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

Abstract

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. Soc. 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 (131I) in a solid phase reaction with iodogen and used in double-label studies with 125I-labeled antibody. Although free CB10 bound very tightly to gel filtration media such as Sephadex G-25, the CB10-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Homogeneous immunoconjugates for boron neutron-capture therapy: Design, synthesis, and preliminary characterization

“…1). Compounds based on this structure have been used in antibody modification experiments in our laboratory (15). The second boron-rich oligophosphate used in this study, designated (G4) 5 , contains monosubstituted nido-carboranes attached to the oligophosphate backbone via a short ether-based tether.…”

“…The homogeneous boron-rich oligophosphate reagents are extremely hydrophilic and can be fitted readily with unique reactive sites (including primary amino groups and thiol residues). Experiments directed toward the sitespecific modification of immunoproteins with these reagents were focused initially on their conjugation to thiol residues on F(abЈ) fragments derived from intact IgG molecules (15). Unfortunately, both the production and conjugation of F(abЈ) immunoprotein fragments have proven to be problematic in our hands, prompting our search for alternative immunoprotein carriers and conjugation methods.…”

Recent Advances in the Synthesis of High Boron-Loaded Nucleic Acids for BNCT

“…An interesting class of high boron-loaded carriers was proposed on the basis of oligomeric phosphate diesters with a regular NA-like backbone, in which acyclic diol replaces the sugar moiety, and carborane cages stand instead of nucleobases (Kane et al, 1993a;Kane et al, 1993b;Chen et al, 1994;Drechsel et al, 1994;Kane et al, 1996;Guan et al, 1998;Nakanishi et al, 1999;Lesnikowski et al, 2004) (Figure 2). In common with oligonucleotides, oligophosphates are produced mainly by a solid-phase phosphoramidite method.…”

Synthesis and screening of bifunctional radiolabelled carborane-carbohydrate derivatives

“…Iodinated nido-carborane derivatives are attractive platforms for developing molecular imaging probes because the B-I bond formed is more robust than aryl C-I bonds, [22][23][24] thereby curtailing de-iodination in vivo. Not surprisingly, there has been an increase in the number of groups developing targeted radiopharmaceuticals using radiohalogenated nido-carboranes [21,22,[24][25][26][27][28][29][30][31][32][33][34][35].…”