Bioconjugate Chem. 1990, I , 314-318
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Misonidazole Conjugates of the Colorectal Tumor Associated Monoclonal Antibody 17-1A Deborah L. Pierce,? Ned D. Heinde1,'ItJ Keith J. Schray,? Michele M. Jetter,+ Jacqueline G. Emrich,$ and David V. Woo$ Institute for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, and Department of Radiation Oncology & Nuclear Medicine, Hahnemann University, Philadelphia, Pennsylvania 19102. Received October 16, 1989 ~
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The radiation sensitizer misonidazole has been linked to the monoclonal antibody 17-1Awhich recognizes a nonshed antigen of a human gastrointestinal tumor. Linkage was accomplished through a hemisuccinate of misonidazole attached by a mixed anhydride coupling and gave a conjugate whose plasma half-life (for drug cleavage) was ca. 70 h. The degree of substitution on the antibody could be precisely regulated by varying the reactant ratios. The binding avidities of the resulting conjugates to the SW1116 colorectal tumor cells decrease logarithmically with increasing drug load. Four to six misonidazoles per antibody represented the optimum drug loading on this system. Enzymatic cleavage of the conjugate drug union took place at both the ester and the amide linkages with the former scission predominating.
Radiation sensitizers such as misonidazole, desmethylmisonidazole, and metronidazole enhance radiation lethality on hypoxic tumor cells in culture but have been much less successful in clinical trials because of their marked dose-limiting neuropathies and minimal tumor affinities (1-6). The tumor-delivery successes reported for the use of monoclonal antibody conjugates in diagnosis (radioimmunoimaging) and in drug targeting (immunochemotherapy) offer a solution to enhancement of the clinical utility of sensitizers (7-9). In an earlier study we reported the conjugation of misonidazole to the anti-colorectal carcinoma monoclonal antibody 19-9 (10). This antibody was derived from a hybridoma 1116-NS-19-9, produced by fusion of the 653 variant of P3X63 Ag8 murine myeloma cells with splenocytes from a mouse immunized with the human colorectal carcinoma cell line SW1116 (11). These studies showed that an optimum number of 10-12 misonidazoles could be attached to the IgG with retention of approximately 40% immunorecognition. However, since the antigenic determinant recognized by 19-9 is found not only on the cell but is also shed into the blood of patients with colorectal cancer, only a fraction of the sensitizerantibody conjugate would bind at the tumor target (12). The antibody 17-1A recognizes a nonshed antigen and was originally derived from the injection of the SW1083 human colon carcinoma line into BALB/c mice with subsequent fusion of the splenocytes with P3x63/Ag8 myeloma cells (11,121. 17-1A recognizes a membrane receptor on colon carcinoma, primary pancreatic cancer, and metastatic adenocarcinomas and has already been used to deliver toxins and boron compounds for neutron therapy to tumors (7, 13, 14). In the following discussion, the synthesis, characterization, and in vitro evaluation of misonidazoleantibody conjugates with 17-1A are described. Antibodydrug conjugates have been prepared with retained binding avidity and sufficient resistance to plasma-enzyme hy-
* To whom requests for reprints should be addressed. Lehigh University.
* Hahnemann University. f
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drolysis to permit targeting. This preliminary investigation indicates that monoclonal transport of sensitizers may constitute an effective way to enhance radiation therapy of hypoxic tumors. EXPERIMENTAL PROCEDURES
Materials. Misonidazole was obtained from Dr. Peter Sorter of Hoffmann-La Roche, Nutley, NJ. The monoclonal antibody 17-1A was obtained from Centocor, Malvern, PA. Human plasma employed in the hydrolysis studies was donated by one of the authors (D.L.P.). Conjugation of Misonidazole Hemisuccinate to 171A. Ratios of sensitizer molecules/IgG from 44 to 1could be obtained by altering the reaction ratios of the sensitizer and the antibody as well as the contact times of the reactants. Table I shows the relationship between reactant ratios and loading factors at a fixed reaction time (15 h). A typical preparation which resulted in a drug load of six sensitizers per antibody is described. Individual stock solutions were prepared of misonidazole hemisucccinate (IO),triethylamine, and isobutyl chloroformate each at a 0.05 M concentration in anhydrous mmol) of acetonitrile. Aliquots of 0.031 mL (1.55 X the triethylamine and the misonidazole hemisuccinate stock solutions were mixed at 4 OC for 15 min to form a light yellow reaction mixture. To this was added 0.031 mL (1.55 X mmol) of isobutyl chloroformatestock solution with the resulting colorless mixture incubated at 4 OC for 1 h. This reaction solution was added dropwise to a solution of 4.65 mg of 17-1A (3.1 X mmol) in 4.5mL of 0.1 M phosphate buffered saline, pH 7.7 (PBS). The reaction was allowed to proceed for 15 h at 4 "C during which time the pH decreased to 7.5, whereafter the product was isolated and purified as described below. This particular conjugate had a loading factor of 6 and a binding avidity of 62%. Purification of the Antibody Conjugates. The requisite antibody conjugate was isolated from the crude reaction mixture by chromatography on Sephadex G-25 (Pharmacia PD-10 columns, Pharmacia Laboratory Separation Division, Uppsala, Sweden), equilibrated, and 0 1990 American Chemical Society
Bioconjugate Chem., Vol. 1, No. 5, 1990
Misonidazole Conjugates of Antibody 17-1A
Table I. Reactant Ratios, Loading Factors, and Binding Avidities of Misonidazole Hemisuccinate Conjugation to 17-1A conjugate 1 2 3 4 5c
6 7
reactant ratioa 2 01 35:l 5 01 1001 9 01 150:l 500:l
loading factorb 1.4 5
6 12 14 21 44
1107
76 avidity
88 67 62 9.3 6.0 4.7
