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A Model System Involving Anti-Concanavalin a for Antibody Targeting of Diphtheria Toxin Fragment A1

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A Model System Involving Anti-Concanavalin a for Antibody Targeting of Diphtheria Toxin Fragment A1 [CANCER RESEARCH 40, 3564-3569, October 1980] 0008-5472/80/0040-OOOOS02.00 A Model System Involving Anti-Concanavalin A for Antibody Targeting of Diphtheria Toxin Fragment A1 D. Gary Gitliland2 and R. John Collier3 Department of Microbiology. College of Letters and Science ¡D G. G.. R J. C.¡,and The Molecular Biology Institute [R. J. C.J. University of California. Los Angeles, California 90024 ABSTRACT other highly toxic proteins catalyze potentially lethal reactions involving target macromolecules or macromolecular structures Results obtained in a model system strongly suggest that within the cytoplasmic compartment of eukaryotic cells. For antibodies to cell surface determinants may be used to direct example, DTA4 (M.W. 21,145) transfers the ADP ribose moiety the toxic potential of the A chain of diphtheria toxin (DTA). The of NAD+ into covalent linkage with EF-2, given by the equa A chain (M.W. 21,000) was covalently attached to antibody tion: against concanavalin A (anti-Con A) by means of a disulfide- NAD* + EF-2 ^ ADP-ribosyl-EF-2 + nicotinamide + H* containing cross-bridge. This DTA-SS-(anti-Con A) conjugate was toxic for 3T3 cells containing Con A on their surface but This results in cessation of protein synthesis and subsequent was not toxic in the same concentration range for: (a) cells cell death (6). A recent report indicates that introduction of a lacking Con A; (b) Con A-treated cells washed with buffer single molecule of DTA into a eukaryotic cell is sufficient to kill containing «-methyl-D-mannoside; (c) cells containing wheat the cell (31 ), and the same may well be true of the A chains of germ agglutinin on their surface; or (d) Con A-treated mutant many other toxins. Chinese hamster ovary cells containing altered, toxin-insensi An important property of DTA and other A moieties is that tive elongation factor 2. Conjugates containing DTA disulfide they are normally unable to enter cells unless attached to their linked to anti-wheat germ agglutinin antibody or to nonspecific respective B chains. For example, the toxicity of DTA for intact rabbit immunoglobulin G were not toxic for cells coated with cells or animals is approximately a million-fold lower than that Con A. The results suggest a new approach to the construction of whole diphtheria toxin, which consists of DTA disulfide linked of antibody-directed, tumor-specific chemotherapeutic agents. to the B chain (M.W. 39,000). The B chains of diphtheria toxin Conjugates containing DTA disulfide linked to antibody against and other toxins generally serve to attach the toxins to specific specific cell surface antigens may also be generally useful as cell surface receptors (6), but little is known beyond this about specific selective agents for the isolation of mutant cell lines. how they may function to promote transfer of the A chains into the cytosolic compartment. Recent studies have indicated that various heterologous INTRODUCTION proteins which bind to cell surfaces and which are unrelated to Many attempts have been made to use antibodies against toxins may serve as surrogate B moieties in promoting the specific cell surface antigens to selectively kill populations of entry of DTA or the ricin A chain. Thus, DTA disulfide linked to mammalian cells which express these antigens (2, 8-11, 14, either Con A or Wistaria floribunda lectin has been shown to 16, 19, 20, 24-27). One approach has been to conjugate a be toxic for mammalian cells in culture (12, 28). Similar results cytotoxic agent, such as chlorambucil (10, 11), 131I(25), or have been reported for conjugates containing the A chain of whole diphtheria toxin (19, 20, 26) to cell surface-directed ricin toxin linked either to Con A (30) or to the B sunbunit of antibody. Two major difficulties with this approach have been human chorionic gonadotropin (22, 23). Here, we report the encountered: (a) nonspecific toxicity for cells lacking antigen; synthesis and characterization of a conjugate containing DTA and (b) the lack of high-titer antibody preparations against disulfide linked to antibody against Con A (anti-Con A). This specific cell surface antigens. The recent application of hybrid- conjugate is lethal only for cells containing Con A on their oma technology has offered a solution to the latter difficulty surface. The results indicate that DTA bound at the cell surface and has aroused renewed interest in the subject of antibody via an antibody molecule can gain access to the cytosol of targeting. Furthermore, advances in our understanding of mammalian cells and strongly suggest that antibody may act structure and activity in certain highly toxic proteins (e.g., as a vehicle for targeting of the A moieties of toxins. diphtheria toxin and ricin toxin) have offered a rational means of minimizing the problem of nonspecific toxicity. Here, we MATERIALS AND METHODS report results strongly suggesting that it will be possible to use antibodies to direct the actions of the A chains of such toxins Anti-Con A and anti-WGA were from Vector Laboratories to specific populations of cells. (Burlingame, Calif.) and were purified by an affinity chromatog- The A chains of diphtheria toxin, ricin, abrin, and certain raphy technique described previously (17). The preparations gave precipitin bands on Noble agar plates at a 512-fold 1This work was supported by USPHS Grant AI-07877 from the National dilution when tested at a lectin concentration of 0.5 mg/ml. Institute of Allergy and Infectious Disease and by Grant MV-51 from the American Con A (Grade IV), nonimmune rabbit IgG, 1-ethyl-3-(3-dimeth- Cancer Society. 2 Recipient of USPHS Grant CA09506, awarded by the National Cancer Institute, Department of Health, Education, and Welfare. ' The abbreviations used are: DTA, diphtheria toxin Fragment A; EF-2. elon ' To whom requests for reprints should be addressed. gation factor 2; Con A. concanavalin A; WGA, wheat germ agglutinin; Dulbecco's Received March 10. 1980; accepted July 7, 1980. PBS, Dulbecco's phosphate-buffered saline; CHO, Chinese hamster ovary. 3564 CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. Toxic Antibody Conjugate ylaminopropyOcarbodiimide HCI, a-methyl-D-mannoside, and Cystamine coupling was initiated by addition of solid 1-ethyl-3- glucose-free galactose were from Sigma Chemical Company (3-dimethylammopropyl)carbodiimide HCI to a final concentra (St. Louis, Mo.). WGA was the generous gift of Dr. Joel Shaper tion of 7.5 mg/ml, and the reaction mixture was maintained at (Department of Pharmacology and Oncology, The Johns Hop pH 4.7 for 30 min. After addition of 1 M sodium acetate (0.2 kins University School of Medicine, Baltimore, Md.). DTA was ml), the preparation was dialyzed exhaustively against Buffer prepared from diphtheria toxin (Connaught Laboratories, To A. Cystaminyl-(anti-Con A) had 7.1 cystamine residues/anti ronto, Ontario, Canada) as described (5) and was heated to body molecule, determined with 5,5'-dithiobisnitrobenzoic acid 80°for 10 min to inactivate any residual traces of toxin. 14C- as described (16). 125I-DTA(1 mg/ml; 6.0 ml) was reduced by Labeled amino acids were from New England Nuclear (NEC- addition of 0.2 ml of 1 M dithiothreitol, pH 7.0, and incubation 445; Cárdena, Calif.). 4-(2-Hydroxyethyl)-1-piperazineethane- for 1 hr at room temperature. The protein was desalted on a sulfonic acid and Dulbecco's PBS were from Grand Island Sephadex G-25 column (2.6 x 11 cm) equilibrated with Buffer Biological Company (Grand Island, N.Y.), and cystamine dihy- A. Peak fractions were pooled and mixed with cystaminyl-(anti- drochloride was from Aldrich Chemical Company (Milwaukee, Con A) (2.4 mg/ml; 3.0 ml). Final concentrations of 126I-DTA Wis.). Gentamicin was from Microbiological Associates (Be- and cystaminyl-(anti-Con A) were 1.6 x 10~5 M and 2.6 x 10~6 M, respectively, in a total volume of 18 ml. Nearly all of thesda, Md.). Anti-Con A activity in column fractions was measured indi the cystaminyl-(anti-Con A) was converted to higher molecular rectly using human erythrocytes (107/ml) treated with succinyl- weight products containing DTA (see autoradiograph, Fig. 1ß). Con A (100 fig/ml) for 4 hr at 4°and then washed in Buffer A We have also found a commercially available disulfide-contain- [10 mW 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ing cross-linking reagent, /V-succinimidyl-3-(2-pyridyldithio)- buffer (pH 7.8), 0.2 M NaCI, 10 ¿tgofgentamicin per ml, and 1 propionate (Pharmacia Fine Chemicals, Inc., Piscataway, fig of phenylmethylsulfonylfluoride per ml]. Serial 2-fold dilu N. J.), to be equally effective in the synthesis of DTA-SS-(anti- tions of column fractions were assayed for ability to agglutinate Con A) conjugates. Crude conjugate was concentrated to a succinyl-Con A-coated cells (200 /il) in 96-well microtiter final volume of 9 ml on an Amicon YM-10 membrane and dishes (Cooke Laboratory Products, Alexandria, Va.) purified on Sephacryl S-200 as detailed in the legend to Chart 125I-DTAwas prepared according to the solid-state lactoper- 1. oxidase method of David (7) and diluted with unlabeled DTA to a final specific activity of 100 mCi/mmol. RESULTS Swiss 3T3 cells were maintained in Dulbecco's modified DTA was linked to anti-Con A by a cystamine-coupling tech Eagle's medium supplemented with 5% fetal bovine serum and nique (12) which incorporates a disulfide bridge into the linkage 10 /tg of gentamicin per ml. CHO K1 c cells, a cloned derivative between the 2 proteins. The presence of the disulfide may be of the CHO K1 line of CHO cells, and CHO RE1.22c, a necessary for release of free DTA into the cytosol and subse diphtheria toxin-resistant variant of CHO K1c (18), were main quent inactivation of EF-2.
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