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2 Antibodies for Delivering Saporin to Lymphoma in Vitro and in Vivo1

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2 Antibodies for Delivering Saporin to Lymphoma in Vitro and in Vivo1 [CANCER RESEARCH 51. 2353-2361, May 1, 1991] Cooperative Mixtures of Bispecific F(ab')2 Antibodies for Delivering Saporin to Lymphoma in Vitro and in Vivo1 Ruth R. French, Ann E. Courtenay, Susan Ingamells, George T. Stevenson, and Martin J. Glennie2 Lymphoma Research Unit, Tenovus Laboratory, General Hospital, Southampton, United Kingdom ABSTRACT in which one Fab' was specific for the surface idiotype of the guinea pig lymphoblastic leukemia, L2C, and the second Fab' We report that selected combinations of two or more monoclonal bispecific F(ab')i antibodies (BsAbs) far outperform single derivatives in recognized a plant RIP, saporin (7, 8). During this work, a simple and efficient means was developed for producing large the delivery of the ribosome-inactivating protein, saporin, to guinea pig amounts of pure BsAb, in which individual Fab' arms were I.;( leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'7, one from monoclonal anti- joined at their hinges via a stable thioether linkage. The only L2C-idiotype antibody and the other from anti-saporin antibody. The prerequisite for preparing these hybrid molecules was that the latter was either affinity-purified rabbit poh donai or one of a panel of parent antibodies, polyclonal or monoclonal, yielded F(ab')2 five mouse monoclonal antibodies. In vitro cytotoxicity studies showed fragments on enzymatic digestion that could be reduced to Fab' that, though all derivatives were effective, the BsAb made with the fragments expressing free hinge-region sulfhydryl groups for poh donai antibody was always 10 to 20 times more potent than those cross-linking by o-phenylenedimaleimide. BsAb proved highly made with a monoclonal antibody in yielding 50% inhibition of [3H|- efficient at delivering saporin to the L2C cells in vitro and leucine uptake. This superior activity could be matched by selective prevented tumor growth in vivo as efficiently as conventional mixtures of two or more of the monoclonal derivatives. Furthermore, in saporin-containing immunotoxin (8). immunotherapeutic delivery of saporin to tumor, a pair of BsAbs per Throughout this work, BsAb derivatives constructed using formed significantly better than did either individually. Fab' fragments from an affinity-purified rabbit polyclonal anti- Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell saporin antibody performed more efficiently than did similar surface by an appropriate BsAb mixture rather than by a single BsAb. reagents containing monoclonal anti-saporin antibodies. The In contrast, only small differences were recorded in the rate at which specificity for the L2C cells has always been supplied by Fab' saporin was internalized as a result of the same maneuver. We conclude from a high-affinity monoclonal anti-idiotype antibody (9). The that the improved performance of combinations of BsAbs arises from advantage of the polyclonal antibody-containing derivative was their ability to provide multiple linkages between saporin molecules and most evident in immunotherapy experiments, where it was cell surfaces, significantly increasing the functional affinity with which approximately 10 times more effective than a similar derivative saporin is tethered to the cell, but, in this system at least, having only a containing monoclonal anti-saporin antibody. In this work, we minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivat have used a panel of five monoclonal anti-saporin antibodies to ing proteins and perhaps also on unwanted cells, could provide an prepare BsAb derivatives. In vitro and in vivo studies show that, important new strategy in immunotherapy. although none of the individual monoclonal bispecific deriva tives matches the polyclonal reagent, a cocktail of all five, or selected pairs, of the monoclonal derivatives performed equally INTRODUCTION as well as did the original polyclonal reagent. The mechanism Conventional immunotoxins, prepared by covalently cou of this improved performance is investigated. pling plant or bacterial toxins to appropriate antibodies, have been shown to be selectively toxic to unwanted cells both in MATERIALS AND METHODS vitro and in vivo (1, 2). As an alternative approach to targeting Materials. Cell culture was performed in supplemented DMEM cytotoxic compounds, Raso and Griffin (3) and Raso (4) dem containing glutamine (200 ITIM),pyruvate (100 HIM), penicillin and onstrated the potential of bispecific antibodies with dual speci streptomycin (100 lU/ml), Fungizone (2 mg/ml), and 10% PCS (My- ficity for target cells and the RIP3 ricin as a means of delivering oclone; Gibco, Ltd., Paisley, Scotland). The RIP saporin was purified toxic substances to neoplastic cells. They showed that BsAbs from the seeds of Saponaria officinalis by water extraction as described having specificity for ricin A chain and the surface immuno- previously (10). globulin of a lymphoma line could deliver toxin to the target Leukemia Cells. The L2C lymphoblastic leukemia cells of strain 2 cells and prevent further protein synthesis. Other workers have guinea pigs were used throughout this investigation (11, 12). The line is maintained by continuous passage in vivo; animals receiving an developed and modified this concept to include different types inoculum of IO5cells die 13 to 15 days later with a WBC count close of BsAbs for targeting various pharmacological agents, includ to 250,000/jil. Cells were recovered by flotation on Ficoll-Hypaque ing Vinca alkaloids (5, 6). More recently, we have constructed BsAb F(ab')2 derivatives (Lymphoprep; Nycomed as., Oslo, Norway) as described previously (12). Cell viability was always >90% as judged by trypan blue exclusion. Received 10/1/90; accepted 2/19/91. Antibodies and Antibody Fragments. For this study, five mouse mono The costs of publication of this article were defrayed in part by the payment clonal antibodies, anti-sap-1 to -5, were raised against saporin using of page charges. This article must therefore be hereby marked advertisement in the method of Köhlerand Milstein (13) as described by Fazekas de St. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work has been supported by Tenovus of Cardiff, the Cancer Research Groth and Scheidegger (14). Hybridoma cells secreting anti-saporin Campaign, and Italfarmaco, Milan, Italy. antibody were identified by ELISA and cloned by limiting dilution in 2To whom requests for reprints should be addressed. microculture plates. The other monoclonal antibody used to construct 'The abbreviations used are: RIP, ribosome-inactivating protein; BsAb, bi the BsAb was an anti-idiotype antibody, anti-Id-1, which is a mouse specific antibody; PBS, phosphate-buffered saline: BSA. bovine serum albumin: IgGl specific for the IgM idiotype of the L2C lymphoblastic leukemia ICso, 50% inhibitory concentration; K,, association (equilibrium) constant; DMEM, Dulbecco's modified Eagle's medium; PCS, fetal calf serum; ELISA, (9). enzyme-linked immunosorbent assay; IgGl, immunoglobulin Gl; IgM, immu- All hybridoma lines secreting these antibodies were expanded as noglobulin M: SH, sulfhydryl. ascitic tumors in Pristane-primed BALB/c x CBA F, mice. 2353 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. BISPECIFIC ANTIBODIES FOR THE DELIVERY OF SAPORIN TO LYMPHOMA Polyclonal antisera reacting with saporin and guinea pig l;;il>V (15) been precoated overnight with 7S IgG isolated from rabbit anti-saporin were raised in rabbits and rats, respectively, using standard immuniza antibody (20 Mg/ml in sodium carbonate buffer, pH 9.6). Test samples tion protocols (7). and antibody standards were then added, and antibody bound to the The 7S IgG fractions of ascitic fluid and polyclonal antiserum were saporin was detected with the appropriate dilution of horseradish isolated by precipitation in 2 M ammonium sulfate, followed by ion peroxidase-labeled rabbit anti-mouse immunoglobulin (Nordic Labo exchange chromatography on Trisacryl-M-DEAE (9). F(ab')2 frag ratories, Ltd., Maidenhead, Berkshire, United Kingdom). Each stage ments from both monoclonal and polyclonal IgG were prepared by of the assay consisted of a 90-min incubation at 37°C,followed by five limited proteolysis with pepsin at pH 4.2 as described previously (7, 9). washes in PBS to remove unbound reagent. Knowing the starting Preparation of BsAb. Heterodimeric F(ab')2 molecules containing concentration of antibody, we could then calculate the amount of two different mouse Fab' fragments or a mixture of mouse and rabbit antibody bound to saporin in the equilibrium mixture and plot the Fab' fragments were constructed as described by Glennie el al. (7). results by the method of Scatchard (18) to determine the K,s. For these Briefly, F(ab')2 molecules from the required antibody were reduced to calculations, it was assumed that each IgG molecule had the capacity obtain Fab' fragments with hinge region SH groups, Fab'(SH). The to bind two molecules of saporin. SH groups on one of the Fab'(SH) species were then fully alkylated In the second procedure, the binding of 125I-saporin to immobilized with excess o-phenylenedimaleimide to provide free maleimide groups. monoclonal antibody was determined. Flexible 96-well plates (Titertek; The two preparations, Fab'(mal) and Fab'(SH), were then
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