Advances in Brief Anti-CD19 Antibodies Inhibit the Function of the P-gp Pump in Multidrug-resistant B Lymphoma Cells

After chemotherapy, tumor cells with multidrug resistance (MDR) often emerge. MDR is attributable to the expression of membrane transport proteins that inhibit the cellular influx and increase the efflux of many chemotherapeutic drugs. One such protein is P-glycoprotein (P-gp), which functions as an ATP-dependent active transporter. Recently, an anti-P-gp monoclonal antibody (MAb) that inhibits P-gp has been described. Previous studies from our laboratory using the antiCD19 B-cell lymphoma-reactive MAb, HD37, have suggested that HD37 may also influence MDR. To test this directly, we used Namalwa/MDR1 cells to study the effect of HD37 on the efflux of rhodamine 123 from these cells. We found that HD37 and three other anti-CD19 MAbs inhibited the efflux of rhodamine 123 from Namalwa/MDR1 cells with ;50% of the efficiency of the well-known chemosensitizer, verapamil. In contrast, MAbs against seven other molecules expressed on these cells were ineffective. The inhibitory activity of HD37 did not require an Fc portion; F(ab*)2 fragments were effective, but Fab* fragments were not, suggesting that higher avidity binding and/or cross-linking of CD19 are necessary. We could find no evidence that HD37 recognizes a cross-reactive epitope on P-gp, modulates P-gp from the cell surface, or enhances the ATPase activity of membranes from treated cells. Introduction After chemotherapy, MDR tumor cells often emerge and, because of this, patients with relapsed cancers have a poor prognosis. The phenomenon of MDR is often attributable to the expression of membrane transport or “pump” proteins, which inhibit the cellular influx and increase the efflux of many chemotherapeutic drugs (1, 2). One such protein is a Mr 170,000 P-gp, a member of the ATP-binding cassette transporter family, that functions as an ATP-dependent active transporter. A variety of agents have been developed to prevent MDR, including analogues of transported drug substrates (e.g., verapamil), inhibitors of ATP binding/utilization (e.g., olygomycin), and MAbs that recognize epitopes on the P-gp molecules (e.g., MRK16; Ref. 3) and block its activity (e.g., UIC2; Ref. 4). Although agents such as verapamil and olygomycin have side effects in humans, many MAbs are well tolerated, although anti-P-gp MAbs have not yet been used in humans. CD19 is a B cell-specific glycoprotein that is expressed on virtually all normal and neoplastic cells of the B-cell lineage and which plays a role in signaling through the B cell receptor complex and other cell surface molecules (e.g., CD72; Ref. 5). Previous studies have shown that immunotoxins prepared with anti-CD19 MAbs render Namalwa/MDR1 tumor cells more sensitive to chemotherapeutic agents and reduce the levels of expression of P-gp on surviving cells (6, 7). Because immunotoxins enter cells through routes that avoid the P-gp pump and kill them by inhibiting protein synthesis, they could also result in a decrease in P-gp synthesis in surviving cells and, hence, decrease MDR. Our previous studies have shown that a dimer of the “naked” anti-CD19 MAb, HD37, can both signal CCA in lymphoma cells and potentate the cytotoxic effects of several chemotherapeutic agents in severe combined immunodeficiency mice with human lymphoma xenografts (8). These data suggested that HD37 itself, even without a toxin component, might be a chemosensitizer. In the present study, we explored this possibility by determining whether anti-CD19 MAbs would inhibit the efflux of rhodamine 123 from the P-gp Namalwa/MDR1 cells. We compared the effect of HD37 and other anti-CD19 MAbs to those of both verapamil and a unique anti-P-gp MAb, UIC2, which has been reported to inhibit P-gp activity (4, 9). We found that HD37 and three other anti-CD19 MAbs, used at 100-fold lower concentrations than those required to signal CCA (10), decreased the efflux of rhodamine 123 from the Namalwa/ MDR1 cells, and that this effect did not occur using MAbs against seven other non-CD19 molecules expressed on the Namalwa/MDR1 cells. We have excluded the possibility that the HD37 MAb recognizes an epitope on P-gp, that it modulates P-gp from the cells, or that it alters ATP levels in cells. By exclusion, we hypothesize that it alters another biochemical function of P-gp and/or that the signaling pathways used by CD19 and P-gp share a common intermediary. If this should be the case in lymphoma cells from patients, HD37 might be useful as an antitumor agent, not only because of its ability to deliver toxins and (as a dimer) induce CCA, but also because it acts as a chemosensitizer. Materials and Methods Cell Lines, Antibodies, and Reagents. The human Burkitt’s lymphoma cell line, Namalwa, was infected with a human MDR1 gene-containing retrovirus (Namalwa/MDR1) and was a gift from Dr. R. O. O’Connor at ImmunoGen (Boston, MA; Refs. 6 and 7). Both the parental P-gp Namalwa cells and the transfected P-gp Namalwa/MDR1 cell lines were maintained in culture in complete RPMI 1640 containing 10% heat-inactivated FBS Received 6/30/99; revised 10/11/99; accepted 10/18/99. 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. 1 Supported in part by NIH Grant CA64679. 2 To whom requests for reprints should be addressed, at Cancer Immunobiology Center, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75235-8576. Phone: (214) 648-1200; Fax: (214) 648-1204; E-mail: [email protected]. 3 The abbreviations used are: MDR, multidrug resistance; CCA, cell cycle arrest; FBS, fetal bovine serum; MAb, monoclonal antibody; MDR1, multidrug resistance human gene; MRP, multidrug resistance protein; P-gp, P-glycoprotein; FACS, fluorescence-activated cell sorter. 3920 Vol. 5, 3920–3927, December 1999 Clinical Cancer Research Research. on July 14, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from supplemented with 20 mM HEPES, 100 units/ml penicillin, 100 mg/ml streptomycin, and 100 mM L-glutamine. A-498 cells (a human kidney carcinoma cell line from American Type Culture Collection) were used as a P-gp, CD19 control. Cells were cultured in Eagle’s MEM with 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 90% Eagle’s balanced salt sodium, supplemented with 10% FBS. The cells were detached from culture flasks by treatment with a 0.25% trypsin solution, washed two to three times with medium, and phenotyped by immunofluorescence and FACS analysis. These cells were also used in the rhodamine 123 efflux assay as described below. Cells were grown in a humidified atmosphere of 5% CO2 and air, and viability was determined by trypan blue exclusion. Cell lines were maintained in culture for 6 weeks and were then replaced with frozen stock. The cellular phenotype was determined using a panel of MAbs. MAbs and Other Reagents The following MAbs or hybridomas were used: (a) antiCD19: HD37 from Dr. D. Dorken, Germany (purified in our laboratory); BU12 from Dr. D. Flavell, University of Southampton, Southampton, United Kingdom; (4G7) from Dr. R. Levy, Division of Oncology, Stanford University, Stanford, CA; and FMC63 from Dr. M. LeTarte at the University of Alberta, Alberta, Canada; (b) anti-CD20: 1F5 from Bristol Meyers, Seattle, WA and the chimeric anti-CD20 (IDEC-C2B8 or Rituxan) from Genentech, San Francisco, CA; (c) anti-CD21: G28-5 from American Type Culture Collection, Rockville, MD; (d) anti-CD22: RFB4 from Dr. G. Janossy, Royal Free Hospital, London, United Kingdom; (e) antiCD40: G29-5 from Bristol Myers, Seattle, WA; (f) anti-CD79a, CD79b: ZL9-1, ZL7-4 from Dr. M. Glennie, Tenovus Research Lab, Southampton General Hospital, Southampton, United Kingdom; (g) goat anti-human IgM (m chain) from Sigma, St. Louis, MO; (g) 3F12, an IgG1 isotype-matched control MAb, was a gift from Dr. E. Hansen, University of Texas Southwestern Medical School, Dallas, TX; (h) anti-P-gp: 4E3, from Signet Laboratories, Dedham, MA; MRK16 from Kamya Biochemical Co., Seattle, WA; UIC2 from Immunotech, Miami, FL; and (i) FITC-goat anti mouse IgG (H1L): GAMIg from Kirkegaard & Perry Laboratory, Inc., Gaithersburg, MD. All antibody preparations used in this study were extensively dialyzed against PBS, and concentrations were normalized in RPMI 1640. Verapamil and rhodamine 123 were purchased from Sigma. DIOC2 was purchased from Molecular Probes (Eugene, OR) The affinity constant (Ka) for the antiCD19 MAb was determined by Scatchard analysis as described previously (11).