SYNTHESIS AND CHARACTERIZATION OF OLIGOMERIC NIDO-CARBORANYL PHOSPHATE DIESTER CONJUGATES TO ANTIBODY AND ANTIBODY FRAGMENTS FOR POTENTIAL USE IN BORON NEUTRON-CAPTURE THERAPY OF SOLID TUMORS

Antibodies conjugated to oligomeric carboranyl compounds have a high potential as target species for boron neutron capture therapy (BNCT) of solid tumors. As a first step toward developing conjugates with BNCT capabilities, an oligomeric nido-carboranyl phosphate diester (Kane, R. R., Dreschel, K., and Hawthorne, M. F. (1993) J. Am. Chem. Sec. 115, 8853-8854), CB10 (10 nido-carboranes containing 90 boron atoms) with a pseudo-5'-terminal amino group, was conjugated to the anticarcinoembryonic antigen antibody T84.66 and its F(ab') fragment. The homobifunctional linker disuccinimidyl suberate (DSS) was coupled to CB10 via its 5'-terminal amino group followed by removal of excess linker with organic solvent extraction and conjugation with intact antibody. Similarly, the heterobifunctional linker, m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), was coupled to CB10 and conjugated to the hinge region sulfhydryl of the F(ab') fragment of T84.66. The extent of reaction was monitored by the mobility shift of CB10-antibody conjugate on native polyacrylamide gels and the increased susceptibility of the CB10-antibody conjugate to staining with silver nitrate. CB10 was also labeled with radioiodine (I-131) in a solid phase reaction with iodogen and used in double-label studies with I-125-labeled antibody. Although free CB10 bound very tightly to gel filtration media such as Sephadex G-25, the CE10-antibody conjugate passed through freely. After separation of CB10-antibody conjugate from free CB10 on Sephadex G-25, molar incorporations of CB10 were calculated. At a molar ratio of 10:1 (CB10:T84.66), greater than 90% of T84.66 and 30% of its F(ab)' fragment were conjugated to CB10. The amount of CB10 covalently incorporated into mT84.66 ranged from 1.2 to 6.2 (moles per mole), with retention of immunoreactivity in the range of 80-90%. Biodistribution studies in Balb/C mice revealed high uptake of free CB10 or CB10-mT84.66 conjugate in the liver followed by rapid clearance presumably via a dehalogenation or biliary clearance mechanism. Tumor uptake at 48 h was 6.6% ID/g for CB10-mT84.66 conjugate compared to 33% ID/g in mT84.66 controls. These studies demonstrate reliable methods for the routine conjugation of oligomeric nido-carboranyl phosphate diesters to both antibody and antibody fragments, but suggest that the resulting conjugates are captured by the liver rendering them inefficient for tumor-targeting. Current chemical studies are being directed toward the synthesis of a variety of oligomeric carboranyl phosphate diester trailers expected to provide more acceptable biodistributions.