Improved biodistribution and radioimmunoimaging with poly(ethylene glycol)-DOTA-conjugated anti-CEA diabody

Diabodies are single chain antibody fragments (scFvs) that spontaneously form bivalent dimers of molecular size 50-55000. Radiolabeled diabodies are almost ideal tumor targeting agents due to their high avidity (bivalent) binding to tumor antigens and small size (50-55000) that leads to improved tumor-to-blood ratio compared to intact antibodies (150000). However, due to their high retention and metabolism in the kidney, radioiodine is the current radiolabel of choice for diabodies since radioiodine is rapidly excreted from the kidney once metabolized. We have previously shown that In-111-DOTA-diabody gives higher tumor uptake in nude mouse xenografts than I-125-diabody, but has extremely high kidney retention since its In-111-labeled metabolites are retained by and only slowly excreted from the kidney. When a diabody is conjugated to a bifunctional PEG-3400 derivative followed by reaction with cysteinyl-DOTA, the resulting product has an apparent molecular size of 75000 and a Stokes radius of 35 angstrom on size exclusion chromatography, compared to a Stokes radius of 25 angstrom for intact diabody. When radiolabeled, the conjugate gives high yields of In-111-labeled product, retains high immunoreactivity, and gives improved biodistributions (30-40%ID/g, 12-48 h) compared to In-111-DOTA-diabody (12-13%ID/g, 6-12 h). We show that the improved biodistribution is due to an increase in Stokes radius caused by the linear PEG-3400 since conjugation of diabody with multiple (PEG)(12) linkers followed by reaction with cysteinyl-DOTA does not reduce kidney accumulation. We also show that In-111-cysteinyl-DOTA-PEG3400-diabody gives excellent tumor images in the nude mouse xenograft model and that I-125-PEG3400-diabody gives equivalent images to I-125-minibody (molecular size, 80000), but improved tumor-to-liver ratios, suggesting that this imaging agent can be used to image liver metastases.