Sep 9, 1982 - gates with horse anti-AFP and monoclonal mouse anti-AFP were capable .... from a plot of y/A - X vs. y, in which A is the starting concentration.
Proc. Natl Acad. Sci. USA
Vol. 79, pp. 7896-7899, December 1982 Medical Sciences
Chemotherapy by intravenous administration of conjugates of daunomycin with monoclonal and conventional anti-rat a-fetoprotein antibodies (immunotargeted chemotherapy/a-fetoprotein-secreting hepatoma)
YUTAKA TSUKADA*, ESTHER HuRwrrZt, RINA KASHIt, MICHAEL SELAt, NOZOMU HIBI*, AKIHIKO HARA*,
AND HIDEMATSU HIRAI* *Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan; and tDepartment of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel Contributed by Michael Sela, September 9, 1982
ABSTRACT Monoclonal antibodies to rat a-fetoprotein (AFP) were produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with rat AFP. The monoclonal antibodies as well as horse anti-rat AFP were coupled via a dextran bridge to daunomycin. Both types of conjugates were tested in vitro and in vivo for their anti-tumor activity. They were equally cytotoxic to rat AH66 hepatoma cell line in culture. Rats challenged with hepatoma cells were treated with the conjugates either by intraperitoneal or intravenous injections. Daunomycin conjugates with horse anti-AFP and monoclonal mouse anti-AFP were capable of delaying the tumor development more efficiently than the controls of antibodies or free drug, mixtures of drug with antibodies, and a conjugate of drug and normal immunoglobulin. The specific conjugates were considerably more effective when the treatments were given intravenously. The specific conjugates produced 60% long-term survival, whereas the controls delayed only slightly tumor development.
in vivo to that of the conjugate made with conventional antibodies. Chemotherapeutic studies were performed with both types of conjugates in the treatment of rat hepatoma. Although previously the tumor and the treatments were delivered by the same route, intraperitoneally (i.p.) (1), treatments now also were given intravenously (i.v.). The systemic treatment with the specific carriers ofthe i.p. tumor produced 60% long-term survival, suggesting high efficacy and successful targeting at the tumor sites.
MATERIALS AND METHODS Specific Antibodies to AFP. The preparation ofhorse anti-rat AFP and the purification of the specific antibody by affinity chromatography have been described (10, 11). Monoclonal antirat AFP was prepared by hybridization (2) according to a procedure described by Eshhar et al. (12). Spleen cells were taken from mice that were immunized with rat AFP according to the following schedule. The mice were injected into the footpad twice (10 days apart) with 10 ,Ag of rat hepatomal AFP (13) in complete Freund adjuvant. Six and 5 days prior to the hybridization they received 10 ,g of AFP, iLv., and i.p., respectively. Positive cultures were detected by solid-phase indirect radioimmunoassay (14), by using as a second antibody "2I-labeled goat anti-mouse F(ab')2 (specific activity, 2 X 10 cpm/mg). They were cloned subsequently in agar or by limiting dilutions. The clones were grown in vitro in culture or in CD2 mice. Tumor Cells. The rat ascites cell line AH66 was used throughout the experiments. It was maintained by i.p. passage in syngeneic Donryu rats or cultivation in vitro (15, 16). Preparation of Daunomycin-Immunoglobulin Conjugates. Daunomycin hydrochloride (Farmitalia, Milano, Italy) was bound to horse and monoclonal antibodies to rat AFP or to normal horse immunoglobulin via a dextran bridge as described (17). The Pharmacological Activity of the Conjugates. Inhibition by drug and drug conjugates of [3H]thymidine (specific activity, 25 Ci/nmol; 1 Ci = 3.7 X 10'° becquerels; Radiochemical Centre, Amersham, England) incorporation into AH66 cells was taken as a criterion of the pharmacological activity (18). The Antigen-Binding Activity of the Antibodies and Their Drug Conjugates. The antigen binding was tested by the following procedure (19). "2I-labeled rat AFP with a specific activity of 2 x 107 cpm/nmol [ref. 20 (appendix, procedure B)] was incubated with dilutions of the antibody in 100 ,ul of 10% horse serum for 24 hr; it then was centrifuged through 1 ml of 21% polyethylene glycol at 1,050 X g for 10 min. The super-
One of the major limitations of effective cancer chemotherapy is that effective killers of neoplastic cells also are usually toxic to proliferating normal cells. One way of improving the selectivity of chemotherapeutic agents is the utilization of antibodies directed against the tumor cells as specific carriers ofthe drugs. It was demonstrated recently that horse antibodies to rat afetoprotein (AFP) linked to an antineoplastic drug, daunomycin, maintained both its cytotoxic potential as a drug and its ability to bind AFP (1). This conjugate inhibited in vitro and in vivo the development of rat hepatoma AH66 in a considerably more efficient manner than free daunomycin, anti-AFP, a mixture of daunomycin and anti-AFP, and a conjugate of daunomycin and
normal horse immunoglobulin. The preparation of tumor-reactive monoclonal antibodies by the hybridoma technique (2) has stimulated much interest in their use for the targeting of cytotoxic agents to tumor cells or tissues. Several groups have prepared monoclonal antibodies against tumor-associated antigens and have used them as carriers of the toxic moiety of plant or bacterial toxins (3-7) and recently, of adriamycin (8). Hybridoma antibodies against human AFP also have been prepared (9) and suggested both for clinical uses as diagnostic aids and as a tool in the evaluation of the antigenic structure of this group of proteins. It was of interest to test a monoclonal antibody to AFP as a specific drug carrier against an AFP-secreting hepatoma. We have prepared hybridoma antibodies to rat AFP and attached them, via a dextran bridge, to daunomycin. The activity of this daunomycin-anti-rat AFP conjugate was compared in vitro and
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Abbreviations: AFP, a-fetoprotein; i.p., intraperitoneal(ly); i.v., intravenous(ly). 7896
Proc. Natl. Acad. Sci. USA 79 (1982)
Medical Sciences: Tsukada et al.
7897
natant was removed and the radioactivity in the precipitate was counted in a gamma counter. The equilibrium constants were determined by solid-phase radioimmunoassay as described (1), by using '25I-labeled antibodies. In Vitro Studies. The hepatoma cells were maintained in culture in the presence ofthe specific antibody-drug conjugates or the controls for 48 hr. The growth and viability of cells were determined by trypan blue exclusion. In Vivo Studies. Hepatoma cells (104 cells per rat) were transplanted i.p. Treatments were injected by the same route or i.v., beginning with the third day after the tumor challenge.
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a
RESULTS Monoclonal Antibodies to Rat AFP. The highest reacting antibody-producing culture obtained from the hybridization was cloned and perpetuated in vitro and in vivo. Immunoglobulin was precipitated from the ascites fluid of hybridoma-312growing mice by 45% saturated (NH4)2SO4. The equilibrium association constants (20) of the hybridoma and the horse antibodies to rat AFP were determined by using "2I-labeled antibodies and by studying their binding to AFP attached to paper discs. Similar constants were obtained for both types of antibodies and with secreted (Fig. 14A or with membrane-bound (Fig. 1B) AFP. Daunomycin-Immunoglobulin Conjugates. Daunomycin was linked through polyaldehyde-dextran to monoclonal antiAFP and to horse anti-AFP. The drug activities of the conjugates, as measured by the inhibition of [methyl-3H]thymidine incorporation over a period of 24 hr, were between 60% and 100% of that of the original drug. The Antibody-Binding Activity of Daunomycin-Specific Antibody Conjugates. The binding activity of the intact antibodies and of their daunomycin conjugates was measured as indicated above by using the labeled antigen. The results depicted in Fig. 2 demonstrate that the complete antigen-binding activity was sustained.
0 x
5
10 y x 10-11
15
1 2 y x 0-5
FIG. 1. The association constants of the antibodies to rat AFP. The equilibrium association constants were determined by the binding of the '251-labeled horse and '251-labeled monoclonal antibodies to AFP. (A) The antigen was secreted and the constants were 7.4 x 108 M-1 and 7.1 x 108 M-1 for horse anti-AFP (e) and monoclonal anti-AFP (o), respectively. (B) The antigen was membrane-bound AFP, and the constants were 8.1 x 108 M-1 and 4.7 x 108 MW1 for horse anti-AFP (o) and monoclonal anti-AFP (o), respectively. The constants were derived from a plot of y/A X vs. y, in which A is the starting concentration of the antibody, X is the molar concentration of bound antibody, and y is the number of antibody molecules bound per antigen. -
