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

The application of immunoprotein-based targeting strategies to the boron neutron-capture therapy of cancer poses an exceptional challenge, because viable boron neutron-capture therapy by this method will require the efficient delivery of 103 boron-10 atoms by each antigen-binding protein. Our recent investigations in this area have been focused on the development of efficient methods for the assembly of homogeneous immunoprotein conjugates containing the requisite boron load. In this regard, engineered immunoproteins fitted with unique, exposed cysteine residues provide attractive vehicles for site-specific modification. Additionally, homogeneous oligomeric boron-rich phosphodiesters (oligophosphates) have been identified as promising conjugation reagents. The coupling of two such boron-rich oligophosphates to sulfhydryls introduced to the CH2 domain of a chimeric IgG3 has been demonstrated. The resulting boron-rich immunoconjugates are formed efficiently, are readily purified, and have promising in vitro and in vivo characteristics. Encouragingly, these studies showed subtle differences in the properties of the conjugates derived from the two oligophosphate molecules studied, providing a basis for the application of rational design to future work. Such subtle details would not have been as readily discernible in heterogeneous conjugates, thus validating the rigorous experimental design employed here.

Synthesis and in vivo murine evaluation of Na4[1-(1′-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy

Reaction of the normal isomer of [B20H18]2− and the protected thiol anion, [SC(O)OC(CH3)3]−, produces an unexpected isomer of [B20H17SC(O)OC(CH3)3]4− directly and in good yield. The isomer produced under mild conditions is characterized by an apical–apical boron atom intercage connection as well as the location of the thiol substituent on an equatorial belt adjacent to the terminal boron apex. Although the formation of this isomer from nucleophilic attack of the normal isomer of [B20H18]2− has not been reported previously, the isomeric assignment has been unambiguously confirmed by one-dimensional and two-dimensional 11B NMR spectroscopy. Deprotection of the thiol substituent under acidic conditions produces a protonated intermediate, [B20H18SH]3−, which can be deprotonated with a suitable base to yield the desired product, [B20H17SH]4−. The sodium salt of the resulting [B20H17SH]4− ion has been encapsulated in small, unilamellar liposomes, which are capable of delivering their contents selectively to tumors in vivo, and investigated as a potential agent for boron neutron capture therapy. The biodistribution of boron was determined after intravenous injection of the liposomal suspension into BALB/c mice bearing EMT6 mammary adenocarcinoma. At low injected doses, the tumor boron concentration increased throughout the time-course experiment, resulting in a maximum observed boron concentration of 46.7 μg of B per g of tumor at 48 h and a tumor to blood boron ratio of 7.7. The boron concentration obtained in the tumor corresponds to 22.2% injected dose (i.d.) per g of tissue, a value analogous to the most promising polyhedral borane anions investigated for liposomal delivery and subsequent application in boron neutron capture therapy.

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco1

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency1

The mobility of elements within plants contributes to a plant species' tolerance of nutrient deficiencies in the soil. The genetic manipulation of within-plant nutrient movement may therefore provide a means to enhance plant growth under conditions of variable soil nutrient availability. In these experiments tobacco (Nicotiana tabacum) was engineered to synthesize sorbitol, and the resultant effect on phloem mobility of boron (B) was determined. In contrast to wild-type tobacco, transgenic tobacco plants containing sorbitol exhibit a marked increase in within-plant B mobility and a resultant increase in plant growth and yield when grown with limited or interrupted soil B supply. Growth of transgenic tobacco could be maintained by reutilization of B present in mature tissues or from B supplied as a foliar application to mature leaves. In contrast, B present in mature leaves of control tobacco lines could not be used to provide the B requirements for new plant growth. 10B-labeling experiments verified that B is phloem mobile in transgenic tobacco but is immobile in control lines. These results demonstrate that the transgenic enhancement of within-plant nutrient mobility is a viable approach to improve plant tolerance of nutrient stress.

The Boron Requirement and Cell Wall Properties of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells1

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (≥7 μm) to the boron-deficient cells. C. album cells grown in the presence of 100 μm boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 μm boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 μm boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.

Preparation and properties of nido-carborane-specific monoclonal antibodies for potential use in boron neutron capture therapy for cancer.

As the first step of a research program aimed at developing a bispecific monoclonal antibody system for the delivery of boron-rich molecules to tumor cells for boron neutron capture therapy, monoclonal antibodies (mAbs) were produced against an anionic nido-carborane derivative, 4-[7,8-dicarbadodecahydroundecaborat(-1)-7-yl]butanoic acid. Two IgG subclass mAbs, designated HAW101 and HAW102, were identified that specifically bound the anionic nido-carborane hapten, as well as a variety of other anionic nido-carborane cage derivatives. By using surface plasmon resonance technology, the affinity constants of HAW101 and HAW102 were determined to be 1.9 x 10(9) and 6.8 x 10(8) M-1, respectively. A diverse array of 7-substituted and 7,8-disubstituted anionic nido-carborane derivatives reacted with the mAb HAW101 in competition ELISA, whereas anionic closo-polyhedral boranes showed negligible binding, suggesting a role for the open nido-carborane cage structure. These results suggest that mAbs such as HAW101, which bind anionic nido-carboranes, are useful in the development of bispecific mAbs for specific targeting and enhanced boron delivery to tumor sites.