(CANCER RESEARCH 48. 926-931, Februar) 15, 1988] Immunochemotherapy of a Murine Thymoma with the Use of Idarubicin Monoclonal Antibody Conjugates Geoffrey A. Pietersz, Mark J. Smyth, and Ian F. C. McKenzie' Research Centre for Cancer and Transplantation, Department of Pathology, The University of Melbourne, Parkville, Victoria, 3052, Australia ABSTRACT Idarubicin is a derivative of daunomycin and is characterized by the absence of the methoxyl group at the C-4 position. It has been reported to have greater therapeutic effect than daunomycin. Idarubicin was chemically coupled to a monoclonal antibody to the murine Ly-2.1 alloantigen and the cytotoxicity of the drug-monoclonal antibody conju gate «TV«* free idarubicin was tested in vitro against I v-2f and Ly-2~ tumor cell lines. Some loss of idarubicin activity occurred upon conjuga tion to the monoclonal antibody; however, antibody activity was preserved and selective cytotoxicity for the I .y-2*cell line was observed. By contrast free idarubicin was equally cytotoxic for all cell lines (Ly-2* and Ly-2~). Idarubicin-anti-Ly-2.1 conjugates were tested for their capacity to inhibit solid tumor growth in (Ly-2.r, Ly-2.2+) (C57BL/6 x BALB/c)F, mice while their nonspecific effects were monitored by histológica!examina tion. The idarubicin-anti-Ly-2.1 conjugates when injected i.v. or directly into the tumor were observed to inhibit tumor growth more effectively than idarubicin or anti-Ly-2.1 alone, with smaller tumors being com pletely eradicated within several days of the completion of treatment. INTRODUCTION The concept of targeting antineoplastic agents to tumors using MoAbs2 is currently being evaluated (1,2). Generally the approach involves producing conjugates of antibody and a toxic agent, capable of selectively localizing and damaging tumor cells. Major attention has been directed towards constructing immunotoxins from A chains of plant and bacterial toxins and antibodies (3-5), such that their antigen binding and internali- zation leads to cell death. In practice, many MoAbs believed to be specific for tumors are also reactive with subpopulations of normal cells and consequently it may be unrealistic to utilize such potent toxins because of their potential damage to normal tissues. A safer alternative to plant toxins has been to couple antibodies to conventional anticancer drugs such as adriamycin (6), vindesine (7), chlorambucil (8, 9), melphalan (10), and methotrexate(ll, 12). Daunomycin, an anthracycline antineoplastic agent has been coupled, via the amino group of the sugar moiety, to antibodies either directly (6) or via a dextran (13) or polyglutamic acid (14, 15) intermediary. The amino group is thought to be essen tial for activity and to avoid the use of this group, a coupling procedure using the C-14 was devised (16). Since then dauno mycin has been coupled via C-14 to polyaspartic acid (17) and to MoAbs (18). We have used a similar procedure to couple a more toxic analogue of daunomycin, idarubicin (4-demethoxy- daunomycin) (Fig. \A) and in this study we report the sel jctive and potent in vitro and /'// rivo antitumor action of idr.rubicin conjugated to a murine anti-Ly-2 antibody. Received 8/5/87; revised 11/10/87; accepted 11/17/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed. •' I lie abbreviations used are: MoAb, monoclonal antibody; Ida, idarubicin; PBS, phosphate buffered saline: TFR, transferrin receptor; Br-Ida. bromoidarub- icin; II).,,,. 50% inhibition in |3H)thymidine incorporation of controls; ID,,,. dosage level of 10% lethality. MATERIALS AND METHODS Tumor Cells. The cell lines examined in this study included the (Ly- 2+) murine thymoma ITT(1)75NS E3 variant (E3) (8), the (Ly-2~, TFR-) lymphoma EL4 (19) and the (TFR*) human cell line CEM (20). Cells were maintained in vitro in Dulbecco's modified Eagle's medium or RPMI 1640 (Flow Laboratories, Sydney, Australia), supplemented with 10% heat-inactivated newborn calf serum (Flow), 2 mM glutamine (Commonwealth Serum Laboratories, Sydney, Australia); 100 ¿ig/ml streptomycin (Glaxo, Melbourne, Australia) and 100 IU/ml penicillin (CSL). The E3 tumor was maintained in vivo by serial passage into (Ly- 2.r, Ly-2.2*) (C57BL/6 x BALB/c)F,, (CBF,) mice. Cells from the ascites fluid were washed and centrifuged (400 g x 5 min) twice in PBS (pH 7.3), resuspended in PBS, and injected s.c. into the abdominal wall of mice. These developed into palpable tumors prior to treatment. Mice were subjected to a series of i.v. or intratumor treatments and the subsequent size of tumors measured daily with a caliper square, meas uring along the perpendicular axes of the tumors. Data were recorded as mean tumor size (product of two diameters ±standard error). Mice. CBA and CBF! mice were produced in the Department of Pathology, University of Melbourne. Experimental groups of eight to ten mice all of the same sex and age were used in each experiment. Monoclonal Antibodies. MoAbs used were: (a) Anti-Ly-2.1 (IgGi) reactive with the murine Ly-2.1 specificity (21) and (b) A3C6 (anti- TFR) (IgGi) reactive with the human transferrin receptor; this MoAb was made by M. Pannacio and satisfies the criteria for an anti-TFR MoAb (unpublished results). The MoAbs were isolated from ascites fluid by precipitation with 40% ammonium sulfate, dissolution in PBS and dialysis with the same buffer. These crude preparations were either adsorbed onto protein A-Sepharose (Pharmacia Inc., Piscataway, NJ), washed extensively with PBS (pH 7.3) and eluted with 0.2 M glycine/ HCl (pH 2.8), or passed through an Affigel blue column. Following neutralization MoAbs were then dialyzed against PBS, aliquoted, and stored at -70°C. When tested by sodium dodecyl sulfate-polyacryl- amide gel electrophoresis these MoAb preparations were demonstrated to be 90-95% pure. Preparation and Quantitation of Conjugates. Intact anti-Ly-2.1 or anti-TFR MoAbs (1-2 mg/ml in borate buffer, pH 8.0) were mixed with molar excesses (5- to 50-fold) of Br-Ida dissolved in (N,N)- dimethylformamide at 10 mg/ml. The reaction was maintained at room temperature for 4 h, before centrifuging (400 g x 5 min) to remove any precipitate caused by a loss of antibody charge and solubility. Free Br ida and other unreacted starting materials were removed by gel filtration chromatography using a Sephadex G-25 column (PD-10 Pharmacia) and the conjugates were then passed through a column of Porapak Q to remove any adsorbed drug (22). The amount of Ida incorporated in drug-MoAb conjugates was determined by absorbance spectrophotom- etry at 483 nm (E4g3= 3.4 x IO3M"1cm"') and protein estimation (23). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fil tration (S 300. Pharmacia) clarified that no high molecular weight aggregates were contaminating the Ida-MoAb preparation. Antibody Activity. A rosetting assay using sheep anti-mouse iinnui- noglobulin was used to determine the antibody activity of Ida-MoAb conjugates compared with free MoAb which had undergone the same procedures used in the coupling method (24). Drug Activity, (a) 24-h inhibition assay: 100 u\ of cells (2-5 x IO6/ ml) were added to a flat-bottom microtiter plate and incubated for l h at 37°C.Free Ida (dissolved in PBS) and Ida-MoAb conjugates were filtered sterile and dilutions were performed in sterile PBS; 50 u\ of free Ida or conjugate were added to the cells using duplicate wells/ sample; control wells received 50 n\ of PBS and the cells were cultured at 37°C, 7% CO2 for 24 h. (b) 30-min inhibition assay: 200 /J of cells 926 Research. on October 17, 2021. © 1988 American Association for Cancer cancerres.aacrjournals.org Downloaded from