity in 100% of mice. In marked contrast, up to 800 Ci (29.6 MBq) 90 Y-DOTA-biotin could be safely administered by the pretargeting approach with only minor toxicity, and 89% of the mice were cured. These data suggest that anti-CD20 pretargeting shows great promise for improving current therapeutic options for B-cell lymphomas and warrants further preclinical and clinical testing. IntroductionNon-Hodgkin lymphomas afflict 58 000 Americans each year and are rapidly increasing in incidence. 1 Only one third of patients with B-cell lymphoma are cured with conventional chemotherapy and radiotherapy; therefore, innovative new treatments are a high priority for this malignancy. Monoclonal antibodies (mAbs) directed against tumor-associated antigens have emerged as effective new reagents for lymphomas and are being tested extensively in the laboratory and in clinical trials. Although many B-cell surface antigens have been targeted with antibodies, to date anti-CD20 antibodies have been the most widely tested and have achieved the best clinical results. [2][3][4][5] CD20 is a 35 000-kd nonglycosylated phosphoprotein expressed on the surface of nearly all mature B-lymphoid cells and on 95% of B-cell lymphomas. 6 The CD20 antigen appears to have many favorable attributes that commend its use as an immunotherapeutic target. CD20 is not shed into the bloodstream, is not rapidly internalized, and is expressed at a high surface density on the vast majority of lymphomas. 6-8 Rituximab, a chimeric anti-CD20 antibody, induces remissions in 50% to 70% of patients with newly diagnosed follicular lymphomas, 48% to 60% of patients with relapsed follicular lymphomas, 30% to 35% of those with relapsed diffuse large B-cell lymphomas, 30% to 35% of patients with relapsed mantle cell...

Radiolabeled anti-CD20 antibodies produce responses in 60% to 95% of patients with relapsed non-Hodgkin lymphoma (NHL); however, absorbed radiation ratios between tumors and normal organs are relatively low, and many patients have relapses. In this study we compared the abilities of anti-CD45 (BC8) and anti-CD20 (1F5) antibodies to target human Ramos lymphoma xenografts in athymic mice. When direct radioiodination was performed with conventional methods, BC8 delivered 2-to 4-fold more radioiodine to tumors than 1F5, with tumor-to-normal organ ratios as high as 20:1 using radiolabeled BC8 compared with a maximal ratio of 9.8:1 using radioiodinated 1F5. To optimize the biodistribution of radioactivity, we performed studies following a pretargeting method using streptavidin (SA)-conjugated BC8 and 1F5. Injection of a synthetic clearing agent decreased the circulating level of conjugates by 80% to 90% within 1 hour. Pretargeting with BC8-SA resulted in a 2-to 4-fold greater tumor uptake of radiolabeled biotin than with 1F5-SA, with maximal tumor-to-normal organ ratios of more than 80:1 and approximately 16:1, respectively. Therapy experiments demonstrated that 400 Ci (14.8 MBq) or more of yttrium-90-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin cured 100% of mice treated with BC8-SA and more than 90% of mice pretargeted with 1F5-SA, with complete remission occurring 8 to 10 days sooner in mice receiving BC8-SA. After treatment with 200 Ci (7.4 MBq) 90 Y-DOTA-biotin, 70% of the mice treated with BC8-SA were cured, but no mice were cured using 1F5-SA. Doses up to 800 Ci (29.6 MBq) 90 Y-DOTA-biotin were delivered with minor toxicity using either antibody conjugate. These lymphoma xenograft data suggest that pretargeted radioimmunotherapy using either anti-CD20 or anti-CD45 conjugates is highly effective and minimally toxic. (Blood.

The results demonstrate the potential of chicken antibody for in vivo radioimmunodetection and localization of tumors.