Cancer Therapy: Preclinical The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma Cells In vitro and In vivo

Purpose: We investigated the antitumor effect of murine/human chimeric CD138specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM. The cell surface proteoglycan CD138 (syndecan-1) is an integral membrane protein acting as a receptor for the extracellular matrix. Within the normal human hematopoetic compartment, CD138 is expressed on differentiated plasma cells and is a primary diagnostic marker of multiple myeloma (MM; ref. 1). The large extracellular domain of CD138 binds via its heparin sulfate chains to soluble extracellular molecules, including the growth factors epidermal growth factor, fibroblast growth factor, and hepatocyte growth factor, and to insoluble extracellular molecules, such as collagen and fibronectin (2, 3). CD138 also mediates cell-cell adhesion through interactions with heparinbinding molecules. Studies of plasma cell differentiation show that CD138 is a differentiation antigen (4) and a coreceptor for MM growth factors (5). Several monoclonal antibodies (mAb; i.e., B-B4, BC/B-B4, BB2, DL-101, 1 D4, MI15, 1.BB.210, 2Q1484, 5F7, 104-9, 281-2) specific for CD138 have been reported. B-B4, 1D4, and MI15 antibodies, which bind to similar or closely related epitopes, Authors' Affiliations: LeBow Institute for Myeloma Therapeutics and Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute Harvard Medical School, Boston, Massachusetts; First Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan; ImmunoGen, Inc., Waltham, Massachusetts; and Biotest AG, Dreieich, Germany Received 11/5/08; revised 1/28/09; accepted 2/9/09; published OnlineFirst 6/9/09. Grant support: NIH grants CA50947, CA78373, and CA10070; a Biotest Fellowship; and The LeBow Family Fund to Cure Myeloma. 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. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Kenneth C. Anderson, Department of Medical Oncology, Dana-Farber Cancer Institute, Mayer 557, 44 Binney Street, Boston, MA 02115. Phone: 617-632-2144; Fax: 617-632-2140; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2867 4028 Clin Cancer Res 2009;15(12) June 15, 2009 www.aacrjournals.org Research. on July 17, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from recognize both the intact CD138 molecule and the core protein (with the heparin sulfate chains removed) and target the same or closely related epitopes. B-B4 preferentially binds to membrane-bound versus soluble CD138 (6). It is a murine IgG1 mAb that binds to a linear epitope between residues 90 to 95 of the core protein on human syndecan-1. Consistent with the expression pattern of CD138, B-B4 strongly reacts with the MM cell line RPMI8226, but not with endothelial cells. Moreover, B-B4-saporin immunotoxin is highly cytotoxic to RPMI8226 cells (7). mAbs are emerging as an important cancer therapy (8), particularly for the treatment of hematologic malignancies. For example, anti-CD20 mAb rituximab, which induces lysis and apoptosis of normal and malignant human CD20-positive B cells by complement-dependent cytotoxicity and antibodydependent cellular cytotoxicity, is now broadly used to treat CD20-positive cancers. Additional mAb-based therapies have been developed for treatment of non–Hodgkin lymphomas, including the radioimmunotherapeutics 90Y-ibritumomab tiuxetan and I-tositumomab (9). These treatment options have distinctive mechanisms of action, with rituximab mediating complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity whereas radioimmunoconjugates represent a targeted systemic radiation approach. The recent progress in therapeutic mAbs has both established these agents as a cornerstone of targeted therapies for cancer and renewed interest in the development of mAbs conjugated to potent cytotoxic agents. The first of these new targeted agents to be approved by the U.S. Food and Drug Administration was gemtuzumab ozogamicin (10), an immunoconjugate of an anti-CD33 mAb chemically linked to the potent cytotoxic calicheamicin, which can achieve complete remission in ∼15% of patients with acute myelogenous leukemia in first relapse (11, 12). More recent efforts are directed at improving selectivity and potency of the immunoconjugates. One such advance is the development of the antibody-maytansinoid conjugates, which are structurally composed of a mAb that binds to a target antigen and a small maytansinoid cytotoxic agent stably linked to the antibody. Such conjugates are much more selective in their cytotoxicity than the parent cytotoxic maytansine. Here, we show the antitumor efficacy of three novel anti-CD138 antibody-maytansinoid conjugates, nBT062-SMCC-DM1, nBT062SPDB-DM4, and nBT062-SPP-DM1, which vary in the linkage between the maytansinoid moiety and mAb. The nBT062SMCC-DM1 linkage contains a thioether bond, which is not cleavable by disulfide exchange, whereas the nBT062-SPDBDM4 and nBT062-SPP-DM1 conjugates contain disulfide linkages, which can be cleaved by disulfide exchange, resulting in liberation of active maytansinoid agent. The anti-CD138 antibody nBT062 is a murine/human chimeric form of B-B4, with identical specificity for CD138 as the parent murine antibody. The observed preclinical antitumor activity of the nBT062-maytansinoid conjugates provides the framework for clinical development of these agents to improve patient outcome in MM. Materials and Methods Cell culture. Dexamethasone-sensitive (MM.1S) and resistant (MM.1R) humanMM cell lines were kindly provided by Dr. Steven Rosen (Northwestern University). RPMI8226, MOLP-8, and U266 human MM cell lines were obtained from American Type Culture Collection. Doxorubicin-resistant (RPMI-DOX40) and Melphalan-resistant (LR5) cells were kindly provided by Dr. William Dalton (Lee Moffitt Cancer Center). OPM1, INA-6, and OPM2 plasma cell leukemia cell lines were kindly provided by Dr. Edward Thompson (University of Texas Medical Branch). All MM cell lines were cultured in RPMI 1640 (Sigma), and bone marrow (BM) stromal cells were cultured in DMEM (Sigma) containing 10% fetal bovine serum, 2 mmol/L L-glutamine (Life Technologies), 100 units/mL penicillin, and 100 mg/mL streptomycin (Life Technologies). The interleukin-6 (IL-6)–dependent INA-6 cell line was cultured in the presence of 1.0 ng/mL of human recombinant IL-6 (R&D Systems). Blood samples collected from healthy volunteers were processed by Ficoll Paque centrifugation to obtain peripheral blood mononuclear cells (PBMC). Patient MM and normal donor plasma cells were obtained from BM samples after informed consent was obtained per the Declaration of Helsinki and approval by the Institutional Review Board of the Dana-Farber Cancer Institute. Normal donor BM mononuclear cells were separated by Ficoll Paque density sedimentation, and plasma cells were purified (>95% CD138+) by positive selection with anti-CD138 magnetic activated cell separation micro beads (Miltenyi Biotec). Tumor cells were purified from the BM of patients with MM using the RosetteSep negative selection system (StemCell Technologies). RosetteSep antibody cocktail is added to BM samples, and CD138-negative cells are crosslinked to RBC (rosetted) with RosetteSep reagents, followed by incubation for 20 min at room temperature and separation by Ficoll density centrifugation, as described previously (13). Immunofluorescence. Cells grown on glass coverslips were fixed in cold absolute acetone for 10 min. After fixation, cells were washed in PBS and then blocked for 60 min with 5% fetal bovine serum in PBS. Slides were then incubated with anti-CD138 antibody (Santa Cruz Biotechnology) for 24 h at 4°C, washed in PBS, and incubated with FITCconjugated goat anti-mouse IgG for 1 h at 4°C. Slideswere analyzed using Nikon E800 fluorescence microscopy, as previously described (14, 15). Growth inhibition assay and proliferation assay. The growth inhibitory effect of nBT062-SMCC-DM1, nBT062-SPDB-DM4, nBT062-SPP-DM1, and dexamethasone on growth of MM cell lines, PBMCs, and BM stromal cells (BMSC) was assessed bymeasuring 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (Chemicon International) dye absorbance, Translational Relevance CD138 is expressed on differentiated plasma cells and is a primary diagnostic marker of multiple myeloma (MM). In this study, we investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against MM cells in vitro and in vivo. We first examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCCDM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We then examined in vivo activity of these agents in murine of human MM xenograft model and SCID-hu mice model, in which human MM cells injected into fetal bone chips implanted s.c. in severe combined immunodeficient mice. Importantly, nBT062-SMCC-DM1, nBT062-SPDB-DM4, and nBT062-SPP-DM1 all have antitumor activity against MM cells and can overcome the protective effects of cytokines and bone marrow stromal cells. Our results, therefore, provide the preclinical framework for clinical trials of these agents to improve patient outcome in MM. 4029 Clin Cancer Res 2009;15(12) June 15, 2009 www.aacrjournals.org Anti-CD138 Therapy for Multiple Myeloma Research. on July 17, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from as previously described (16). One antibody molecule has attached to it ∼3.5 molecules DM4. The molecular weight of the antibody is not significantly increased by the attachment of the DM4 molecules. The ability for nBT062-SPDB-DM4 to mediate antigen-dependent bystander killing of proximal CD138-negative cells was evaluated. CD138-positive MMOPM2 cells (1 × 10 per well) and CD138-negative Namalwa cells (3 × 10 per well) were plated separately or mixed in 96-well round-bottomed plates and exposed to nBT062-SPDB-DM4 for 120 h. Cell viability was then assessed using WST-8 reagent. To evaluate growth inhibitory effects of immunoconjugates against MM cells in the BM milieu, MM cells (2 × 10 per well) were cultured for 48 h in BMSC (1 × 10 per well) coated 96-well plates (Costar) in the presence or absence of the drugs. DNA synthesis was measured by [H]thymidine (PerkinElmer) uptake, with [H]thymidine (0.5 μCi/well) added during the last 8 h of 48-h cultures. All experiments were done in quadruplicate. Cell cycle analysis. MMcells (1 × 10) were incubatedwith or without agents, washedwith PBS, permeabilized by a 30-min exposure to 70% ethanol at −20°C, incubated with propidium iodide (50 μg/mL) in 0.5 mL PBS containing 20 units/mL RNase A (Roche Diagnostics) for 30 min at roomtemperature, and analyzed forDNA content byusing flowcytometry. Detection of apoptotic cells and caspase inhibitor. MM cells (1 × 10) were incubated with or without agents, washed with PBS, stained with PE-conjugated Apo 2.7 antibody (7A6, Beckman Coulter, Inc.), and analyzed on RXP Cytomics software on an Epics flow cytometer (Beckman Coulter, Inc.). Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk; Bachem Bioscience, Inc.), a pan-caspase inhibitor was added in culture medium to reach a final concentration of 50 μmol/L. OPM1 cells were treated with or without z-VAD-fmk for 1 h before drug