Single-Chain Fv-Streptavidin Substantially Improved Therapeutic Index in Multistep Targeting Directed at Disialoganglioside GD2

T/NT ratio was calculated as follows: (%ID/g in tumor)/(%ID/g in tissue). Athymic nude mice xenografted with established human NB LAN-1 (0.5- to 0.8-cm diameter) were injected intravenously with 5F11-SA, followed in 24 h by intravenous injection of sCA biotin-LC-NM-(Gal-NAc)₁₆, and then 4 h later injected intravenously with ∼2.5 μg ¹¹¹In-DOTA-biotin. Mice were killed 24 h after ¹¹¹In-DOTA-biotin for biodistribution studies. Four dose levels of 5F11-SA were tested: 100, 300, 900, and 2,400 μg (n = 5 mice per group). Dose of sCA was 50% (weight ratio) of 5F11-scFv-SA dose. T/NT ratio was summarized for each organ as arithmetic mean ± SEM.

T/NT ratio was calculated as follows: (%ID/g in tumor)/(%ID/g in tissue). Athymic nude mice xenografted with established human NB LAN-1 (0.5- to 0.8-cm diameter) were injected intravenously with 5F11-SA, followed in 24 h by intravenous injection of sCA biotin-LC-NM-(Gal-NAc)₁₆, and then 3 h later injected intravenously with ∼2.5 μg ¹¹¹In-DOTA-biotin. Fixed dose of 900 μg of 5F11-scFv-SA was used at 5 doses of sCA (n = 4–8 mice per group). Mice were killed 24 h after ¹¹¹In-DOTA-biotin for biodistribution studies. T/NT ratio was summarized for each organ as arithmetic mean ± SEM.

T/NT ratio was calculated as follows: (%ID/g in tumor)/(%ID/g in tissue). Athymic mice with xenografted NB LAN-1 were injected with standard dose of 900 μg of 5F11-scFv-SA, 450 μg of sCA, and ∼2.5 μg of ¹¹¹In-DOTA-biotin. Blood samples were obtained for kinetic studies. At 2, 19, 52, 104, and 144 h, groups of mice (n = 5) were killed and their organ radioactivity was measured. %ID/g for blood and various organs was calculated and T/NT ratio from 2 to 144 h after ¹¹¹In-DOTA-biotin injection was presented for each organ as arithmetic mean ± SEM.

NA = organ masses were not available for these calculations.

Absorbed radiation dose was calculated and is presented as mean ± SD. Model 1 assumes complete local β-ray absorption: Bladder absorbed dose was calculated without using dynamic bladder model. For walled organs (bladder, large intestine, small intestine, stomach), absorbed dose was calculated based on cpm of organs containing their respective contents. Model 2 uses spheric organ mass assumption: For ¹³¹I, there was nearly complete local absorption of β-radiation and correction was not necessary.

↵* Mean ± SEM = arithmetic mean ± SEM across cell lines in each tumor group.

↵^† GD2 (% LAN-1) = relative mean fluorescence, indirect fluorescent staining with anti-GD2 antibody 3F8. It is calculated as mean fluorescence channel of cell line divided by mean fluorescence channel of NB cell line LAN-1.

H = human, M = murine.

Mice (n = 4 or 5 per group) xenografted with GD2-negative human lymphoma (Daudi), leukemia (MOLT-4), colon carcinoma (Colo205), rhabdomyosarcoma (HTB82), and GD2-positive NB (SK-N-LP, SK-N-JD, LAN-1) as well as C57Bl/6 mice autografted with GD2-negative melanoma (B16), and GD2-positive melanomas (B16D14, B16D14Kb, B16D14KbKd) were studied using 900 μg of 5F11-scFv-SA, 450 μg of sCA, and ∼2.5 μg of ¹¹¹In-DOTA-biotin. At 20–28 h, mice were killed and their organ radioactivity was measured. %ID/g for blood and various organs was calculated and T/NT ratio was presented for each organ.