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 p otential 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 anticarcinoemb ryonic antigen antibody T84.66 and its F(ab') fragment. The homobifunc tional linker disuccinimidyl suberate (DSS) was coupled to CB10 via it s 5'-terminal amino group followed by removal of excess linker with or ganic solvent extraction and conjugation with intact antibody. Similar ly, the heterobifunctional linker, m-maleimidobenzoyl-N-hydroxysuccini mide (MBS), was coupled to CB10 and conjugated to the hinge region sul fhydryl of the F(ab') fragment of T84.66. The extent of reaction was m onitored by the mobility shift of CB10-antibody conjugate on native po lyacrylamide gels and the increased susceptibility of the CB10-antibod y conjugate to staining with silver nitrate. CB10 was also labeled wit h radioiodine (I-131) in a solid phase reaction with iodogen and used in double-label studies with I-125-labeled antibody. Although free CB1 0 bound very tightly to gel filtration media such as Sephadex G-25, th e CE10-antibody conjugate passed through freely. After separation of C B10-antibody conjugate from free CB10 on Sephadex G-25, molar incorpor ations 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 conju gated to CB10. The amount of CB10 covalently incorporated into mT84.66 ranged from 1.2 to 6.2 (moles per mole), with retention of immunoreac tivity 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 liv er followed by rapid clearance presumably via a dehalogenation or bili ary clearance mechanism. Tumor uptake at 48 h was 6.6% ID/g for CB10-m T84.66 conjugate compared to 33% ID/g in mT84.66 controls. These studi es demonstrate reliable methods for the routine conjugation of oligome ric nido-carboranyl phosphate diesters to both antibody and antibody f ragments, but suggest that the resulting conjugates are captured by th e liver rendering them inefficient for tumor-targeting. Current chemic al studies are being directed toward the synthesis of a variety of oli gomeric carboranyl phosphate diester trailers expected to provide more acceptable biodistributions.