Preparation and characterization of anti-tenascin monoclonal antibody-streptavidin conjugates for pretargeting applications

Radioimmunopretargeting is based on the separate injection of a modified mA b and the radionuclide and most frequently exploits the very high avidity o f biotin for streptavidin (SA). Currently, we are evaluating the therapeuti c potential of directly labeled monoclonal antibody (mAb) 81C6, reactive wi th the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this s etting, the synthesis of 81C6 mAb-SA conjugates was required. In the curren t study, we have evaluated five methods for preparing both murine 81C6 (m81 C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yiel d, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C 6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-3-acetylthioacetate, dithiothreito l or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulf osuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-su ccinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA als o was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer , as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate wit h excellent biotin-binding capacity and immunoreactivity. The (I-125-labele d m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity af ter a 72 h incubation in human glioma cyst fluid in vitro. Although the con jugate was stable in murine serum in vivo, its biotin-binding capacity decl ined rapidly, consistent with high endogenous biotin levels in the mouse. A fter injection of the radioiodinated conjugate into athymic mice with subcu taneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.