Specific Killing of Human T-Leukemia Cells by Immunotoxins Prepared with Ricin A Chain and Monoclonal Anti-Human T-Cell Leukemia Antibodies1

In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of specific killing of human T-leukemia cells in vitro. These immunotoxins were prepared by conjugating ricin A chain (RIA) with our recently generated murine monoclonal anti bodies, SN1 and SN2, the latter of which was obtained from a hybridoma clone N6/D11 described previously by Negoro and Seon (Cancer Res., 42: 4259-4262, 1982), directed to two unique human T-cell leukemia antigens. We have shown previ ously that these monoclonal antibodies do not react with non-Tleukemia cells nor with various normal cells including normal Tcells, thymocytes, and bone marrow cells (Proc. Nati. Acad. Sci. U. S. A., 80: 845, 1983; Cancer Res., 42: 4259, 1982). Control conjugate was also prepared by conjugating RIA with a murine monoclonal immunoglobulin G (IgG), the isotype of which is the same as that of SN1 and SN2, i.e., IgGI-*. In initial experiments, the cytotoxic activity of an SN1 IgGiRIA conjugate preparation and the control IgG conjugate preparation against leukemic Tcell lines and normal B-cell lines was tested by two different test procedures, i.e., by measuring direct killing of the cells and by measuring inhibitory activity against protein synthesis in the cells. In each test, the SN1 conjugate showed specific cytotoxic activ ity against T-leukemia cells, whereas the control conjugate was not cytotoxic against either T-leukemia cells or normal B-cells. Nearly complete killing of T-leukemia cells and inhibition of protein synthesis in T-leukemia cells were observed at the concentra tions of 10~8 to 10~7 M of the SN1 lgG:RIA conjugate. In subsequent experiments, another preparation of SN1 IgGrRIA conjugate and an SN2 IgGiRIA conjugate preparation were tested individually and together for their inhibitory activity against protein synthesis in T-leukemia cells and control cells. With T-leukemia cells, specific inhibition was observed for both SN1 lgG:RIA and SN2 IgGiRIA. The combined use of these conjugates did not display a synergistic effect. Nevertheless, the combined use of different immunotoxins directed to different antigen molecules will be important in clinical use, since uncul tured tumor cells derived freshly from patients, in general, display heterogeneity with respect to the expression of tumor-associ ated antigens. These immunotoxins may be useful for the in vitro eradication of tumor cells in the bone marrow taken from patients with T-cell leukemia. Such tumor cell-free bone marrow speci mens will be very useful in autologous marrow transplantation to leukemia patients who have received aggressive high-dose chemotherapy and radiotherapy. INTRODUCTION Antibody carriers of toxins, termed immunotoxins, have drawn considerable attention recently because of their promising poten tial in tumor therapy. Preparation of useful immunotoxins requires appropriate specific antibodies; thus, monoclonal antitumor an tibodies will be very useful. We have generated recently and characterized several MCAbs3 that are directed to human T-cell leukemia antigens (14, 23). These MCAbs react with T-ALL cells but do not react with various non-T-leukemia cells nor with various normal cells including normal Tand B-cells, thymocytes, and bone marrow cells. In the present study, we selected 2 of these MCAbs, i.e., SN1 and SN2, for preparing immunotoxins [MCAb SN2 was obtained from hybridoma clone N6/D11 de scribed previously (14)]. These MCAbs define unique individual human T-cell leukemia antigens (14, 23) and are directed to different antigen molecules as determined by specific immunoprecipitation followed by SDS:PAGE." Of the toxins, we chose ricin, a plant toxin derived from the castor bean, since the usefulness of the antibody:RIA conjugates for the in vitro killing of tumor cells has been reported already by several groups of investigators (6, 8-11, 15, 18, 28). The ricin molecule consists of 2 subunits, an A and a B chain, which are linked by a disulfide bond (reviewed in Ref. 16). The B chain is a lectin which is specific for galactose residues present on the surface of a wide variety of cells. The binding of the ricin B chain to mammalian cells allows the entry of the entire ricin molecule, and conse quently the RIA, into the cytoplasm where the A chain is split from the B chain and acts on the 60S subunit of the ribosome to cause irreversible inhibition of protein synthesis and cell death. Isolated ricin B chain binds to cell surfaces but is nontoxic, while isolated RIA is not significantly toxic because of its inability to efficiently bind to cell surface and to traverse the cell membranes. Thus, conjugates of RIA with appropriate monoclonal antitumor antibodies may provide immunotoxins specifically targeted on the tumor cells that react with the antibodies. Our present immunotoxins of anti-T-leukemia MCAbs and RIA showed specific killing of T-leukemia cells in vitro. Clinical utili zation of such immunotoxins will be important in view of the facts that T-cell leukemia is associated with poor prognosis (1, 2, 7,17, 20, 22, 29) and that autologous marrow transplantation using leukemia cell-free bone marrow has promising potential in the cure of patients with T-cell leukemia (see "Discussion"). ' This was supported in part by USPHS Grant CA19304 awarded by the National Cancer Institute. 2A unit of the New York State Department of Health. Received March 22, 1983; accepted September 28. 1983 3The abbreviations used are: MCAb, monoclonal antibody; ALL, acute lymphoblastic leukemia; SDS. sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; RIA, ricin A chain; SPDP. N-succinimidyl-3-(2-pyridyldithio)propionate; PBS, phosphate-buffered saline, RPMI, Roswell Park Memorial Institute; PCS, fetal calf serum; TCA, trichloroacetic acid. *B. K. Seon and S Negoro. Human T-cell leukemia-associated cell surface glycoprotein defined by monoclonal antibody, manuscript in preparation.