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 Â¡D G. G.. R J. C.Â¡,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 Â«-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.

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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 80Â°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; CÃ¡rdena, 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. 1ÃŸ). Con A (100 fig/ml) for 4 hr at 4Â°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 Â¿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. Anti-Con A was first derivatized with tained in Ham's nutrient mixture F-12 with 10% fetal bovine varying amounts of cystamine in the presence of a water- serum and 10 /Â¿gofgentamicin per ml. soluble carbodiimide to determine optimal conditions for con Inhibition of protein synthesis was measured by a modifica jugate synthesis. Five preparations of anti-Con A, containing tion of the procedure of Moehring and Moehring (18). Cells (8 0, 2.3, 5.4, 7.2, and 8.2 mol cystamine per mol antibody, were x 10") in 1.0 ml of Dulbecco's modified Eagle's medium made (Fig. 1, legend). Each was as active in indirect hemag- supplemented as above containing 5 mg galactose per ml glutination of human erythrocytes as was unmodified anti-Con instead of glucose were seeded in 8-ml flint glass scintillation A. (1?5l-DTA)-SS-(anti-Con A) hybrids were formed when the vials and incubated for 24 hr in a CO2 incubator. Cells were cystaminyl-(anti-Con A) preparations were reacted with an 8- cooled to 4Â°and washed twice with 2.0 ml of cold Dulbecco's fold molar excess of reduced 1?6I-DTA under conditions pro PBS. A freshly prepared Con A solution (1 ml; 10 /tg/ml in moting disulfide interchange. No hybrids were observed when Buffer A) was added, and cells were incubated for 2 hr at 4Â° unmodified anti-Con A was used. The hybrids found between (Layer 1). After a second wash with four 2-ml aliquots of 125I-DTA with cystaminyl-(anti-Con A) (2.3 mol cystamine per Dulbecco's PBS, appropriate dilutions of conjugate were mol antibody) exhibited several bands of higher molecular added in 0.9 ml of serum-free medium (Layer 2). Cells were weight than anti-Con A. That these high-molecular-weight incubated for an additional 1 hr at 4Â°and supplemented with bands contained 125I-DTA was confirmed by autoradiography 0.1 ml fetal bovine serum. After incubation at 37Â° for 24 hr, (Fig. 16). The higher-molecular-weight conjugate bands gave cells were assayed for ability to incorporate 14C-labeled amino more intense autoradiographic images than the lower-molecu acids into trichloroacetic acid-precipitable material as de lar-weight bands, which implied that some antibody molecules scribed (18). Each point is the average of triplicate values, and were derivatized with more than 1 DTA/mol antibody (most levels of toxicity reported were reproducible in at least 3 clearly seen in Fig. 1, Lane 5). It was also evident that the separate experiments. average number of attached DTA molecules increased as the DTA was coupled to anti-Con A by a cystamine-coupling number of cystamine residues per antibody molecule was technique which has been described previously (12). All steps raised (Fig. 1, A and B, Lanes 5-8). Each of the preparations were performed at 4Â°or on ice. Anti-Con A (5.1 mg/ml; 2.0 was as active as native anti-Con A in indirect hemagglutination. ml) was applied to a 2.4- x 9-cm Sephadex G-25 column Cystaminyl-(anti-Con A) containing 7.2 mol cystamine per equilibrated with 0.2 M NaCI, 0.02% sodium azide, and 1 /xg mol antibody was used in a batch scale preparation of the DTA conjugate (see "Materials and Methods"). The conjugate was phenylmethylsulfonylfluoride per ml. Peak fractions were pooled (2.4 mg/ml; 4.0 ml), mixed with cystamine dihydrochlo- separated from unreacted DTA by molecular exclusion chro- ride (45 mg), and adjusted to pH 4.7 by addition of 0.1 N HCI. matography on Sephacryl S-200 (Chart 1). The final product

OCTOBER 1980 3565

inhibition of protein synthesis in cultured mammalian cells. Toxicity is expressed as the concentration of conjugate re -150,000 quired to inhibit protein synthesis by 50% after a 24-hr expo sure. We tested the conjugate on Swiss 3T3 cells to rule out the possibility that toxicity could be attributed to contaminating traces of whole diphtheria toxin. This cell line and other lines of murine origin are essentially unaffected by diphtheria toxin due to a blockage of toxin entry at an unidentified step; their EF-2 is fully sensitive. In a typical assay, 3T3 cells were preincubated with Con A for 2 hr at 4Â°and then washed thoroughly to remove unbound Con A. After addition of DTA-SS-(anti-Con A), the cells were -42,000 incubated for an additional 24 hr and then assayed for ability to incorporate 14C-labeled amino acids into trichloroacetic acid- precipitable material. As shown in Chart 2A, DTA-SS-(anti-Con A) was lethal for cells pretreated with Con A (2 nw) and was at least 400-fold more toxic than diphtheria toxin. No toxicity was observed with -21,000 comparable concentrations of DTA or cystaminyl-(anti-Con A) alone (Chart 2) or with either DTA and unmodified anti-Con A 12345678 9 or S-carboxymethylated DTA and cystaminyl-(anti-Con A) (data not shown). The following observations confirmed that the B conjugate bound specifically to Con A on the cell surface, (a) -150,000 The conjugate was not toxic for cells pretreated with WGA instead of Con A or cells preincubated with Con A in the presence of a-methyl-o-mannoside. (b) No killing was observed if cells were not pretreated with Con A (Chart 2A). Also, toxic activity was dependent on the concentration of Con A to which cells were exposed (data not shown). DTA conjugated to anti- WGA or to nonimmune rabbit IgG was not toxic for cells pretreated with Con A, which indicated that binding was me diated by the antigen binding sites of the immunoglobulins and not by binding of the Con A moiety to saccharide residues on -42,000 the immunoglobulin molecule. Finally, it was demonstrated that toxic activity of the conju gate could be attributed to the ADP-ribosylation of EF-2. The conjugate was tested on a mutant line of CHO K1c cells, CHO RE1.22c. CHO RE1.22c cells contain EF-2 which functions -21,000 normally in protein synthesis but cannot be ADP-ribosylated 12345678 Fig. 1. A. synthesis and purification of C25l-DTA)-SS-(anti-Con A) monitored on sodium dodecyl sulfate-polyacrylamide gels (6.8%). Lane ÃŒ,reduced DTA 20 (nonradioactive). Under oxidizing conditions, DTA dimer (M.W. 42,000) is formed (Lanes 3 to 8). Lane 2. anti-Con A; Lane 3, batch scale preparation of (125I-DTA)- 15 SS-

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be required only in catalytic amounts. Shearer ef al. (25) 100- prepared a cytotoxic conjugate between antibody against hu man colonie cancer cells and glucose oxidase, while Flickinger and Trost (8) have reported enhanced cytotoxicity of phospholipase C when coupled to antibody against Friend leukemia cells. Another enzyme, diphtheria toxin, has attracted particular attention in this regard because of its remarkable potency. Moolten ef al. (20) reported that glutaraldehyde-cross-linked conjugates between whole diphtheria toxin and anti-SV40 an tibody were effective in promoting tumor regression in hamster. Three doses of the toxin-antibody conjugate administered at weekly intervals (2 to 14 /Â¿g/dose) induced complete regres sion of established TRD14 lymphomas in 12 of 28 hamsters. However, nonspecific toxicity of the conjugate was sufficiently high (2 to 14 ^ig/0.1 minimal lethal dose) to preclude testing at higher doses. A rational solution to the problem of nonspecific toxicity is suggested by the properties of the A chain of diphtheria toxin. DTA alone is virtually nontoxic, because it lacks the intrinsic receptor-binding activity of the toxin. However, since ADP- ConcenTrotion, moles/liter ribosyltransferase activity is retained, DTA is endowed with the Chart 2. A. inhibition of protein synthesis in 3T3 cells by conjugate. Toxicity was assayed as described in 'Materials and Methods. Briefly, cells were potential for toxic activity if the molecule can be introduced into incubated for 2 hr at 4Â°with Layer 1. washed, and incubated for 24 hr with Layer the cytosol (19). Although the events necessary for membrane 2. â€¢ â€¢:Layer 1, Con A; Layer 2. (I25l-DTA)-SS-

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. D. G. Gilliland and R. J. Collier experiments indicated that cystamine derivation of anti-Con A truly tumor specific. Recent work with monoclonal antibodies and subsequent coupling to DTA had no effect on antigen against certain types of tumors has given promising results. binding activity. For example, Herlyn ef al. (13), Koprowski ef al. (15), and The experiments described here provide convincing evi Wiktor and Koprowski (29) have reported the isolation of hy- dence that antibody can serve as a specific and functional bridoma cell lines that produce monoclonal antibody against vector for attachment and entry of DTA into mammalian cells. antigens on human colorectal and melanoma cells, and certain DTA-SS-(anti-Con A) was toxic for cells containing Con A on of these hybridomas appear to secrete antibodies that are their surface but not for cells which had not been treated with highly specific for the target tissue. With methods in hand for Con A or for Con A-treated cells which had been washed in efficient coupling of DTA and the evidence presented here that buffer containing a-methyl-o-mannoside. The conjugate was antibodies can mediate the toxic potential of DTA, there should not toxic for cells treated with an immunologically unrelated be no significant barrier to the preparation and testing of DTA antigen (WGA), and no toxic activity was observed if DTA conjugates of such monoclonal antibodies in cell culture and coupled to nonimmune IgG was added to Con A-coated cells. animal models. The mode of entry of toxin A chains into the cytosol and the role of the receptor in entry are unknown, regardless of whether REFERENCES the A chain is attached to a homologous or a heterologous B 1 Boquet. P.. Silverman. M. S., Pappenheimer. A. M., Jr.. and Vernon. N. B. moiety. Several models have been put forward. One hypothesis Binding of Triton X-100 to diphtheria toxin, crossreacting material 45, and proposed for native diphtheria toxin is that the B moiety, their fragments. Proc. Nati. Acad. Sei. U. S. A.. 73: 4449-4453, 1976. 2. Burstein, S., and Knapp. R. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. A Model System Involving Anti-Concanavalin A for Antibody Targeting of Diphtheria Toxin Fragment A

D. Gary Gilliland and R. John Collier

Cancer Res 1980;40:3564-3569.

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