[CANCER RESEARCH 49. 4829-4834. September I. 1989] Elimination of Chemoresistant Multiple Myeloma Clonogenic Colony-forming Cells by Combined Treatment with a Plasma Cell-reactive Monoclonal Antibody and a P-Glycoprotein-reactive Monoclonal Antibody1 Alex W. Tong,2 Jennifer Lee, Ru-Mun Wang, William S. Dalton, Takashi Tsuruo, Joseph W. Fay, and Marvin J. Stone Cancer Immunology Research Unit [A. W. T., J. L., R. M. W., M. J. S.] and Bone Marrow Transplantation Unit ¡J.H'. F.], Charles A. Sommons Cancer Center, Baylor University Medical Center, Dallas, Texas 75246; the Department of Medicine, University of Arizona Cancer Center, Tucson, Arizona 85724 [W. S. D.]; and the Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan /T. T.J ABSTRACT Patients with multiple myeloma (MM) commonly become refractory to chemotherapy despite a favorable response to induction treatment. We examined the effectiveness of a previously characterized plasma cell- reactive monoclonal antibody, MM4, in eliminating MM clonogenic colony-forming cells (CCC) with a multidrug-resistant (MDR) pheno- type. Experiments were performed using MM cell lines that exhibit 6 (RPMI 8226/DOX,,)- and 40 (RPMI 8226/DOX,,,)-fold resistance to doxorubicin (DOX). Both lines were selected from the chemosensitive MM line RPMI 8226/S and were cross-resistant to mitoxantrone, acro- nycine, etoposide, and vincristine. Surface marker analysis conducted in this study showed that DOX« and DOX«, overexpressed the MDR1 gene product pi 70. Both MDR lines remained reactive to the plasma cell- reactive monoclonal antibodies MM4 and PCA-1 and expressed the relevant cytoplasmic immunoglobulin light chain. Treatment with MM4 and rabbit complement ((.") was equally cytotoxic to RPMI 8226/S (80 ±5.6% (SD)|, DOX,,|74 ±8.5|, and DOX,,, cells |75 ±11.3%], based on short-term chromium release studies. Furthermore, MM4 + C' de leted up to 3 logs of CCC colonies from chemosensitive and MDR lines (RPMI 8226/S, 99.87 ±0.11%; DOX«99.91 ±0.08%; DOX«,, 99.55 ± 0.44%). By comparison, the P-glycoprotein-reactive monoclonal antibody MRK-16 and C' inhibited tumor colony formation of MDR cells (8226/ DOX6, 95.71 ±2.51%; 8226/DOX*,, 99.61 ±0.43%) but affected that of chemosensitive cells only slightly (8.9 ±17.8%). In an attempt to optimize the depletion of myeloma CCC, MM4 was used together with MRK-16. This approach resulted in uniform depletion of myeloma clon ogenic colony-forming cells from the chemosensitive (98.32 ±1.53%, n = 4) and MDR lines (8226/DOX,,, 98.83 ±0.08%, n = 4; 8226/DOX«* 99.29 ±0.62, n = 7) but did not result inenhanced CCC depletion. When DOX«, cells were mixed with normal bone marrow (BM) in the ratio of 90:10 (BM:MM), either MM4 or MRK-16 and C' depleted MM colonies (98.8 ±0.71% and 98.10 ±1.0%, respectively) without affecting the majority of BM progenitor cells. These observations suggest that either MM4 or MRK-16 is useful for depleting MDR myeloma clonogenic colony-forming cells. INTRODUCTION MM1 represents a malignant monoclonal proliferation of plasma cells which is currently incurable (1, 2). Chemotherapy Received 12/5/88; revised 3/31/89; accepted 6/8/89. 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. 1Supported in part by grants from the Charles A. Sammons Cancer Center Research Fund, the Baylor Research Foundation Oncology/Immunology Re search Fund, and the Tri Delta Cancer Research Fund. 2To whom requests for reprints should be addressed, at Cancer Immunology Research Unit, Charles A. Sammons Cancer Center, Baylor University Medical Center. 3500 Gaston Avenue. Dallas. TX 75246. 'The abbreviations used are: MM. multiple myeloma; MoAb, monoclonal antibody; CCC, clonogenic colony-forming cell: MDR. multidrug resistant; DOX. doxorubicin/Adriamycin: FBS, fetal bovine serum; BM, bone marrow; FITC, fluorescein isothiocyanate; PCD. plasma cell dyscrasia; CFU-GM. colony forming unit-granulocytic/monocytic; CFU-GEMM, colony forming unil-granulocyte/ erythrocyte/monocyte/macrophage; BFU-E, burst-forming unit erythrocyte; GCT, giant cell tumor; clg: cytoplasmic immunoglobulin; CALLA, common acute lymphocytic leukemia antigen: MFI, mean fluorescence index; pl70, M, 170,000 p-glycoprotein; VAD. vincristine/Adriamycin/dexamethasone. can induce objective remission in 30-60% of patients but is largely ineffective at relapse (3). Patients with refractory disease commonly develop resistance to previously administered drugs as well as to drugs of different chemical structure and mecha nism of action [3-5]. This phenomenon, known as pleiotropic drug resistance (6), severely limits the effectiveness of conven tional chemotherapy in treating patients with refractory MM. In recent studies, experimental approaches such as immuno- therapy or intensive experimental chemotherapy have been considered as possible treatment alternatives (3, 7, 8). However, their efficacy in destroying chemoresistant tumor cells has not been clearly defined. Recently, Dalton et al. (4) have developed a number of DOX- resistance sublines from the well-characterized MM cell line RPMI-8226/S (9). DOX is frequently used in combination chemotherapy for the treatment of the alkylating agent-resistant myeloma (3, 10). Myeloma patients treated with DOX eventu ally relapse and became resistant to multiple drugs (1,3, 10). Two RPMI 8226 sublines, selected through continuous culture with DOX, exhibited a stable phenotype that was 6 (RPMI- 8226/DOX6)- or 40 (RPMI 8226/DOX4,,)-fold resistant to DOX (4, 5). Both were cross-resistant to other chemotherapeu- tic agents, including mitoxantrone, acronycine, etoposide, and vincristine, and overexpressed the Mr 170,000 membrane P- glycoprotein or p 170, a product of the MDR1 gene (4, 5). Nevertheless, these MDR cells retained plasma cell features of the chemosensitive parent line (4). Recent clinical studies in dicate that certain patients with refractory MM also express the MDR] phenotype (11, 12). These observations indicate that RPMI 8226/DOX, and RPMI 8226/DOX4»cells can serve as valuable in vitro models for the study of refractory MM. With the use of clonogenic assays, Karp et al. ( 13) and Durie et al. (14) showed that tumor CCCs derived from primary culture of MM patient BM were closely related to the self- renewing myeloma stem cell in vivo. The capacity to eliminate this clinically important tumor subset may be a potentially useful therapeutic adjunct. Previously, we showed that the parallel myeloma colony-forming subset from established MM cell lines can be deleted selectively from normal human BM (15), with the use of the plasma cell-reactive MoAb MM4 and C'. In this study, we compared the susceptibility of MDR DOX,, and DOX4I, myeloma cells to MM4 + C' treatment with their chemosensitive counterpart. Similar cell-killing experiments also were conducted with the P-glycoprotein-reactive MoAb MRK-16 (16), since pl70 was overexpressed on certain MDR myelomas (5, 11, 12) but has a limited expression on normal human tissues (17, 18). MATERIALS AND METHODS Human Cell Lines and Culture Conditions. The RPMI 8226 human myeloma cell line was obtained from the American Type Culture 4829 Research. on January 22, 2022. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from