Comparison of Biodistributions and Therapeutic Efficacies of Pretargeted Radioimmunoconjugates Targeting the CD20, CD22, and DR Molecules on Human B Cell Lymphomas.

Radioimmunotherapy (RIT) using anti-CD20 monoclonal antibodies (Ab) produces response rates of 60–95% in relapsed non-Hodgkin’s lymphoma (NHL) patients; however, tumor-to-normal organ ratios of absorbed radiation are low and many patients relapse. The efficacy of RIT is limited by non-specific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled antibodies. Pretargeted RIT (PRIT) using streptavidin (SA)-Ab conjugates followed by a clearing agent and radiolabeled biotin can augment the efficacy of RIT and decrease toxicity compared with conventional RIT. Although PRIT using anti-CD20-SA Abs have been studied with promising results, targeting multiple antigens may increase efficacy. Since successful clinical trials have been conducted with directly radiolabeled anti-DR and anti-CD22 Abs, we initiated in vitro and in vivo studies comparing pretargeted anti-CD20 Ab-SA conjugate (1F5/SA) with pretargeted anti-CD22-SA (HD39/SA) and anti-HLA-DR-SA (Lym-1/SA) conjugates in three different human B-lymphoma cell lines, RAMOS (Burkitt), RAJI (Burkitt) and FL-18 (transformed follicular). Using standard flow cytometry techniques all three Ab-SA conjugates bound to ≥ 97% of FL18 cells. Cell binding for 1F5/SA, Lym-1/SA, and HD39/SA was 99%, 99%, and 83% to RAJI cells, respectively, and 99%, 22%, and 85% to RAMOS cells. The blood half-life of each conjugate in vivo was measured by injecting groups of 4 mice i.v. with 0.7nmol (150μg) of 125I labeled Ab-SA conjugate and drawing 10μl of blood at various time points to determine the percent injected dose per gram (% ID/g). The half-lives of 1F5/SA, Lym-1/SA and HD39/SA were 18.38, 14.92 and 16.23 hours, respectively. When 5.8nmol (50μg) of a clearing agent (synthetic biotin-N-acetyl-galactosamine) was given 24 hours post 125I-Ab-SA injection, the % ID/g in blood fell by more than 80% of the initial dose within a half-hour. Blood, tumor and non-specific organ uptake was determined by biodistribution experiments in mice (Balb/c nu/nu) bearing human lymphoma xenografts. Athymic mice with s.c. RAMOS, RAJI, or FL-18 xenografts received 1.4nmol (300μg) of either 1F5/SA, HD39/SA, or Lym-1/SA i.v. followed 24 hours later by 5.8nmol (50μg) clearing agent to remove non-localized conjugate from circulation, and 3 hours later by an 111In labeled DOTA-biotin ligand (1μg). The biodistributions of each conjugate were evaluated by sacrificing mice at 24 and 48 hours after 111In-DOTA-biotin. At 24 hours, the ID/g was 18.2±13.6% in FL18 xenografts for pretargeted Lym-1/SA, 18.2±17.4% ID/g for 1F5/SA and 3.3±0.7% ID/g for HD39/SA. Conversely, at 24 hours pretargeted Lym-1/SA uptake in RAJI tumors was 10.8±2.1% ID/g, and 1F5/SA and HD39/SA RAJI tumor localization was 5.2±1.9% ID/g and 2.2±0.5% ID/g. respectively. 1F5/SA had superior uptake (7.1±3.3% ID/g) in RAMOS xenografts compared with Lym-1/SA (3.5±1.5% ID/g) and HD39/SA (2.7±1.0% ID/g). These data suggest a strong correlation between in vitro cell binding results and in vivo biodistributions for all three Ab-SA conjugates in all three human lymphoma cell lines. Using these agents in combination may result in a synergistic effect that has the potential to increase the efficacy of PRIT over using any one of the agents alone. Biodistribution and therapy studies using the Ab-SA conjugates in combination in tumored mice are on-going.