Antibody-directed cytotoxic agents: Use of monoclonal antibody to ...

Proc. Natl. Acad. Sci. USA

Vol. 77, No. 8, pp. 4539-4543, August 1980 Biochemistry

Antibody-directed cytotoxic agents: Use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells (diphtheria toxin/ricin toxin/targeting agents/chemotherapy)

D. GARY GILLILAND*, ZENON STEPLEWSKIt, R. JOHN COLLIER**, KENNETH F. MITCHELLO, TONG H. CHANGt, AND HILARY KOPROWSKIt *Department of Microbiology and *The Molecular Biology Institute, University of California, Los Angeles, California 90024; and tThe Wistar Institute of

Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104 Contributed by Hilary Koprowski, May 2,1980

ABSTRACT We have constructed cell-specific cytotoxic agents by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-lA was modified by attachment of 3(2pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linke conjugates that retained the original binding specificity of the antibody moiety. The conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents. One approach to the construction of cell-specific cytotoxic agents is to couple highly toxic proteins, such as diphtheria toxin and ricin toxin, onto antibodies against cell surface antigens. Although encouraging results have been obtained with this approach (1-5), progress has been hampered by difficulties in obtaining sufficient quantities of cell surface-directed antibodies and by problems of nonspecific toxicity. Recently, the application of hybridoma technology has provided a way to circumvent the former problem. Monoclonal antibodies directed against a variety of cell surface determinants have been isolated and are readily obtainable in quantity (see, for example, refs. 6-8). Moreover, Koprowski and his colleagues (9-12) have shown that certain hybridomas obtained against human tumor cells produce monoclonal antibodies that react with tumorassociated antigens, which are rare or nonexistent on other types of cells. This has prompted speculation that such antibodies might be used as target-specific vehicles for cytotoxic agents. Concurrent studies on structure-function relationships in toxic proteins have revealed information that suggests a rational approach to the problem of nonspecific toxicity. Diphtheria toxin (DT; Mr t 60,000) and ricin toxin (RT; Mr 62,000) may each be separated into two functionally distinct chains (13-15), designated A (for activity) and B (for binding). The A chains are enzymes that catalyze reactions that inactivate specific components of the protein-synthesis machinery within the cytosolic compartment (elongation factor 2 for DT A chain, ref. 16; 60S ribosomal subunits for RT A chain, ref. 17). The B chains bind the toxin molecules to specific receptors on the cell surface and promote transfer of the A chains into the cytosol (16). Little is known about the actual mechanism of transfer, but it is clear that the A chains are virtually nontoxic for whole cells unless attached to B chains. -

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If whole DT or RT were coupled to antibodies by conventional crosslinking methods, at least a fraction of the resulting conjugates would be expected to retain the toxin B moiety in functional form. Significant nonspecific toxicity may therefore result from binding of the conjugate via the toxin B moiety to ubiquitous toxin receptors on cells. However, if one constructed an antibody conjugate that contained only the toxin A chain, problems of nonspecific toxicity might be significantly reduced or eliminated. The feasibility of this approach is supported by recent studies that demonstrated toxicity of conjugates containing toxin A chains covalently coupled to certain unrelated cell surface ligands, such as lectins, hormones, or antibodies (18-25). In the present study we report the use of a monoclonal antibody to direct the actions of DT and RT A chains (DTA and RTA, respectively) onto cells in culture that contain the target antigen. The antibody used was directed against a tumor-associated antigen on colorectal carcinoma cells (9). We demonstrate the ability of toxin A chains to kill tumor cells in culture

when conjugated to monoclonal antibodies. MATERIALS AND METHODS Cell Lines. Human cell lines. Eight different cell lines were used. SW 1116, SW 948, and SW 1083 are colorectal carcinoma (CRC) cell lines obtained from A. Leibovitz (Scott and White Clinic, Temple, TX). Lung carcinoma line MBA-9812 and normal lung fibroblast line HS-0853 were obtained from the Cell Culture Laboratory (Naval Biosciences Laboratory, Oakland, CA). The MRC-5 line of normal fibroblasts was from Flow Laboratories (Rockville, MD). Melanoma cell lines WM 9 and WM 56 were established at the Wistar Institute. Mouse cell lines. BALB/c myeloma P3x63Ag8 cells (abbreviated P3) were obtained from C. Milstein. Hybridoma cell line. HIybridoma cell line 1083-17-IA (hereafter designated 17-1A) produces colorectal carcinomaspecific antibodies and has been characterized (9, 11). Ascitic fluid was produced by inoculation of BALB/c mice intraperitoneally with 107 hybridoma 17-lA cells. The fluid was collected from mice that developed tumors after 2-3 weeks and centrifuged at 300 X g. The supernatant was stored at -200C. Purified 17-1A antibody was prepared from ascitic fluid with Staphylococcus aureus protein A-Sepharose (Pharmacia). Toxins. Fragment A (DTA) was prepared from DT (Connaught Laboratories, Willowdale, ON, Canada) as described (26) and was heated to 800C for 10 min to inactivate any residual traces of toxin. Enzymic activity of DTA was assayed by Abbreviations: DT, diphtheria toxin; DTA, diptheria toxin A chain; RT, ricin toxin; RTA, ricin toxin A chain; SPDP, N-succinimidyl 3(2-pyridyldithio)propionate; PDP, 3-(2-pyridyldithio)propionate group.

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Biochemistry: Gilliland et al.

ADP-ribosylation of wheat germ elongation factor 2 with [14C]NAD as substrate (27). RT was purified from castor beans (A. H. Hummert Seed Co., St. Louis, MO) by affinity chromatography on Sepharose 4B, as described by Nicolson and Blaustein (28) and modified by Cawley et al. (29). RTA was purified from whole RT by reduction with 2-mercaptoethanol and chromatography on Cellex-D (Bio-Rad) as described (15). RTA was freed of residual traces of RT by repeated cycling on a Sepharose 4B column. Radioimmunoassay. The assay was performed on live target cells at approximately 5 X 105 cells per well, as described for melanoma cells (10). Target cells were incubated with the conjugates or control proteins for 1 hr at room temperature. The cells were washed three times and then incubated with radiolabeled (125I-labeled) rabbit anti-mouse F(ab')2. After three final washes, radioactivity in the cell pellet was determined in a Packard y spectrometer. Cytotoxicity. Cytotoxicity was measured by inhibition of protein synthesis as described (18). Briefly, 5 X 104 cells in 1 ml of medium were plated in 8-nil flint glass vials (ICN), incubated for 24 hr, and then treated with an appropriate concentration of a conjugate or control protein. After an additional 24-hr incubation, the medium was aspirated and replaced with a medium containing low amounts of amino acids (0.5 ml) with 0.2 uCi of 14C-labeled amino acid mixture per ml (1 Ci = 3.7 X 1010 becquerels). Reagents. N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was from Pharmacia, dithiothreitol was from Vega Biochemicals (Tuscon, AZ), and all other reagents were as described (18). Synthesis of (DTA)-SS-(17-1A) Antibody Conjugates. 17-1A antibody (7.0 ml, 2 mg/ml) was dialyzed against Dulbecco's phosphate-buffered saline (GIBCO) (pH 7.4) containing antibiotic/antimycotic solution (GIBCO, 5.0 ml/liter). SPDP (0.186 ml, 20 mM) in absolute ethanol was added to the antibody with vigorous mixing. The mixture was allowed to react for 30 min at room temperature and then dialyzed against two 1-liter changes of the same buffer. After dialysis the antibody preparation was analyzed for 2-pyridyldisulfide content as described (30) and found to contain 4.3 such residues per antibody molecule. DTA (3.0 ml, 2.5 mg/ml) was reduced by addition of 0.3 ml of 1.0 M dithiothreitol (pH 7.0) for 30 min at room temperature and desalted on Sephadex G-25 (2.6 X 12 cm column) equilibrated with the buffer described above. Peak fractions from the column were pooled (11.0 ml, 0.53 mg/ml) and mixed with PDP-(17-1A) antibody (7.0 ml, 2.1 mg/ml) [PDP is the 3-(2-pyridyldithio)propionate group]. Final concentrations of DTA and 17-1A antibody were 15 and 5.5 MM, respectively. The final molar ratio of DTA to 17-1A antibody in the reaction mixture was 2.8. The crude conjugate preparation (18.0 ml) was concentrated to a final volume of 9.0 ml by ultracentrifugation on an Amicon YM-10 membrane and purified by chromatography on Sephacryl S-200 as described in the legend to Fig. 1. (DTA)-SS-(17-1A) was also prepared by a different method involving cystamine-modified antibody (24) and was purified on Sephacryl S-200 as above. Synthesis of (RTA)-SS-(17-1A) Antibody Conjugate. (RTA)-SS-(17-1A) was synthesized as described for (DTA)SS-(17-1A) with the following modifications: 17-1A antibody (4 ml, 2.5 mg/ml) was treated with SPDP (0.167 ml, 20 mM in absolute ethanol) to give 8.9 mol of PDP per mol of antibody. RTA (7.0 ml, 1.1 mg/ml) was reduced, desalted, and mixed with PDP-(17-1A) to give a final concentration of 12 and 3.9 ,uM for RTA and 17-1A, respectively. RTA was in 3.1-fold molar excess to antibody in the reaction mixture. The crude conjugate mixture was concentrated to 10 ml on an Amicon

Proc. Natl. Acad. Sci. USA 77 (1980)

YM-10 membrane before chromatography on Sephacryl S-200 as described in the legend to Fig. 1.

RESULTS of (DTA)-SS-(17-1A) and and Purification Synthesis (RTA)-SS-(17-1A) Antibody Conjugates. Hybridoma 1083-17 was produced by fusion of mouse myeloma cells with splenocytes from mice immunized with the human colorectal carcinoma cell line SW 1083 (9). The hybridoma and its clone, 1083-17-1A (17-1A), secrete antibodies that bind specifically to human colorectal carcinoma cell lines (eight of nine tested). These antibodies do not bind to other normal and malignant human cells, including melanomas, fibrosarcomas, astrocytomas, myelomas, or normal embryonic fibroblasts (9, 11). The antigenic determinant recognized by 17-1A antibody on tumor cells in culture is also recognized by the antibody on tumor cells freshly isolated from patients with colorectal carcinoma (9). Conjugates between monoclonal antibody secreted by clone 1083-17-1A (17-1A antibody) and DTA (Mr 21,000) or PTA (Mr 30,000) were prepared with SPDP, a heterobifunctional reagent that permits attachment of the toxin A chain to antibody molecule via a disulfide bridge (31). Briefly, 17-1A antibody was derivatized with SPDP, and the resulting PDP-antibody was treated with a 5-fold molar excess of freshly reduced and desalted A chain. Under these conditions the intrinsic sulfhydryl group of the A chain reacts with the pyridyldisulfide groups of the modified antibody, resulting in displacement of thiopyridine and formation of a disulfide-containing intermolecular bridge. Evidence obtained in another system (unpublished data) strongly suggests that the presence of a disulfide is necessary for expression of toxic activity. With A chain in molar excess to antibody in the conjugation mixture, nearly all of the antibody was converted into conjugate. Unconjugated DTA or RTA was removed from the crude conjugate mixtures by molecular exclusion chromatography on Sephacryl S-200, as described in the legend to Fig. 1. In the purification of (DTA)-SS-(17-1A), the first large peak of A2so-absorbing material that eluted from the column (fractions 31-40) contained the A chain-antibody conjugate. Unconjugated DTA dimer (Mr 42,000) eluted in fractions 43-46, and DTA monomer (Mr 21,000) in fractions 47-60. The (DTA)-SS-(17-1A) conjugate contained ADP-ribosylation activity but was devoid of free

DTA dimer, as judged by electrophoresis on NaDodSO4/ polyacrylamide gels. The conjugate was composed of six to eight distinct bands (lanes 4-9, Fig. 1B), which were larger than antibody alone (>150,000 daltons). Evidence presented in another system (24) suggests that each of these bands corresponds to a population of antibodies that contain integral numbers of attached DTA molecules. Thus, band 1 corresponds to antibody with one DTA moiety attached; band 2, to antibody with two attached toxin A molecules; and so on. Band 0 corresponds to traces of unconjugated antibody. A similar protocol was used in purification of the (RTA)SS-(17-1A) conjugate. Because RTA did not couple as efficiently as DTA to PDP-antibody, higher levels of derivatization of 17-1A antibody with SPDP were required to obtain comparable coupling efficiencies. The purified (RTA)-SS-(17-1A) preparation shown (lane 13) had slightly more unconjugated antibody than the corresponding DTA conjugate. Specific Binding of (DTA)-SS-(17-1A) and (RTA)-SS-(17-1A) Conjugates to Target Cells. Binding of the A-chain conjugates to various cell lines was assayed to ensure that modified 17-1A antibody retained activity and specificity for colorectal carcinoma cells. Cells were first incubated with a conjugate or control protein. The cells were then washed and incubated with 125I-labeled antibody directed against the Fab region of the



4541

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FIG. 2. Binding of conjugates and control proteins to colorectal carcinoma cell lines SW 1116 (A) and SW 948 (B). *, 17-1A antibody; *, (RTA)-SS-(17-1A) conjugate; 0, (DTA)-SS-(17-1A) conjugate; a, unconjugated RTA; 0, unconjugated DTA.

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FIG. 1. (A) Sephacryl S3200 chromatography of crude (DTA)SS-(17-1A) conjugate mixture. Crude (DTA)-SS-(17-1A) (9.0 ml) was chromatographed on a Sephacryl S-200 column (2.6 X 106 cm; 22.1 ml/hr flow rate) equilibrated with Dulbecco's phosphate-buffered saline. Each fraction (6.2 ml)'was analyzed for ADP-ribosylation activity and by NaDodSO4polyacrylamide gel electrophoresis. Fractions 30-40 were pooled, concentrated on an Amicon YM-10 membrane, and used in cytotoxicity assays after filter sterilization. (B) NaDodSO4polyacrylamide gel electrophoresis (7.5%) of Sephacryl S-200 column fractions. Lane 1, unmodified 17-lA antibody. Lane 2, DTA. DTA dimer ran atMr 42,000; DTA monomer (Mr 21,000) ran with the tracking dye. Lane 3, crude (DTA)-SS-(17-1A) conjugate. Lanes 4-9, column fractions 32-36, respectively, from Sephacryl S-200 purification of (DTA)-SS-(17-1A). Lanes 10 and 11, column fractions 43 and 44, respectively. Lane 12, purified and concentrated (DTA)SS-(17-1A) conjugate. Lane 13, purified and concentrated (RTA)SS-(17-1A) conjugate (6.5% gel).

17-lA antibody, and cell-bound radioactivity was measured. (DTA)-SS-(17-1A), (RTA)-SS-(17-1A), and unmodified 17-lA antibody each bound to colorectal carcinoma cell lines SW 1116 and SW 948 (Fig. 2). As expected, no binding was detected if P3 antibody (that from the parental myeloma) or either of the unconjugated A chains was added to cells. Unmodified 17-lA antibody apparently bound more efficiently than (RTA)-SS(17-1A), which in turn bound to a greater extent than (DTA)SS-(17-1A). This difference may result from steric hindrance of the binding of l25I-labeled anti-F(ab')2 to DTA- or RTAmodified 17-lA antibody, or hindrance of 17-lA binding to the cell surface because of the attached A chain, or both causes. Specificity was demonstrated by measuring conjugate binding to cell lines that lack antigen. As shown in Fig. 3, (DTA)SS-(17-1A) bound to both SW 948 and SW 1116 but did not bind to four other cell lines that do not express antigen: MRC-5 (human embryo fibroblast), WM 56 (melanoma), MBA 9812 (lung carcinoma), and HS-0853 (normal lung fibroblast).

These results correlate with the known binding specificity of 17-1A antibody (9). The higher level of binding of the conjugate to SW 948 cells relative to SW 1116 cells reflects a difference in the number of cells in the assay, not in the amount of antigen expressed by SW 948 cells. Cytoxicity of Conjugates for Colorectal Carcinoma Cells. Cytotoxicity of (DTA)-SS-(17-1A) and (RTA)-SS-(17-1A) for colorectal carcinoma cells was tested by a protein-synthesis inhibition assay (18). Briefly, cells were assayed for ability to incorporate '4C-labeled amino acids into trichloroacetic acidprecipitable material after a 24-hr exposure to conjugate or control protein. Both (DTA)-SS-(17-1A) and (RTA)-SS-(17-1A) were toxic for SW 1116 cells, giving 50% inhibition at a concentration of about 10-9 M (Fig. 4A). (DTA)-SS-(17-1A) prepared with cystamine-modified 17-lA antibody (24) was as toxic as (DTA)-SS-(17-1A) prepared with PDP-antibody. The coupling efficiency for RTA to cystaminyl-antibody was low and precluded testing of this conjugate. (DTA)-SS-(17-1A) and (RTA)-SS-(17-1A) were at least 100-fold more toxic than controls, which included unconjugated DTA, RTA, and 17-lA antibody. Perhaps the most convincing control experiment is shown in Fig. 4B. The A-chain conjugates were not toxic for melanoma cells (WM 56) at concentrations as high as 10-7 M although these cells were as sensitive as SW 1116 cells to whole DT. Cytotoxicity was therefore dependent on the presence of A

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2.0 0.2 0.5 1.0 2.0 Hybrid, Mg/ml FIG. 3. Specificity of conjugate binding to human cell lines. *, SW 948, colorectal carcinoma; *, SW 1116, colorectal carcinoma; v, MRC-5, normal fibroblast; O, WM 56, melanoma; A, MBA-9812, lung carcinoma; 0, HS-0853, lung fibroblasts. (A) (DTA)-SS-(17-1A); (B) (RTA)-SS-(17-1A). 0.2 0.5 1.0


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Proc. Natl. Acad. Sci. USA 77 (1980) Table 1. Protein-synthesis inhibition by conjugates* Cells (DTA)-SS-(17-1A) (RTA)-SS-(17-1A) Line 10-8 M 10-7 M 10-8 M 10-7 M Origin

A _'_

100 0

9080-

X

- 700

80

60-

t

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Colorectal carcinoma Lung carcinoma Melanoma

SW 948 SW 1116

80 87

96 96

81 92

94 96

MBA 9812 WM 9 WM 56

7 6 11

27 3 0

2 0 0

0 8 8

1

0

0

0

0

0

Embryonal fibroblasts MRC 5 0 Normal lung fibroblasts HS 0853 0 * Results are given as % inhibition.

401 301 20.

10

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1-12

10-l

0

10-10

10-9

10-8

B

carcinoma cells (SW 403, SW 1083, and SW 1116) under conditions where the melanoma cell line WM 56 was unaffected. The reason for the nonspecific toxicity of the PDP-(17-LA) antibody preparation for SW 403 and SW 1083 cells is not clear, but the preparation was not toxic for melanoma cells, to which the antibodies do not bind.

DISCUSSION 0

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Protein, M FIG. 4. Protein-synthesis inhibition of human cell lines by conjugates. (A) Colorectal carcinoma line SW 1116; (B) melanoma line WM 9. v, DTA; v, RTA; a, 17-1A antibody; 0, PDP-(17-1A) antibody; 0, cystaminyl-(17-1A) antibody; A, (DTA)-SS-(17-1A), prepared with cystaminyl-antibody; *, (DTA)-SS-(17-1A), prepared with PDP-antibody; X, (RTA)-SS-(17-1A), prepared with PDP-antibody; O. DT.

cell surface antigen capable of binding 17-1A monoclonal antibody. Further evidence of specificity of the conjugates for cells that contain antigen is summarized in Table 1. The conjugates were not toxic for lung carcinoma, melanoma, human embryo fibroblast, and normal lung fibroblast cell lines at concentrations at which nearly 100% inhibition of protein synthesis was observed in colorectal carcinoma cell lines SW 948 and SW 1116. Specificity of the toxic activity correlated with the binding specificity of the conjugates and unmodified 17-lA antibody for these cell lines (Figs. 2 and 3). These results implied that it was possible to specify conditions under which most, if not all, cells expressing antigen were killed but cells lacking antigen were not affected. This was effectively shown in the plating efficiency experiment described in Table 2. (DTA)-SS-(17-1A) at 5 ,ug/ml killed virtually all the colorectal

There have been numerous attempts to use antibodies to target the toxic activity of cytotoxic drugs and enzymes to specific cell types, including tumor cells (1-5, 32-39). One major difficulty with this approach has been obtaining high-titer antibody against specific cell surface antigens. Purification of antibody specific for tumor antigens, for example, has relied on fractionation of antisera prepared against whole tumor cells by repeated absorption with normal syngeneic cells (see, for example, ref. 13). In practice, this technique yields small quantities of relatively low-titer antibody. The development of hybridoma cell lines that secrete monospecific antibody has effectively circumvented this problem. Large quantities of monospecific antibody produced by hybridomas against a variety of cell surface antigens are now available. A more recent development is the isolation of hybridomas, such as the 1083-17-1A clone, that produce antibody highly specific for tumor-associated antigens. Koprowski et al. (11) have isolated several hybridoma clones, including 1083-17-lA, 1116 NS 19, and 1116 NS 29, that secrete antibody to epitopes on colorectal carcinoma cells; these antigens have not been detected on normal tissues or other tumor cell lines tested. Antibodies against human tumor-associated antigens have many potential applications, including use as diagnostic aids in evaluation of the progress of clinical cancers, use as agents for control of tumors by means of antibody-dependent cell-mediated cytotoxicity (40), and use as highly specific targeting veTable 2. Plating efficiency (no. of colonies per well) 50 ng/ml 5 Ag/ml Cell Con- (DTA)-SSPDP(DTA)-SS- PDPlines trol* (17-1A) (17-1A) (17-1A) (17-1A) SW 403 64 34 62 34 0 SW 1083 36 2 13 17 28 34 SW 1116 38 33 0 36 WM 56 29 27 25 24 22 Colorectal carcinoma (SW 403, SW 1083, and SW 1116) and melanoma (WM 56) cells were inoculated in 24-well Linbro plates at 100 cells per well. After overnight culture, the medium was removed and replaced with medium containing (DTA)-SS-(17-1A) conjugate or its intermediate PDP-(17-1A) (2 ml/well). After 10 days of culture, the number of colonies per well was calculated. * No conjugate or its intermediate.


Biochemistry: Gilliland et al. hides for cytotoxic drugs and enzymes. The experiments reported here suggest that at least the last of these'is feasible. We have tested the possibility that monoclonal antibodies against a specific cell surface epitope might be used to direct the actions of the A chains of DT and RT onto cells that express a cognate epitope. (DTA)-SS-(17-1A) and (RTA)-SS-(17-1A) were shown to be highly toxic for colorectal carcinoma cells in culture that contained the 17-1A antigen but were virtually not toxic for a variety of other cell lines that lack this antigen. Moreover, high levels of killing were observed at readily attainable concentrations of conjugate (1-10 nM). Thus, at the very least, conjugates such as (DTA)-SS-(17-1A) and (RTA)SS-(17-1A) should be useful as selective agents to isolate mutant cultured cells that lack a particular antigen. Whether they will be useful as therapeutic agents in vivo will await testing in animal models. A major advantage of using conjugates containing A chains is the potential for achieving lower levels of nonspecific toxicity than have been observed with antibody conjugates of whole toxins. We found that the A chain conjugates tested killed virtually 100% of cells expressing antigen under conditions where cells lacking antigen were unaffected. Furthermore, by a variety of means it may be possible to enhance the specific toxicity of A chain-antibody conjugates without increasing nonspecific toxicity. Certain classes, subclasses, or fragments [e.g., F(ab')2] of antibody may be more efficient than others in promoting productive internalization of covalently coupled toxin A chains. Also, there may be significant differences among various cell surface antigens in promoting entry of bound antibody. Finally, the specific toxicity of antibody conjugates possibly could be enhanced by replacing the toxin A chains with longer, mutant forms of DT, such as CRM45 (41), which lacks the receptor binding site but contains a hydrophobic domain that has been postulated to facilitate entry of the A domain into cells. We thank Patricia Bernard, Suzanne Morris, and Denise Owens for expert technical assistance. We also thank Dr. Leon Wofsy for suggesting the use of SPDP as a coupling reagent. This work was supported by grants from the National Institute of Allergy and Infectious

Diseases (AI-07877), the American Cancer Society (MV-51), the National Cancer Institute (CA-10815 and CA-21124), and the Division of Research Resources (RR-05540) and funds from the W. W. Smith Foundation. D.G.G. was supported by U.S. Public Health Service Grant CA09506, awarded by the National Cancer Institute. 1. Thorpe, P. E., Ross, W. C., Cumber, A. J., Hinson, C. A., Edwards, D. C. & Davies, A. J. S. (1978) Nature (London) 271, 752755. 2. Moolten, F. L., Capparell, N. J. & Cooperband, S. R. (1972) J. Natl. Cancer Inst. 49, 1057-1062. 3. Moolten, F. L., Capparell, N. J., Zajdel, S. H. & Cooperband, S. R. (1975) J. Natl. Cancer Inst. 55,473-477. 4. Flickinger, R. A. & Trost, S. R. (1976) Eur. J. Cancer 12, 159160. 5. Shearer, W. T., Turnbaugh, T. R., Coleman, W. E., Aach, R. O., Philpott, G. W. & Parker, C. W. (1974) Int. J. Cancer 14, 539-547. 6. Koprowski, H., Gerhard, W. & Croce, C. M. (1977) Proc. Natl. Acad. Sci. USA 74,2985-2988. 7. Gerhard, W., Croce, C. M., Lopes, D. & Koprowski, H. (1978) Proc. Natl. Acad. Sci. USA 75, 1510-1514.

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8. Wiktor, T. J. & Koprowski, H. (1978) Proc. Natl. Acad. Sci. USA 75,3938-3942. 9. Herlyn, M., Steplewski, Z., Herlyn, D. & Koprowski, H. (1979) Proc. Natl. Acad. Sci. USA 76, 1438-1442. 10. Koprowski, H., Steplewski, Z., Herlyn, D. & Herlyn, M. (1978) Proc. Natl. Acad. Sci. USA 75,3405-3409. 11. Koprowski, H., Steplewski, Z., Mitchell, K. F., Herlyn, M., Herlyn, D. & Fuhrer, J. P. (1979) Somatic Cell Genet. 5,957-972. 12. Steplewski, Z. (1980) Transplant. Proc., in press. 13. Collier, R. J. & Kandel, J. (1971) J. Biol. Chem. 246, 14961503. 14. Drazin, R., Kandel, J. & Collier, R. J. (1971) J. Biol. Chem. 246, 1504-1510. 15. Olsnes, S. & Pihi, A. (1973) Biochemistry 12,3121-3126. 16. Collier, R. J. (1975) Bacteriol. Rev. 39,54-58. 17. Benson, S., Qisnes, S., PihN A., Skorve, J. & Abraham, K. A. (1975) Eur. J. Biochem. 59,573-580. 18. Gilliland, D. G., Collier, R. J., Moehring, J. M. & Moehring, T. J. (1978) Proc. Natl. Acad. Sci. USA 75,5319-5323. 19. Uchida, T., Yamaizumi, M., Mekada, E., Okada, Y., Tsuda, M., Kurokawa, T. & Sugino, Y. (1978) J. Biol. Chem. 253, 63076310. 20. Yamaguchi, T., Kato, R., Beppu, M., Terao, T., Inoue, Y., Ikawa, Y. & Osawa, T. (1979) J. Natl. Cancer Inst. 62, 1387-1395. 21. Oeltmann, T. N. & Heath, E. C. (1979) J. Biol. Chem. 254, 1022-1027. 22. Oeltmann, T. N. & Heath, E. C. (1979) J. Biol. Chem. 254, 1028-1032. 23. Miskimins, W. K. & Shimizu, N. (1979) Biochem. Biophys. Res. Commun. 91, 143-151. 24. Gilliland, D. G. & Collier, R. J. (1980) Cancer Res., in press. 25. Masuho, Y., Hara, T. & Noguchi, T. (1979) Biochem. Blophys. Res. Commun. 90,320-326. 26. Chung, D. W. & Collier, R. J. (1977) Biochim. Biophys. Acta 483,

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