Single-chain Fv-streptavidin substantially improved therapeutic index in multistep targeting directed at disialoganglioside GD2.

UNLABELLED: Multistep targeting can improve the therapeutic index of antibody-based targeting, particularly relevant to pediatric tumors where acute toxicity and late effects of treatment are major concerns. Neuroblastoma is uniquely suited for such investigations because of its abundance of surface ganglioside GD2. METHODS: 5F11scFv (scFv = single-chain variable fragment) was constructed from the variable regions of the heavy (V(H)) and kappa-light (V(L)) chain complementary DNA (cDNA) of anti-GD2 IgM hybridoma 5F11 and ligated to full-length streptavidin cDNA for expression in Escherichia coli. Purified 5F11-scFv-streptavidin (5F11-scFv-SA) was a homotetramer and showed comparable avidity to 5F11 IgM and a 30-fold improvement over monomeric scFv. Biodistribution of 5F11-scFv-SA was studied in nude mice xenografted with neuroblastoma LAN-1. Twenty-four hours after intravenous injection of 300-900 microg 5F11-scFv-SA, 150-450 microg of a thiogalactoside-containing clearing agent, (Gal-NAc)(16)-alpha-S-C(5)H(10)-NH-LC-N-Me-biotin (molecular weight, 8652), were administered intravenously, followed by approximately 2.5 microg (1.85-3.7 MBq) (111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin ((111)In-DOTA-biotin) intravenously 4 h later and clocked as time 0. RESULTS: Tumor uptake (percentage of injected dose per gram [%ID/g]) at 2 h was 7 %ID/g and decayed with a half-life of 72 h, whereas blood %ID/g rapidly decreased to <1/500 of that of tumor after the first 24 h. The tumor-to-nontumor (T/NT) ratio at 72 h was high (median, 106; range, 3.4 [kidney] to 1660 [blood]). When the area under the radioactivity curve was computed, the T/NT organ ratio was favorable (4.8 for kidney and 162 for blood). When human and murine tumors were surveyed, the T/NT ratio of (111)In-DOTA-biotin uptake correlated with their levels of GD2 expression as assayed by flow cytometry. Biotinylated polypeptides (bovine serum albumin and vasointestinal peptides) achieved selective tumor targeting when the multistep strategy was applied. CONCLUSION: Improvement in the T/NT ratio using pretargeting strategy may increase the efficacy and safety of scFv-based approaches in cancer therapy. Additionally, since biotinylated polypeptides can be rendered tumor selective, a large repertoire of agents can potentially be explored.

Pretargeting radioimmunotherapy of a murine model of adult T-cell leukemia with the alpha-emitting radionuclide, bismuth 213.

We used a pretargeting technique to treat a nonobese diabetic/severe combined immunodeficient murine model of human adult T-cell leukemia with an anti-Tac antibody-streptavidin (HAT-SA) conjugate, which recognizes CD25, followed by bismuth 213 ((213)Bi)-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)- biotin. In the 3-step pretargeting radioimmunotherapy protocol, HAT-SA (140 or 400 microg) was administered intravenously (i.v.) to bind to the interleukin 2 receptor alpha (IL-2R alpha; CD25)-expressing tumor cells. After 24 hours, 100 microg of a synthetic clearing agent was administered i.v. to remove unbound circulating HAT-SA conjugate from the circulation. Four hours later, (213)Bi-DOTA-biotin was administered i.v. for therapy. Tumor growth was significantly inhibited in 3 trials by using 250 microCi (9.25 MBq) of (213)Bi-DOTA-biotin with a pretargeting technique as monitored by serum levels of soluble IL-2R alpha and/or human beta-2-microglobulin (P <.05, t test) and by survival of tumor-bearing mice in the treatment groups (P <.02, log rank test) as compared with the control groups. No prolongation of survival was observed with a nonspecific antibody-SA conjugate or in the absence of the radionuclide. Additionally, no prolongation of survival resulted from administration of (213)Bi directly linked to intact HAT. Furthermore, there was no prolongation of survival when the beta-emitting radionuclide yttrium 90 instead of the alpha-emitting radionuclide (213)Bi was used. The pretargeting approach with (213)Bi inhibited tumor growth more effectively than did immunotherapy with unmodified HAT. The best results were obtained with combination therapy that involved (213)Bi-DOTA-biotin with a pretargeting technique supplemented by 4 weekly doses of HAT. The findings of this study support the use of this combination approach in a clinical trial in patients with IL-2R alpha-expressing leukemias.