[CANCER RESEARCH 52, 4448-4452, August 15, 1992] Cytotoxicity of Streptavidin-blocked Biotinyl-Ricin Is Retrieved by in Vitro Immunotargeting via Biotinyl Monoclonal Antibody1 Bilha Schechter, Ruth Armin,2 and Meir Wilchek Departments of Chemical Immunology [B. S., R. A.J and Membrane Research and Biophysics [M. W.], The Weidmann Institute of Science, Rehorot, Israel 76100 ABSTRACT of the B chain (5, 8). Such A chain immunotoxins were shown to be specifically toxic to antibody-reactive target cells. How The streptavidin-biotin system has been used to immunotarget whole ricin to tumor cells in a system that overcomes ricin-nonspecific cyto- ever, various studies have shown that ricin A chain immuno toxicity. Biotin was linked to ricin via a disulfide-containing reagent, toxins may sometimes be inactive, or not as active as expected sulfosuccinimidyl-2-(biotinamido)ethyl-l,3'-dithiopropionate. The (9), and often require potentiating agents (10, 11). Addition of product, l>iotiiiyl-.Y,.S'-ricin(b-ricin), retained most of its in vitro cyto ricin B chain-antibody conjugate (12, 13) or free B chain toxic activity against human epidermoid carcinoma (KB) cells. Com- (14, 15) has shown to augment weak ricin A immunotoxin plexing b-ricin to streptavidin resulted in >99% loss of its cellular activity, demonstrating the potentiating effect of the B chain on toxicity which is associated with loss of cell-binding activity. The A chain function. The property of B chain in intact ricin im streptavidin-b-ricin complex could, however, be targeted to KB cells via munotoxin that facilitates internalization of the A chain to the the biotinylated monoclonal antibody 108 which is specific to the epi dermal growth factor receptor overexpressed on KB cells. The complex cytosol appears to be, at least in part, independent of galactose did not regain its activity if the specific antibody was not biotinylated or recognition (16). if the biotinylated antibody was of a different specificity. Streptavidin is These observations have initiated new approaches to the use thus used to block b-ricin, presumably due to a storie restraint of the of whole ricin immunotoxin, mainly by introducing new con streptavidin on the ricin B-chain, and to bridge it to biotinyl antibody jugation methods (16-18) or blocking techniques (19) that ob recognizing the target cell. Avidin could not replace streptavidin in this struct the B chain galactose-binding sites. In the present study system since a complex between b-ricin and avidin retained a major part we have used streptavidin to block the cytotoxic activity of (60%) of ricin cytotoxic activity. This is attributed to the nonspecific b-ricin3 in which the biotin group is linked to ricin via a disul- binding of avidin to cells in vitro, including the KB cells. It is suggested that b-ricin is blocked by both streptavidin and avidin, but once the fide bond. Whole ricin can thus be specifically targeted to cells complex gains access to the cell surface, its cytotoxic activity is specif when incorporated in a cytotoxically blocked streptavidin-b- ically retrieved. ricin complex carrying free biotin binding sites, that can inter act with biotinyl groups on specific antibodies associated with INTRODUCTION the cell surface. It is assumed that ricin activity is regained following internalization and release of the ricin A chain. Ricin is a toxic plant glycoprotein composed of two polypep- tides, A chain and B chain, linked by a single interchain disul- fide bond (1). The A chain is a potent inhibitor of protein MATERIALS AND METHODS synthesis by inactivating 60S ribosomal subunits (2). The B Radiolabeling chain consists of two domains, each possessing a lactose bind ing site presumed to be associated with the strong binding af Ricin and b-ricin (0.2 mg/0.2 ml 0.05 Msodium phosphate buffer, pH finity of ricin to cells (3). The B chain binds to D-galactose or 8) were radiolabeled with 0.5 mCi I25I (Amersham International pic, /V-acetylgalactosamine-terminal residues of oligosaccharides on Amersham, United Kingdom) using the chloramine-T method (20). cell surface membranes and facilitates transfer of the toxin A After purification on Sephadex G-25 the specific activity was in the chain into the cytosol (4). Several approaches have been used to range of 0.5-1.0 ¿iCi/Mgprotein, target ricin to tumor cells. Linkage of a tumor-specific antibody to the whole ricin molecule generates an immunotoxin which Cells and Antibodies combines the tumor specificity of the antibody and the potent The KB human tumor cell line derived from oral epidermoid carci toxicity of ricin (5). However, the intact ricin immunotoxin noma was obtained from the American Type Tissue Culture Collection. cannot be used in vivo since it generates nonspecific effects due The cells were maintained in DMEM supplemented with 10% heat- to the B chain galactose-binding activity. However, whole ricin inactivated fetal calf serum, 2 HML-glutamine, penicillin, and strepto immunotoxins are still powerful reagents for the in vitro elim mycin (Biolab, Jerusalem, Israel) at 37°Cin 5% CO2 in air. The cells ination of T-cells (6) or leukemia cells (7) prior to transplanta were harvested by incubation with 0.1% trypsin and 1 HIMEDTA (Bi tion. Such a procedure can be carried out only in vitro since high olab) at 37°Cfor 5 min. The mAb 108 IgG2a hybridoma line, specific concentrations of galactose are needed to inhibit the B chain to the epidermal growth factor receptor overexpressed on KB cells, was binding, to allow conjugate reactivity only via its antibody rec generated by immunizing mice with the KB cells (21). The antibody ognition site. To overcome the in vivo effects of whole ricin fraction was isolated by ammonium sulfate precipitation at 45% satu immunotoxin, the antibody has been attached to the ricin A ration. Monoclonal antibody against CEA, which does not bind to KB chain, generating a conjugate in which the antibody carrying the cells, was used as control antibody. tumor cell specificity is used to replace the cell-binding function Received 2/17/92; accepted 6/8/92. 3 The abbreviations used are: b-ricin, biotinyl-5,5-ricin; NHS-S.S-biotin, sulfo- The costs of publication of this article were defrayed in part by the payment of succinimidyl-2-(biotinamido)ethyl-l,3-dithiopropionate: mAb, monoclonal anti page charges. This article must therefore be hereby marked advertisement in accord body; b-mAb, biotinyl monoclonal antibody: HABA, 2(4-hydroxyazobenzene) ance with 18 U.S.C. Section 1734 solely to indicate this fact. benzoic acid; PBS, 0.15 MNaCI - 0.02 Mphosphate buffer, pH7.2; CEA, carcino- 1This research was supported by a grant from Makor Chemicals, Jerusalem, embryonic antigen; IC$o. concentration of ricin causing 50% inhibition of ['111 leucine incorporation by the cells; DMEM. Dulbecco's modified Eagle's medium; Israel. 2 To whom requests for reprints should be addressed. b-mAb, biotinylated monoclonal antibody. 4448 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1992 American Association for Cancer Research. IMMUNOTARGETING OF BIOTINYL-RICIN VIA STREPTAVIDIN Conjugating Biotin to Ricin and Antibody- Binding of I25l-Streptavidin to b-mAb 108-coated KB Cells Conjugating NHS-5,5-Biotin (Pierce, Rockford, IL) to Ricin. Ricin KB cell monolayers in 24-well plates were incubated for 40 min with (Sigma Chemical Co., St. Louis, MO; 0.93 mg) in 0.3 ml 0.05 Msodium 10 Mg/ml b-mAb 108, mAb-108, or b-mAb anti-CEA in 0.2 ml DMEM bicarbonate at pH 8.5 was mixed with NHS-S.S-biotin (0.15 or 0.4 mg) at room temperature. The monolayers were then washed twice with dissolved in 0.3 ml ice-cooled water. The reaction proceeded for 3 h at PBS and 0.2-ml samples containing 125I-streptavidin in a biotin-free 0°Cafter which the b-ricin was extensively dialyzed against PBS. medium (DMEM supplemented with dialyzed fetal calf serum) were added. After 30 min incubation at 37°Cthe cells were washed and Conjugating Biocytin Hydrazide to Modified Carbohydrates in An tibody. Monoclonal antibodies (mAb; 0.6 mg) were oxidized in 1 ml of dissolved in 0.5 M NaOH, and the cell-bound radioactivity was moni 0.1 M sodium acetate buffer, pH 5.5, containing 10 imi sodium meta- tored as described above. periodate for l h at 4°C.Theprotein was then dialyzed against the same buffer and biocytin hydrazide (Sigma; 0.6 mg) was added together with Immunotargeting in Vitro sodium cyanoborohydride (1.2 mg). The coupling reaction proceeded for l h at 4°Cafter which the b-mAb was dialyzed extensively against Experiments of immunotargeting streptavidin-b-ricin to KB cells us PBS. Retention of antibody binding activity of b-mAb 108 was deter ing b-mAb 108 were carried out in microtiter plates in a biotin-free mined by competing with 125I-mAb 108 for the binding to KB cells with medium. KB cell monolayers were preincubated with 0.1-ml aliquots of b-mAb 108, b-mAb anti-CEA or unbiotinylated mAb 108, at a concen reference to cold mAb 108, as described before (22). tration of 20 Mg/ml. After 30 min incubation at 4°Cthe antibody- The molar substitution ratio (mol biotin coupled/mol protein) was containing medium was replaced by fresh medium and the streptavidin- determined spectrophotometrically according to the method described b-ricin complex (at a 5:1 molar ratio) was added. Incubation proceeded by Green (23). The assay uses the change in absorbance at 500 nm for 18 h at 37°Cafter which the cells were pulsed and processed as which occurs when biotin-binding sites of avidin (S.