A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy

Single-chain Fv antibody fragments from the CD20-specific murine monoclonal antibody B9E9 were genetically engineered as streptavidin fusions [single- chain Pv-streptavidin (scFvSA) fusion protein] for use in pretargeted radio immunotherapy, The scFvSA constructs were expressed as soluble, tetrameric species in the periplasm of Escherichia coil, Expression Levels were affect ed by the order of the variable regions and the length and composition of t he single-chain Fv linker. The best expressor was obtained with the variabl e regions in the heavy chain-light chain configuration separated by a 25-me r Gly(4)Ser linker, This construct produced 250-300 mg of soluble, tetramer ic fusion protein per liter of fermenter culture. The fusion protein (M-r 1 73,600) was purified from crude lysates by iminobiotin affinity chromatogra phy with an coverall yield of about 50% and was analyzed for functionality both in vitro and in vivo. Immunoreactivity of the scFvSA fusion protein an d its nanomolar affinity to CD20-positive Ramos cells were comparable with the B9E9 monoclonal antibody. The fusion protein had a biotin dissociation rate identical to recombinant streptavidin and bound an average of 3.6 biot ins/molecule of a possible 4 biotins/molecule, Labeled fusion protein clear ed from the blood of BALB/c mice with a beta half-life of about 16 h, In nu de mice bearing Ramos xenografts, the fusion protein demonstrated sufficien t tumor localization of functional streptavidin to enable efficient, tumor- specific targeting of a subsequently administered radionuclide-chelate/ bio tin molecule. These results suggest that large quantities or functional scF vSA can be produced for clinical testing as a therapy for non-Hodgkin's lym phoma.