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Bioconjugate Chem. 1996, 7, 532−535
Bispecific Antibody Mediated Targeting of nido-Carboranes to Human Colon Carcinoma Cells F. James Primus,† Roger H. Pak,‡ Karen J. Rickard-Dickson,† Gyo¨rgy Szalai,† James L. Bolen, Jr.,§ Robert R. Kane‡,| and M. Frederick Hawthorne*,‡ Divisions of Immunology and Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569. Received May 31, 1996X
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.
Boron neutron capture therapy (BNCT) is a binary approach to cancer therapy that is based upon the capture of biologically innocuous low-energy neutrons by the nonradioactive isotope boron-10, which results in the formation of energetic, localized, and extremely cytotoxic He and Li nuclei. Successful cancer treatment with BNCT requires the development of boron-containing species that concentrate selectively in tumors, to minimize radiation damage to normal cells. Boron-substituted compounds that may be able to distinguish between normal and neoplastic tissues include boronated porphyrins, nucleosides, amino acids, liposomes, and monoclonal antibodies (MAb) (1). Immunoproteins have been proposed as a general class of boron-delivery agents. Although we (2-4) and others (5, 6) have successfully synthesized boron-rich immunoconjugates that retain immunoreactivity, the in vivo delivery of these conjugates to tumor has been poor, primarily as a result of significant nonspecific uptake in liver. This problem is exacerbated by the requirement that each immunoprotein molecule be conjugated to a large number of boron-10 atoms (on the order of 103) to attain clinically useful tumor-boron concentrations (1). Our own experience has highlighted some of the problems in this area while offering potential solutions to the problem of obtaining high-substitution levels of boron conjugated to antibody molecules (7). Bispecific antibodies (BsMAb) offer an alternative means for site-directed boron-targeting. This approach to immunoprotein-mediated BNCT requires the creation of an antibody with affinity for both a tumor-associated antigen and a hapten found on a boron-rich macromolecule (Figure 1). The BsMAb is initially pretargeted to * Author to whom correspondence should be addressed [telephone (310) 825-7378; fax (310) 825-5490; e-mail mfh@ chem.ucla.edu]. † Division of Immunology, Beckman Research Institute. ‡ University of California. § Division of Biology, Beckman Research Insitute. | Present address: Department of Chemistry, Baylor University. X Abstract published in Advance ACS Abstracts, August 15, 1996.
S1043-1802(96)00050-X CCC: $12.00
Figure 1. Schematic diagram demonstrating the principle of pretargeting with BsMAbs. The antibody is first pretargeted to the tumor by its reaction with a tumor cell surface antigen. The other antigen combining site is then in position to react with a boron-rich macromolecule that is subsequently administered.
tumor sites by virtue of its tumor antigen specificity. A boron-rich macromolecule is then administered and is retained in the tumor by virtue of its binding to the antihapten specificity provided by the tumor-bound BsMAb. The use of BsMAbs for the delivery of diagnostic and therapeutic agents has received heightened interest because of the potential for improving tumor-to-normal tissue differentiation in agent distribution (8, 9). A distinct advantage of BsMAbs when the goal is to deliver elevated quantities of a cytotoxic drug is that modification of antibody caused by direct conjugation methods can be avoided. This feature is of paramount importance in BNCT, since the need to localize high concentrations of boron requires that each antibody molecule deliver hundreds of boron atoms. We initially turned our attention to the creation of an antibody with binding specificity for derivatives of the readily accessible anionic boron-rich cage, nido-7,8dicarbadodecahydroundecaborate(-1) (1, Figure 2). Much of our previous work in this area has utilized derivatives of this nido-carborane cage, a stable negatively charged © 1996 American Chemical Society
Communications
Figure 2. Structures of nido-carboranes and closo-polyhedral borane anions used in the production and characterization of the anti-nido-carborane MAbs.
ion composed of nine B-H and two C-H vertices (along with a B-H-B bridging hydrogen) with the structure of an icosahedral fragment. Importantly, this cage structure has been fashioned into designed boron-rich macromolecules, including oligomeric peptides (10, 11) and phosphate diesters (12, 13). We previously reported the production and characterization of two anti-nido-carborane MAbs, designated HAW101 and HAW102, which were generated by immunization with a nido-carborane attached to keyhole limpet hemocyanin (KLH) carrier protein (14) (nido-KLH; 2, Figure 2). The affinity constants of HAW101 and HAW102 with a nido-carborane attached to bovine serum albumin (nido-BSA; 3, Figure 2) were determined by surface plasmon resonance technology to be 1.9 × 109 and 6.8 × 108 M-1, respectively. The HAW101 MAb was selected for further study because of its higher affinity. Its fine specificity was characterized by examining a panel of boron hapten containing compounds in competition assay (14). The parent unsubstituted nido-carborane 1 (Figure 2) effectively inhibited HAW101 binding to nido-BSA at nanomolar concentrations, demonstrating that the antibody has specificity for the nido-carborane cage structure rather than for side chains used for its attachment to protein carriers. Anionic closo-polyhedral boranes (4 and 5, Figure 2) were not effective inhibitors, indicating the importance of the open nido-carborane cage structure for recognition by the HAW101. By contrast, Liu et al. have produced MAbs reactive with anionic closo-polyhedral boranes for the development of BsMAbs for use in BNCT (15). We selected the carcinoembryonic antigen (CEA) as the target antigen for the tumor specificity component of BsMAbs also possessing nido-carborane reactivity. The CEA is a 180 kDa glycoprotein that is produced by a variety of epithelial-derived cancers including colorectal, lung, ovarian, and breast carcinomas (16). MAbs against CEA have been under extensive clinical evaluation for their ability to target radionuclides and cytotoxic agents for immunodiagnosis and therapy of cancer (17). Other groups have developed CEA-targeted BsMAbs for the tumor-selective delivery of metal chelates (18, 19) or toxins (20). In our studies, the high-affinity anti-CEA MAb, T84.66, which has been shown to localize tumors in human patients, was selected for the preparation of BsMAbs (21). Among the various methods for the preparation of BsMAbs, we chose to create quadromas via the fusion of two hybridomas each synthesizing MAbs with different
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specificities (22). Hybridomas producing the T84.66 antiCEA and HAW101 anti-nido-carborane MAbs were labeled with contrasting lipophilic chromophores and fused with polyethylene glycol.1 Fused cells, or “quadromas”, were separated by fluorescence-activated cell sorting, and the selected cells were grown in culture and cloned. Supernatants from clones were first tested for reactivity with CEA and nido-BSA in separate enzyme immunoassays. In three different cell-sorted fusions, 4-6% (15 total) of the wells positive for cell growth produced supernatants that reacted in both assays. The majority (41-76%) of the clones showed reactivity with only one antigen. A bridging enzyme immunoassay, which assayed for the ability of BsMAbs to link a biotinylated antiCEA idiotype antibody to wells coated with nido-BSA, was used to identify BsMAb activity in clones reactive with both antigens. Only 3 of the 15 clones positive in both individual antigen assays showed reactivity in the bridging assay (Figure 3A). The reason for the high percentage of clones that displayed no reactivity in the bridging assay is unclear, but it may be due to low levels of BsMAb production or to clones that still contained a mixture of the parental hybridomas. The former explanation seems most likely since antibody purified by protein G chromatography from two of the clones had very modest bispecific activity, suggesting that the parental antibodies were the predominant species synthesized by these clones. Nonetheless, antibody from the third clone, H11, contained substantial BsMAb activity. BsMAbs are monovalent toward a given specificity, while their respective parents are bivalent. Since antibody avidity is influenced by the antibody valency, it was important to establish that the BsMAb, like the anti-CEA MAb parent, was capable of localizing to tumor sites in vivo. To remove the parental MAbs produced by the H11 clone, further purification of BsMAb was carried out by ion exchange chromatography,2 as the isoelectric points for the T84.66 and HAW101 parental MAbs were quite different (approximately 5.4 and 7.0, respectively). For localization experiments in vivo, radioiodinated BsMAb and anti-CEA MAb were separately injected into athymic nude mice bearing LS-174T colon carcinoma xenografts. At various time points after injection, tumor as well as normal reference tissues (blood, liver, spleen, kidney, lung) were removed and the antibody uptake was measured. At 24 h the uptake of the BsMAb in tumor was similar to that of the parental anti-CEA MAb (Figure 3B). With time, the amount of radioactivity in tumors from animals injected with the BsMAb decreased at a faster rate than that observed in the group administered the anti-CEA MAb. The biodistribution and clearance patterns among the reference tissues were similar for both groups at the various time points. Thus, the BsMAb retains the tumor-localizing properties of the parental 1 The T84.66 anti-CEA and HAW101 anti-nido-carborane hybridomas were labeled with fluorescent cell linkers PKH26GL (red) and PKH2-GL (green), respectively, according to the manufacturer’s instructions (Zynaxis Cell Sciences, Malvern, PA). The optimal staining concentrations of PKH26-GL and PKH2-GL were 20 and 8 µM, respectively. After culturing overnight, the labeled hybridoma cells were fused with PEG and then cultured overnight. Prior to cell sorting, dead cells were removed by centrifugation over ficoll-hypaque. Cells were sorted under sterile conditions using a FACS IV. The gating window contained 1.0-1.1% of total cells from fused preparations as compared to 0.2% from a mixture of stained hybridoma cells that were not fused but otherwise treated in the same way. Three sorts were carried out on two separate fusions producing 3000-5000 cells/sort (0.3-0.7% of cells in gated area). Sorted cells were placed in bulk culture for 11-13 days prior to cloning at limiting dilution.
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Figure 3. (A) BsMAb activity detected in bridging enzyme immunoassay. Antibody produced by quadromas was purified from ascites by protein G chromatography and then reacted with microtiter wells coated with nido-BSA followed by biotinylated antiCEA idiotype MAb. Binding of the anti-idiotype MAb was detected with a strepavidin-enzyme conjugate. The reactions of clones B5, D6, and H11 with nido-BSA and of clones B5 and H11 with BSA as negative control are depicted. (B) Localization of radioiodinated BsMAb in LS-174T human colon carcinoma xenografts. Error bars ) SEM. (C) Binding of radioiodinated nido-carborane ([125I]-6) to the surface of human colon carcinoma cells mediated by BsMAb. The results shown are the mean of duplicate tubes that were within 5% of one another. This experiment was repeated a second time with identical results.
anti-tumor MAb, although its residence time appears to be somewhat altered. The latter should not have a mitigating effect on the pretargeting properties of the BsMAb since the timing of the administration of the boron-rich macromolecule can be adjusted to the time of optimal antibody accretion in the tumor. With these data in hand, experiments were performed to determine if the BsMAb could target nido-carboranes to cancer cells expressing CEA. CEA-positive human colon carcinoma cells (LS-174T) were sequentially exposed to the ion exchange-purified BsMAb followed by a radioidinated nido-carborane compound [125I]-6 (Figure 4).3 The LS-174T colon carcinoma cells showed enhanced binding of [125I]-6 when previously exposed to the BsMAb as compared to the binding obtained after exposure of the cells to a physical mixture of the respective parental antibodies (Figure 3C). By contrast, only background levels of [125I]-6 were bound to the CEA-negative human breast carcinoma cell line, SK-BR-3, following incubation with either the BsMAb or a mixture of the parental 2 BsMAb was purified from ascites by sequential chromatography over protein G and ABx ion exchanger (J. T. Baker, Phillipsburg, NJ). For ABx chromatography, antibody was equilibrated with 0.01 M MES, pH 6.0, and applied to the ABx using the same buffer. After elution of the parental anti-CEA MAb, bound bispecific antibody was eluted using a gradient consisting of 0.01 M NaOAc, pH 6.0, start buffer, and 1.0 M NaOAc, pH 6.0, limit buffer. Parental anti-CEA MAb could not be detected in the purified BsMAb preparation as determined by isoelectric focusing. The immunoreactivity of the labeled (Iodogen method) T84.66 anti-CEA MAb and BsMAb as determined by binding to solid phase CEA was 80-100% as compared to 4-5% binding to an irrelevant antigen affinity gel. Seven days after LS-174T inoculation into athymic nude mice (NCr-nu), 10 µCi of antibody (1 µg) was administered by tail vein injection. Five to six mice were used for each antibody group at each time point. Animals were euthanized at various time points after injection, and blood, tumor, and normal tissues were removed for determination of radioactivity. All biodistribution studies were carried out under approval of the Institutional Research Animal Care Committee. 3 To tubes precoated with 5% BSA, 2 × 106 LS-174T (CEApositive human colon carcinoma cell line) or SK-BR-3 (CEAnegative human breast carcinoma cell line) cells were added (in 50 µL of PBS + 0.02% NaN3) followed by 33.3 pmol of antibody in an equal volume. After incubation for 1 h at 0 °C, unbound antibody was removed and the cells were exposed to nidocarborane 6 (Figure 4; 0.03 pmol), which had been radioiodinated by the chloramine-T procedure (2).
Figure 4. Immunoreactive iodinated (6) and oligomeric (7) nido-carborane derivatives.
antibodies. Of note, the nonspecific binding of [125I]-6 by both tissue-cultured tumor cell lines was very low. These experiments demonstrate for the first time that a BsMAb possessing reactivity with both a human tumor antigen and a boron-containing cluster species can elicit sitedirected boron labeling of the appropriate antigen-positive cells. We have shown that BsMAbs prepared with an antinido-carborane specificity can selectively localize a boronrich compound to a tumor cell surface. It is anticipated that the high-level boron labeling of tumor cells required in vivo for successful BNCT will be accomplished with the use of boron-rich polymers containing appropriate haptenic groups available for specific reaction with BsMAbs. The feasibility of this approach is suggested by the binding of the HAW101 MAb to oligomeric carboranyl phosphate diesters such as the recently synthesized compound 7 (Figure 4). This compound, which contains 90 boron atoms, is bound by the the antinido-carborane MAb (14) substantially better than any monomeric carborane studied to date (23), and we believe that oligomers of greater length will exhibit similar properties. The availability of BsMAbs with nido-carborane binding capacity, coupled with our ability to
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design and assemble boron-rich macromolecules that are reactive with these BsMAbs, suggests that this approach holds great potential for the specific in vivo loading of tumors with boron. To increase the yield, purity, and targeting properties of the BsMAbs, we have recently cloned the genes for the HAW101 MAb for use in the preparation of engineered BsMAbs. ACKNOWLEDGMENT
This research was supported by NIH Grant CA53870 to M.F.H. Additional support was provided by a NIH predoctoral traineeship to R.H.P. (Grant GM08375), a NIH postdoctoral fellowship to R.R.K. (Grant CA09306), and a NIH Cancer Core Grant (CA33572) to the City of Hope. LITERATURE CITED (1) Hawthorne, M. F. (1993) The role of chemistry in the development of boron neutron capture therapy of cancer. Angew. Chem., Int. Ed. Engl. 32, 950. (2) Varadarajan, A., Sharkey, R. M., Goldenberg, D. M., and Hawthorne, M. F. (1991) Conjugation of phenyl isothiocyanate derivatives of carborane to antitumor antibody and in vivo localization of conjugates in nude mice. Bioconjugate Chem. 2, 102. (3) Paxton, R. J., Beatty, B. G., Varadarajan, A., and Hawthorne, M. F. (1992) Carboranyl peptide-antibody conjugates for neutron-capture therapy: preparation, characterization, and in vivo evaluation. Bioconjugate Chem. 3, 241. (4) Chen, C.-J., Kane, R. R., Primus, F. J., Szalai, G., Hawthorne, M. F., and Shively, J. E. (1994) 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. Bioconjugate Chem. 5, 557. (5) Ferro, V. A., Morris, J. H., and Stimson, W. H. (1995) A novel method for boronating antibodies without loss of immunoreactivity, for use in neutron capture therapy. Drug Des. Discovery 13, 13. (6) Barth, R. F., Adams, D. M., Soloway, A. H., Alam, F., and Darby, M. V. (1994) Boronated starburst dendrimer monoclonal antibody immunoconjugatessevaluation as a potential delivery system for neutron capture therapy. Bioconjugate Chem. 5, 58. (7) Hawthorne, M. F. (1991) Biochemical applications of boron cluster chemistry. Pure Appl. Chem. 63, 327. (8) Nolan, O., and O’Kennedy, R. (1990) Bifunctional antibodies: concept, production, and applications. Biochim. Biophys. Acta 1040, 1. (9) Yuan, F., Baxter, L. T., and Jain, R. K. (1991) Pharmacokinetic analysis of two-step approaches using bifunctional and enzyme-conjugated antibodies. Cancer Res. 51, 3119.
(10) Varadarajan, A., and Hawthorne, M. F. (1991) Novel carboranyl amino acids and peptides: reagents for antibody modification and subsequent neutron-capture studies. Bioconjugate Chem. 2, 102. (11) Kane, R. R., Pak, R. H., and Hawthorne, M. F. (1993) Solution-phase segment synthesis of boron-rich peptides. J. Org. Chem. 58, 991. (12) Kane, R. R., Lee, C. S., Drechsel, K., and Hawthorne, M. F. (1993) Solution-phase synthesis of boron-rich oligophosphates. J. Org. Chem. 58, 3227. (13) Kane, R. R., Drechsel, K., and Hawthorne, M. F. (1993) Automated syntheses of carborane-derived homogeneous oligophosphates. J. Am. Chem. Soc. 115, 8853. (14) Pak, R. H., Primus, F. J., Rickard-Dickson, K. J., Ng, L. L., Kane, R. R., and Hawthorne, M. F. (1995) Preparation and properties of nido-carborane-specific monoclonal antibodies for potential use in boron neutron capture therapy (BNCT) of cancer. Proc. Natl. Acad. Sci. U.S.A. 92, 6986. (15) Liu, L., Barth, R. F., Adams, D. M., Soloway, A. H., and Reisfeld, R. A. (1995) Bispecific antibodies as targeting agents for boron neutron capture therapy of brain tumors. J. Hematother. 4, 477. (16) Sikorska, H., Shuster, J., and Gold, P. (1988) Clinical applications of carcinombryonic antigen. Cancer Detect. Prev. 12, 321. (17) Goldenberg, D. M. (1991) Imaging and therapy of gastrointestinal cancers with radiolabeled antibodies. Am. J. Gastroenterol. 86, 1392. (18) Phelps, J. L., Beilder, D. E., Jue, R. A., Unger, B. W., and Johnson, M. J. (1990) Expression and characterization of a chimeric bifunctional antibody with therapeutic applications. J. Immunol. 145 (4), 1200. (19) Stickney, D. R., Anderson, L. D., Slater, J. B., Ahem, C. N., Kirk, G. A., Schweighardt, S. A., and Frincke, J. M. (1991) Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma. Cancer Res. 51, 6650. (20) Corvalan, J. R. F., Smith, W., and Gore, V. A. (1988) Tumour therapy with vinca alkaloids targeted by a hybridhybrid monoclonal antibody recognising both CEA and vinca alkaloids. Int. J. Cancer, Suppl. 2, 22. (21) Beatty, J. D., Williams, L. E., Yamauchi, D., Morton, B. A., Hill, L. R., Beatty, B. G., Paxton, R. J., Merchant, B., and Shively, J. E. (1990) Presurgical imaging with indium-labeled anti-carcinoembryonic antigen antibody for colon cancer staging. Cancer Res. 50, 922s. (22) Karawajew, L., Micheel, B., Behrsing, O., and Gaestel, M. (1987) Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter. J. Immunol. Methods 96, 265. (23) Kane, R. R., Guan, L., Kim, Y. S., Rickard-Dickson, K. J., Primus, F. J., and Hawthorne, M. F. Unpublished results.
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