title: Pretargeted radioimmunotherapy with a single-chain antibody/streptavidin construct and radiolabeled DOTA-biotin: strategies for reduction of the renal dose.
abstract: Multistep immune targeting holds great promise for radioimmunodiagnosis and therapy of cancer. Pretargeting of the tetrameric single-chain, variable-fragment streptavidin construct of the tetrameric monoclonal antibody CC49 with subsequent administration of radiolabeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-biotin has yielded promising results in TAG-72-expressing tumor xenograft models. A potential limitation of this approach, however, has been high and prolonged renal uptake of radioactivity. The objective of the current study, therefore, was to evaluate the reduction of kidney uptake of radiolabeled DOTA-biotin achieved by each of 4 different methods.
  
  
  METHODS
  A human pancreatic adenocarcinoma xenograft model (HPAC) in nude mice was used. The animals were intravenously injected with the antibody-streptavidin construct and a synthetic clearing agent (biotinylated N-acetyl-galactosamine) 24 and 4 h, respectively, before the administration of (67)Ga-DOTA-biotin. For reduction of the renal uptake, different groups of mice were treated with streptavidin saturated with biotin, with several administrations of lysine or colchicine or with a succinylated antibody-streptavidin construct (resulting in a decreased electrical charge). All animals were sacrificed 24 h after injection of the (67)Ga-DOTA-biotin for biodistribution and quantitative autoradiography (QAR) studies and selected animals underwent microSPECT/microCT studies.
  
  
  RESULTS
  There was marked targeting of the radiolabeled DOTA-biotin to tumor in all groups except in negative-control animals. Only succinylation of the scFv-CC49-streptavidin fusion protein significantly reduced ( approximately 30%) kidney uptake without affecting tumor activity. QAR corroborated these results and demonstrated that radiolabeled DOTA-biotin localized selectively in the renal cortex. Among the other experimental groups, there was no change in kidney uptake of the radiolabeled biotin.
  
  
  CONCLUSIONS
  In contrast to directly labeled antibodies and antibody fragments, administration of the negatively charged amino acid lysine was largely ineffective in pretargeting strategies with a single-chain-immuno-streptavidin fusion protein. Succinylation of the scFv-CC49-streptavidin construct, on the other hand, reduces kidney uptake of subsequently administered radiolabeled biotin, presumably by inhibiting reuptake of the fusion protein in the proximal renal tubules, and, therefore, could significantly reduce renal doses and improve therapeutic indices associated with multistep immune targeting approaches to radioimmunotherapy.
authors.affiliation: Nuclear Medicine Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. Gregor.Forster@Charite.de
authors.forename: Gregor J
authors.initials: GJ
authors.lastname: Förster
authors.forename: Elmer B
authors.initials: EB
authors.lastname: Santos
authors.forename: Peter M
authors.initials: PM
authors.lastname: Smith-Jones
authors.forename: Pat
authors.initials: P
authors.lastname: Zanzonico
authors.forename: Steven M
authors.initials: SM
authors.lastname: Larson
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000532:radiotherapy*; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000627:therapeutic use; D016503:Drug Delivery Systems / Q000379:methods*; D005260:Female; D005710:Gallium Radioisotopes / Q000493:pharmacokinetics* / Q000627:therapeutic use; D007128:Immunoglobulin Fragments / Q000627:therapeutic use; D007668:Kidney / Q000000981:diagnostic imaging / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009928:Organ Specificity; D009942:Organometallic Compounds / Q000493:pharmacokinetics* / Q000627:therapeutic use; D016499:Radioimmunotherapy / Q000379:methods*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics / Q000627:therapeutic use; D011879:Radiotherapy Dosage; D019809:Streptavidin / Q000493:pharmacokinetics / Q000627:therapeutic use; D014018:Tissue Distribution; D014915:Whole-Body Counting
chemical_list: C405616:DOTA-biotin; D005710:Gallium Radioisotopes; D007128:Immunoglobulin Fragments; D009942:Organometallic Compounds; D019275:Radiopharmaceuticals; D001710:Biotin; D019809:Streptavidin