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)

This study investigates the influence of antibody protein dose on the radioimmunodetection of a CEA-producing, human colonic tumor xenograft (GW-39) using affinity-purified goat anti-carcinoembryonic antigen (CEA) antibody and a murine monoclonal anti-CEA antibody (NP-2). Hamsters bearing GW-39 tumors were given an equal mixture of 131I-labelled antibody and 125I-labelled irrelevant IgG at doses varying from 0.01 to 1.0 mg (0.1 to 10 mg/kg body weight) for each antibody preparation. No differences were found in the percentage of antibody in the tumor or the clearance rate from the blood as the antibody dose was increased. The concentration of antibody in the tumor increased proportionally with the antibody dose and maintained a linear relationship with increasing dose, indicating that antigenic sites in the tumor were not saturated with antibody. The concentration of irrelevant IgG in the tumor also increased proportionally as the dose was increased, but the concentration of irrelevant IgG in the tumor was less than the antibody and was removed more rapidly from the tumor than the antibody. By considering the amount of irrelevant IgG in the tumor as a measure of the amount of antibody non-specifically bound, we determined that there was no change in the amount of antibody in the tumor between days 3 and 7, and the amount of antibody increased uniformly for both the tumor and non-tumor tissues, resulting in no improvement in the tumor/non-tumor ratios with increasing antibody dose. External scintigraphy verified that the dose of the antibody did not influence tumor imaging. Thus, tumor accretion of antibody in this model is not dependent principally on the antibody protein dose, and other factors such as accessibility to antigen and antibody metabolism may play important roles in regulating the uptake of antibody in tumor.

Boron neutron capture therapy, a binary form of cancer treatment, has the potential to deliver potent cytotoxic radiation to tumor cells with minimal collateral damage to normal tissues if methods for the selective accretion of elevated concentrations of boron-10 in tumor can be developed. In this regard, a monoclonal antibody with dual specificity, for both anionic boron cluster compounds (nido-carboranes) and a tumor-associated antigen (carcinoembryonic antigen, CEA), was produced. The specific binding of a nido-carborane to CEA-expressing tumor cells was achieved using this bispecific antibody. The ability of this bispecific antibody to concentrate selectively at tumor sites in vivo has also been demonstrated, thus suggesting its potential for sequestering boron-rich compounds in tumors.

A series of 14 monoclonal anti-carcinoembryonic antigen (CEA, Mr 180,000) antibodies (MAbs) that show a strong degree of selective reactivity for human colon carcinomas versus normal adult tissues were used to construct a serological map of the CEA molecule. The MAbs were generated using extracts of colon carcinomas as immunogen and are thus given a COL designation. None of the 14 COL-MAbs tested were reactive with purified non-specific cross-reacting antigen (NCA, Mr 55,000) from normal lung, although some showed reactivity to human granulocytes. All the COL-MAbs tested were reactive with normal fecal antigen-2 (NFA-2, Mr 170,000); however, many of the COL-MAbs demonstrated a higher affinity constant to CEA than to NFA-2. Cross-competition radioimmunoassays classified the 14 COL-MAbs into 5 groups. The chemical nature of the COL-binding domains was tested using chemically or enzymatically treated CEA; all reacted with periodate-treated CEA and deglycosylated CEA, indicating that the COL-reactive epitopes appear to be of a proteinaceous nature. Heat treatment, reduction, alkylation, pepsin digestion or pronase treatment of CEA, however, gave differential results with respect to COL binding. Antibody titration experiments were carried out to define differential reactivities to colorectal carcinoma versus NCA-containing granulocyte extracts; these results were compared with results obtained using several anti-CEA MAbs that have been used in clinical trials. Granulocyte binding and biochemical studies showed that the COL MAbs may distinguish as many as 7 to 10 CEA epitopes.