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


CONCLUSIONS
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.
Cheung
Nai-Kong V
NK
Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. cheungn@mskcc.org
Modak
Shakeel
S
Lin
Yukang
Y
Guo
Hongfen
H
Zanzonico
Pat
P
Chung
John
J
Zuo
Yuting
Y
Sanderson
James
J
Wilbert
Sibylle
S
Theodore
Louis J
LJ
Axworthy
Donald B
DB
Larson
Steven M
SM
D000818:Animals; D005732:Gangliosides / Q000378:metabolism*; D006801:Humans; D007205:Indium Radioisotopes; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D016513:Mice, SCID; D009374:Neoplasms, Experimental / Q000628:therapy*; D019809:Streptavidin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D005732:Gangliosides; D007205:Indium Radioisotopes; C025447:sialogangliosides; D019809:Streptavidin