Mammalian expression and hollow fiber bioreactor production of recombinantanti-CEA diabody and minibody for clinical applications

Genetically engineered radiolabeled antibody fragments have shown great pro mise for the radioimmunoscintigraphy of cancer. Retaining the exquisite spe cificity of monoclonal antibodies yet smaller in molecular size, antibody f ragments display rapid tumor targeting and blood clearance, a more uniform distribution in the tumor, and present a lower potential to elicit an immun e response. However, one of the factors that has limited clinical evaluatio n of these antibody-derived proteins has been the difficulty in expressing and purifying the quantities necessary for clinical trials. This study outlines the capability of mammalian expression for the producti on of recombinant antibody fragments intended for clinical use. Two anti-ca rcinoembryonic antigen antibody fragments, the T84.66/212 Flex minibody (sc Fv-C-H(3)) and the T84.66 diabody (scFv dimer) have been previously express ed and have shown excellent radioimaging properties in tumor bearing animal s. To proceed toward human studies, these high affinity recombinant fragmen ts and a second minibody version, the T84.66/GS18 Flex minibody, were expre ssed using a high-level mammalian expression system. Production of all thre e antibody fragments in a small-scale hollow fiber bioreactor resulted in 1 37-307 mg of crude antibody harvest. A purification protocol that employed ceramic hydroxyapatite and anion exchange chromatography resulted in 50-150 mg of purified T84.66 diabody and T84.66 minibody. The development of this level of research grade material established conditions for clinical produ ction as well as provided material to complete pre-clinical studies and und ertake protein crystallization studies. Scale-up for clinical studies produ ced 3.4 g of the T84.66 minibody in the harvest. A portion of this material was purified yielding 180 mg of highly purified T84.66 minibody intended f or pilot radioimmunoscintigraphy studies of carcinoembryonic antigen (CEA) positive disease. (C) 2001 Elsevier Science B.V. All rights reserved.