ICANCER RESEARCH 46, 1208-1213, March 1986] Elimination of Clonogenic Tumor Cells from Human Bone Marrow Using a Combination of Monoclonal Antibody:Ricin A Chain Conjugates1 M. Bregni,2 3 P. De Fabritiis,2 4 V. Raso, J. Greenberger, J. Upton, L. Nadler, L. Rothstein, J. Ritz,5 and R. C. Bast, Jr.6 Divisions oÃ-Tumor Immunology [M. B., P. D. F., L. N., J. P., R. C. B.], Cancer Pharmacology [V. R.], Medicine [R. C. B.], Radiation Therapy [J. G., L. R.], and Pediatrie Oncology [J. L.], Dana-Farber Cancer Institute; the Department ol Medicine, Brigham and Women's Hospital [L. N., J. R., R. C. B.]; and the Departments of Medicine [L. N., J. R., R. C. B.¡,Radiation Therapy [J. G.¡,Pathology [V. R.], and Pediatrics [J. L], Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT Effective autologous bone marrow transplantation for leukemia and lymphoma is likely to depend upon the selective removal in vitro of malignant cells from normal human bone marrow precur sors. Highly specific cytotoxic conjugates formed by coupling ricin A chain to monoclonal antibodies might prove useful for the selective elimination of malignant cells. Consequently, ricin A chain conjugates have been prepared with several different murine monoclonal antibodies and tested for their ability to eliminate clonogenic Burkitt's lymphoma cells from an excess of human bone marrow. The most active reagents included an antibody:A chain conjugate which bound to the nonpolymorphic chain of the la molecule and another which reacted with the u. heavy chain of cell surface immunoglobulin. Conjugates formed with anti-common acute lymphoblastic leukemia antigen, anti-Mr 26,000 glycoprotein, and anti-B1 were much less active on these Burkitt's cells, contrasting with results of complement-dependent tumor cell lysis. Tumor cell kill was partially inhibited by the addition of greater than 2 x 106 human bone marrow cells/ml but could be potentiated by increasing the concentration of conjugate or by the addition of 10 HIM ammonium chloride. In the presence of ammonium chloride, at least 4 logs of clonogenic tumor cells could be eliminated within 24 h from a 20-fold excess of bone marrow using 10~7 M ricin A chain linked to one or two different antibodies. Similar treatment of normal human bone marrow temporarily inhibited granulocyte-macrophage colony- forming units (cell) formation but did not compromise establish ment of continuous bone marrow cultures. The degree of selec tive elimination of tumor cells with A chain antibody conjugates was comparable to that achieved with 4-hydroperoxycyclophos- phamide or with multiple monoclonal antibodies and complement. INTRODUCTION One requirement for autologous bone marrow transplantation is the complete removal of malignant cells from human bone marrow while sparing normal progenitors. Selective elimination of malignant cells has been achieved through the use of cytotoxic drugs (1) and of antibodies (2), in both animal systems (3, 4) and clinical studies (5, 6). In recent reports, monoclonal antibodies have been used in combination with heterologous complement Received 1/2/85; revised 11/25/85; accepted 12/2/85. 1Supported in part by National Cancer Institutes Grants CA 28740 and CA 29039. 2 Fellow of the Associazione Italiana per la Ricerca sul Cancro. 3 Fellow of Fondazione A. Villa Rusconi. 4 Fellow of the American-Italian Cancer Research Foundation. * Scholar of the Leukemia Society of America, Inc. "Scholar of the Leukemia Society of America, Inc. To whom requests for reprints should be addressed, at Box 3843, Duke University Medical Center, Durham, NC 27710. (2) or have been conjugated with intact ricin (7) or ricin A chain (8-10). Given antibodies of appropriate specificity, selective de struction of tumor cells has been achieved using some systems in vitro. It remains to be determined, however, which antigenic determinants will prove to be optimal targets for conjugates containing ricin A chain and monoclonal antibodies. Recently, we have conjugated ricin A chain to several different monoclonal antibodies which react with well-defined antigenic determinants expressed on Burkitt's lymphoma tumor cell lines. As these lines exhibit high clonogenic efficiency, it has been possible to study elimination of several logs of tumor cells in the presence of a 20-fold excess of irradiated human bone marrow. Optimal conditions for the selective elimination of Burkitt's tumor cells have been defined, and the effect of treatment on normal human bone marrow has been evaluated in short-term and continuous bone marrow cultures. MATERIALS AND METHODS Burkitt's Lymphoma Tumor Cell Lines. Namalwa, Bjab, Bjab 113, CA 46, and JD 38 were used in this study (11). Cells were grown in RPMI 1640 medium (Microbiological Associates, Walkersville, MD) con taining 10% heat-inactivated FBS7 (Gibco Labs., Grand Island, NY), 2 rriM L-glutamine, 1 HIM sodium pyruvate, 10 ITIM hydroxyethylpi- perazineethanesulfonic acid, penicillin (100 units/ml), and streptomycin (100 /jg/ml). Cells were cultured at 37°C in 5% CO2-95% humidified air. Each of the Burkitt's lymphoma cell lines bore multiple antigenic deter minants including la, CALLA, gp26, B1, B2, and cell surface immuno globulin. Monoclonal Antibodies. An lgG1 monoclonal antibody, designated 287, reacts with the n heavy chain expressed on normal and malignant B-cells.e The J2 antibody is an lgG2a, murine monoclonal reagent that reacts with the nonpolymorphic portion of the M, 32,000 A chain of the la complex that is expressed on a majority of B-cell cancers (12). J5 is an lgG2a murine monoclonal antibody (13) directed against the CALLA; J2 and J30 are, respectively, an IgM and an lgG2a reactive with the gp26 cell surface glycoprotein previously described (14). B1 is an lgG2a antibody that recognizes a M, 35,000 B-cell surface antigen (15). Mono clonal antibodies directed against human immunoglobulin light and heavy chain determinants have been developed (16). To evaluate reactivity of these reagents with the cell lines used in the study, indirect immunofluorescence assays were performed. Burkitt's lymphoma cells were incubated for 30 min at 4°Cin medium containing a 1:100 dilution of each antibody in PBS with 5% FBS. Cells were washed twice in the same medium without antibody and incubated for 30 min at 4°Cwith a 1:20 dilution of fluorescein-conjugated goat anti- mouse immunoglobulin (Meloy Lab., Inc., Springfield, VA). Fluorescence intensity was evaluated using an Ortho Cytofluorograph 30-L (Ortho Diagnostics, Westwood, MA). 7The abbeviations used are: FBS, fetal bovine serum; CALLA, common acute lymphoblasticleukemiaantigen;GM-CFU-C,granulocyte-macrophagecolony-form ing units (cell); MEM, minimal essential medium; PBS, phosphate-buffered saline gp26, M, 26,000 glycoprotein. 8L. Nadler, personal communication. CANCER RESEARCH VOL. 46 MARCH 1986 1208 Research. on January 19, 2016. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from