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The Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). SGN-40 treatment also induces a down-regulation of CD40 dependent on an endocytic pathway. Consequently, pretreatment of MM cells with SGN-40 blocked sCD40L-mediated phosphatidylinositol 3'-kinase/AKT and nuclear factor 
B activation. Importantly, pretreatment of MM.1S and MM.1R cells with SGN-40 inhibited proliferation triggered by interleukin 6 (IL-6) but not by insulin-like growth factor-I. In addition, SGN-40 pretreatment of MM.1S cells blocked the ability of IL-6 to protect against Dex-induced inhibition of DNA synthesis. This was associated with a 2–4-fold reduction of IL-6 receptor at protein and mRNA levels in SGN-40-treated MM.1S cells and patient MM cells. Taken together, these results provide the preclinical rationale for the evaluation of SGN-40 as a potential new therapy to improve patient outcome in MM. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because human MM remains incurable, novel biologically based therapies urgently are needed. In other cancers, mAbs and mAb-based reagents have shown clinical efficacy (13) . For example, immunotherapy using antibody-targeting CD20 (rituximab), alone and in combination with chemotherapy, has been effective for management of B-cell lymphoproliferative diseases (14 , 15) . However, few MM patients express CD20 (16) . The mAbs targeting CD40 on patient tumor cells represent an attractive therapeutic strategy for MM, and a variety of carcinomas also highly express CD40, broadening its potential therapeutic application. Many investigators have reported that anti-CD40 mAb can enhance antitumor activity and immunity. For example, murine anti-CD40 mAb blocked IL-6 secretion induced by MM cell adhesion to bone marrow stromal cells; because IL-6 is a key growth and survival factor for human MM cells, blockade of IL-6 secretion abrogates MM cell growth in the bone marrow milieu (4) . In addition, anti-CD40 immunotoxin can effectively kill B-lineage acute lymphoblastic leukemia and non-Hodgkin’s lymphoma (17) . In vivo treatment of severe combined immunodeficiency mice bearing human B lymphomas with anti-CD40 antibodies inhibited tumor progression and enhanced survival (18 , 19) . Funakoshi et al. (20) demonstrated that murine anti-CD40 mAb was more effective at killing tumors in human B-cell lymphoma-xenografted severe combined immunodeficiency mice, in the absence of Fc receptor-bearing effector cells, than was anti-CD20 mAb to kill tumors. Subsequently, van Mierlo et al. (21) reported that systemic in vivo administration of agonistic anti-CD40 antibodies resulted in tumor eradication mediated via dendritic cell-induced CD8+ T-cell responses. Honeychurch et al. (22) most recently showed that anti-CD40 mAb in combination with irradiation results in CD8 T-cell-dependent immunity against B-cell lymphoma in mice, suggesting that combining irradiation with anti-CD40 mAb may provide a more potent therapeutic approach.
In the present study, we evaluated the direct impact of SGN-40 humanized anti-CD40 mAb on MM cell lines and patient cells (CD40+CD138++). SGN-40 was engineered from SGN-14 mouse mAb, which showed significant antitumor activity against human HS-Sultan and IM-9 cell line-xenografted severe combined immunodeficiency mice, without adverse effects on human normal B cells (23)
. We recently showed that SGN-40 triggered antibody-dependent cell-mediated cytotoxicity against CD40-expressing MM cell lines and patient cells, supporting its potential therapeutic use in human MM (24)
. Moreover, the tumoricidal effects of SGN-40 could not be solely attributed to enhancing effector functions of antibody-dependent cell-mediated cytotoxicity. To examine for direct induction of apoptosis, as is induced in malignant B lymphocytes by rituximab, we specifically examined the effects of SGN-40 on CD40+ and CD138+ MM.1S and MM.1R cell lines and on patient MM cells (25)
. SGN-40-induced cytotoxicity against MM cells is associated with up-regulation of cytotoxic ligands of the TNF family FasL, TNF-related apoptosis-inducing ligand (TRAIL), and TNF-. Moreover, SGN-40 suppressed IL-6 receptor (IL-6R) expression at mRNA and protein levels, associated with inhibition of IL-6-mediated, but not insulin-like growth factor I (IGF-I)-mediated, MM cell growth and survival. These data provide the preclinical framework for the evaluation of SGN-40 as a novel therapy to improve patient outcome in MM.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytotoxicity Assay.
MM cells (4 x 104 cells/well) were incubated in 96-well culture plates (Costar, Cambridge, MA) in the presence of SGN-40 for 6 h and then cocultured with or without cycloheximide (CHX; 0.2 µg/ml; Sigma) for an additional 36 h. Cells were pulsed with [3H]thymidine (0.5 µCi/well; NEN Products, Boston, MA) during the last 8 h of 48-h cultures, harvested onto glass filters, and counted using the LKB Betaplate scintillation counter (Wallac, Gaithersburg, MD). To assay for inhibition of IL-6-induced cell proliferation by SGN-40, MM.1S and MM.1R cells were pretreated with 20 µg/ml of SGN-40 or control immunoglobulin overnight. They then were cultured with IL-6 (0, 50, or 200 ng/ml) or IGF-I (0, 20, or 200 ng/ml) for 36 h before pulsing with [3H]thymidine. For IL-6-induced survival experiments, MM.1S cells pretreated with SGN-40 or control immunoglobulin were incubated with Dex (0–10 µM), in the presence or absence of IL-6 (50 ng/ml) or IGF-I (100 ng/ml). All of the experiments were performed in triplicate. Cell viability also was assessed by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (Chemicon International, Temecula, CA) assay, according to manufacturer’s instructions.
Reagents.
The humanized anti-CD40 mAb SGN-40 (IgG1) and control human monoclonal IgG1 were provided by Seattle Genetics. CHX (0.2 µg/ml) and phosphatidylinositol 3'-kinase inhibitor LY 294002 (5 µM) were obtained from Sigma. SB203580 (5 µM) was obtained from Calbiochem (San Diego, CA). Mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 inhibitors PD98059 (10 µM) and U1026 (1 µM) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). All of the antibodies for immunoblotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) except for anti-AKT and anti-pAKT antibody (Cell Signaling) and anti--tubulin mAb (Sigma).
Immunoblotting and Immunoprecipitation.
MM.1S cells and patient MM cells were treated as described previously. Total cell lysates were subjected to 8–10% SDS-PAGE and transferred onto membranes; immunoblot analysis was performed as reported previously (8)
. For immunoprecipitation, 1 mg of lysates was incubated with anti-IL-6R antibody (4 µg) in lysis buffer for 2 h at 4°C.
Reverse Transcription-PCR.
Total RNA samples were isolated using RNeasy kit (Qiagen, Valencia, CA). The first-strand cDNA was synthesized from 2 µg of total RNA priming by oligo(dT) using Superscript reverse-transcription kit (Invitrogen, Carlsbad, CA). Primers were for FasL, 5'-ggtccatgcctctggaatgg-3' and 5'-cacatcgcccagtagtgca-3'; for TRAIL (Apo-2L), 5'-agacctgcgtgctgatcgt-3' and 5'-gaccagttcaccattcctc-3'; for TNF-, 5'-cttctgcctgctgcactttgga-3' and 5'-tcccaaagtagacctgcccaga-3'; and for CD40L, 5'-caccttctctgccagaagataccatttcaa-3' and 5'-ttatgaagactcccagcgtcacgtccacca-3'. IL-6R cDNA was amplified by reverse transcription-PCR using a primer pair (5'-cagctgagaacgaggtgtcc-3' and 5'-gcagcttccacgtcttcttga-3') flanking the transmembrane coding region of IL-6R. The primer pair for gp130 is 5'-atactggagtgactggagtg-3' and 5'-catcttgtgagagtcacttc-3'. Templates initially were denatured at 95°C for 5 min, and then a cycle at 95°C for 45 s, 56°C for 45 s, and 72°C for 1 min was repeated 25 times, followed by a final extension at 72°C for 5 min. The number of amplification cycles chosen for each reaction was determined to be within the linear range of the assay. PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (GADPH) from the same reverse-transcribed cDNA template under the same conditions served as an internal control, with the primer pair of 5'-ccatggagaaggctgggg-3' and 5'-caaagttgtcatggatgacc-3'. Real-time reverse transcription-PCR was performed on a TaqMan ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) using heat-activated TaqDNA polymerase (Amplitaq Gold, Applied Biosystems). Primers for ß-actin are 5'-acgtggacatccgcaaag-3' and 5'-tgcatcctggtcggcaatg-3'.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, SGN-40 did not stimulate proliferation of MM.1S and MM1R cells and CD40-expressing tumor cells from two MM patients (P > 0.1). To further define the cytotoxic effect of SGN-40 against MM cells, MM.1S and MM.1R cells were treated for 6 h with SGN-40 (10 µg/ml) and then cocultured with the de novo protein synthesis inhibitor CHX (0.2 µg/ml) for an additional 48 h. Cell viability was assayed by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide. SGN-40 and CHX triggered 50–60% cell killing in both cell lines (Fig. 1B
, left). Treatment with isotype control immunoglobulin alone or with CHX also did not induce cytotoxicity (Fig. 1B
, right). SGN-40 triggered 20–30% cell cytotoxicity in two patient MM cells. In the presence of CHX at a nontoxic dose (0.2 µg/ml), SGN-40-induced cell cytotoxicity was enhanced significantly (Fig. 1C)
. These results suggested that endogenous production of cytotoxic cytokines might mediate SGN-40-induced cell death in CD40-expressing MM cells; therefore, we investigated whether SGN-40 treatment modulated expression of FasL and other cytotoxic members of the TNF superfamily, i.e., TNF- and TRAIL (Apo-2L). Expression of FasL, TNF-, and TRAIL increased after SGN-40 treatment, although the kinetics of induction of each gene varied (Fig. 1D)
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B, and ERK in MM.1S Cells.B (IB), and extracellular signal regulated kinase (ERK)-1/2 using phospho-specific antibodies. CD40 activation by sCD40L induces phosphorylation of AKT, IB, and ERK (Fig. 2A
, right) as shown previously (8)
. In contrast, SGN-40 did not activate either AKT or IB phosphorylation. Phosphorylation of ERK was observed following SGN-40 stimulation, less potent than triggered by sCD40L. Little, if any, phosphorylation of AKT and IB was observed, even at increasing concentrations of SGN-40 (up to 1000 µg/ml; Fig. 2B
).
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B, whereas sCD40L either alone or together with SGN-40 (5 and 10 µg/ml) induced activation of these signaling proteins (Fig. 3)
. Phosphorylation of ERK-1/2 was similarly induced by SGN-40, either alone or with sCD40L. MM.1S cells then were pretreated with increasing concentrations (5–1000 µg/ml) of SGN-40 for 30 min before stimulation with sCD40L (5 µg/ml). As shown in Fig. 3B
, SGN-40 at either 50 or 1000 µg/ml did not significantly inhibit sCD40L-induced phosphorylation of AKT.
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B and ERK Pathways.
). sCD40L treatment for 20 min to 24 h also did not alter levels of CD40 (Fig. 4A
, Lane 2 compared with Lane 1). In contrast, SGN-40 triggered down-regulation of CD40 after 4 h (Fig. 4A
, Lane 3) and significant inhibition of CD40 expression by 24 h.
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, PS341 did not block down-regulation of CD40 by SGN-40 compared with cells treated with only SGN-40 or DMSO vehicle control. This suggests that down-regulation of CD40 induced by SGN-40 does not occur via proteasomal degradation. To determine whether down-regulation of CD40 was mediated by an endocytic pathway, cells then were preincubated with ammonium chloride (10 mM for 2 h) and then treated with sCD40L or SGN-40 (26)
. Ammonium chloride increases pH of endosomes, thereby inhibiting the endocytic pathway. Pretreatment of cells with ammonium chloride blocked SGN-40-induced down-regulation of CD40 (Fig. 4C)
. This result suggests that down-regulation of CD40 triggered by SGN-40 occurs via a lysosomal and endocytic pathway.
We next examined whether MM.1S cells respond to sCD40L stimulation after antibody-induced down-regulation of CD40. Cells were pretreated with either sCD40L or SGN-40 for 24 h and then either untreated or treated with additional sCD40L (5 µg/ml). Phosphorylation of AKT, IB, and ERK was induced by sCD40L (Fig. 5
, Lanes 1–3) as shown previously. In cells pretreated with sCD40L for 24 h and then treated with additional sCD40L for 7 min, AKT, IB, and ERK were phosphorylated (Fig. 5
, Lanes 4 and 5). In contrast, in cells pretreated with SGN-40 followed by treatment with sCD40L for 7 min, phosphorylation of AKT, IB, and ERK was undetected (Fig. 5
, Lanes 6 and 7). Cells pretreated with SGN-40 and re-treated with additional SGN-40 did not show enhanced AKT, IB, and ERK phosphorylation (data not shown). Thus, 24-h pretreatment of MM.1S cells with SGN-40 blocks sCD40L-induced phosphorylation of AKT and IB and activation of ERK. In contrast, cells treated with sCD40L for 24 h were stimulated by subsequent treatment with additional sCD40L. These data suggest that down-regulation of CD40 by SGN-40 blocked the ability of sCD40L to trigger phosphatidylin-ositol 3'-kinase/AKT, nuclear factor B, and ERK signaling.
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(left), IL-6-induced cell proliferation is significantly blocked in MM.1S and MM.1R cells pretreated with SGN-40 (P < 0.01) compared with the 2.5–3-fold increase in cell proliferation induced by IL-6 in control immunoglobulin-treated cells (Fig. 6A
, left). In contrast, IGF-I induces cell proliferation to a similar extent in SGN-40- and control immunoglobulin-treated MM.1S and MM.1R cells. We next tested whether pretreatment with SGN-40 alters IL-6-induced protection against Dex-induced apoptosis in MM.1S cells. Cells were pretreated with SGN-40 or control immunoglobulin for 24 h, washed, and then incubated with Dex (0–10 µM) for 36 h before assay for [3H]thymidine incorporation. IL-6 (50 ng/ml) completely rescues Dex-induced cell death in control immunoglobulin-treated cells, whereas IL-6-induced protection against Dex was blocked significantly in SGN-40-pretreated cells (P < 0.023; Fig. 6B
, left). In contrast and as shown in Fig. 6B
(right), IGF-I-induced MM cell antiapoptosis was observed in SGN40-treated and control immunoglobulin-treated cells. These results indicate that SGN-40 pretreatment inhibits IL-6-, but not IGF-I-, mediated MM cell growth and survival.
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, activation of these IL-6 downstream signaling molecules was reduced in SGN-40-pretreated MM.1S cells, thereby inhibiting IL-6-induced growth and survival.
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, a 2–4-fold suppression of IL-6R was observed in MM.1S and a patient’s MM cells following 9 h of treatment with SGN-40. To confirm specific down-regulation of IL-6R expression, MM.1S and patient MM cells (MM#1) were incubated with 20 µg/ml of SGN-40 or control immunoglobulin overnight, followed by assessment for IL-6R by flow cytometry. Down-regulation in IL-6R expression was seen only in SGN40-treated, but not in control immunoglobulin-treated, MM.1S cells (Fig. 8B)
. In addition, SGN-40-induced down-regulation of IL-6R expression was prevented by mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 and PD98059 (Fig. 8C)
but unaffected by inhibitors of either phosphatidylinositol 3'-kinase or p38 mitogen-activated protein kinase (Fig. 8C)
. These results suggest that mitogen-activated protein kinase/ERK signaling mediates SGN-40-induced inhibition of cell surface IL-6R expression.
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. The smaller species lacks 94 bp (exon 2), including the coding region of the transmembrane domain of the IL-6R (27)
. As shown in Fig. 9A
, SGN-40 treatment induced time-dependent suppression of both species of IL-6R transcripts in MM.1S cells (Lanes 4–6) and two patients’ MM cells (Lanes 7–9 and Lanes 10–12), whereas no difference in IL-6R transcripts was found in control immunoglobulin-treated MM.1S cells (Fig. 9
, Lanes 1–3). The gp130 and GADPH mRNAs were unchanged by treatment with either SGN-40 or control immunoglobulin. IL-6 mRNA suppression following SGN-40 treatment also was confirmed by real-time reverse transcription-PCR (Fig. 9B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, SGN-40 suppresses expression of IL-6R protein and mRNA associated with inhibition of growth and survival of MM cells induced by IL-6 but not by IGF-I. These data support a new treatment strategy using SGN-40 to improve patient outcome in human MM.
We show that SGN-40 induces apoptosis of CD40+ and CD138+ MM cell lines and patient MM cells in the presence of CHX, in addition to its previously reported tumoricidal activity via antibody-dependent cell-mediated cytotoxicity (24)
. SGN-40-mediated MM cell death is associated with up-regulation of Fas/FasL because the expression of FasL was induced following SGN-40 treatment. Fas-induced apoptosis has been reported in MM.1S cells (28)
. We also found that SGN-40-induced cell death occurs through a caspase-8-dependent pathway (data not shown), consistent with Fas engaging a proapoptotic cascade leading to caspase activation (28, 29, 30)
. Furthermore, SGN-40-induced apoptosis in MM.1S and MM.1R lines is partially inhibited by neutralizing anti-FasL antibody NOK1 (30% ± 5%; data not shown). However, the inability of neutralizing anti-FasL antibodies to abolish completely SGN-40-mediated cytotoxicity suggests additional death signals. Exposure of MM.1S cells to SGN-40 also induced transcriptional activation of other cytotoxic members of the TNF family, such as TRAIL (Apo-2L) and TNF-, although with different kinetics. TRAIL induces MM apoptosis through the rapid activation of caspase-8, caspase-9, and caspase-3 (31)
. Moreover, an individual neutralizing reagent only partially protects against SGN-40-induced cell death, confirming that SGN-40-mediated MM cell apoptosis occurs via targeting more than one cytotoxic ligand of the TNF family. Previous studies in human monocytes and normal plasma cells demonstrate the presence of high intracellular levels of FasL or TRAIL, which rapidly translocates to the cell surface in response to various stimuli (32, 33, 34)
. Whether SGN-40 treatment regulates the redistribution of intracellular cytoplasmic pools of these cytotoxic ligands, in addition to their de novo transcription and translation, remains to be elucidated.
SGN-40-induced cytotoxicity in MM cells is enhanced by inhibition of protein synthesis. In particular, CHX blocks the production of protective antiapoptotic proteins (i.e., FLIP), thereby unmasking the cytotoxic potential of SGN-40. CHX-induced reduction of the short-lived antiapoptotic FLIP protein is associated with enhanced SGN-40-induced apoptosis in MM.1S cells (data not shown). Although the molecular basis for SGN-40-dependent sensitization to apoptosis in MM cells is not understood completely, this requirement of protein synthesis inhibition for efficient killing reveals a mechanism whereby MM cells, via activation of antiapoptotic cascades including AKT, Bcl-2, and Mcl-1, may escape CD40-mediated cytotoxicity. Although the ability of these pathways to block CD40-mediated cell death remains to be verified, previous studies have implicated Bcl-2, Bcl-xL, and AKT in suppression of TRAIL- and Fas-induced apoptosis in MM (35 , 36) .
In vitro studies have demonstrated internalization of CD40 after ligation by mAb (37 , 38) . Our data indicate that SGN-40 caused down-regulation of CD40 via the endosomal endocytic, but not the proteasome-ubiquitin, pathway. It is possible that SGN-40 causes increased endocytosis without allowing for receptor recycling to the membrane, resulting in a net decrease in cell surface levels over time. An increase in receptor turnover leading to down-modulation of HER-2/c-erbB-2 or IGF-I receptor has been reported with specific mAbs against these receptors (39 , 40) . In addition, rituximab therapy leads to down-regulation of CD20 expression at protein and mRNA in cells from treated patients (41) . This down-modulation of targeted receptors may have important implications for scheduling and dosing, not only of anti-CD20 but also of potential novel anti-CD40 therapies.
We found down-regulation of IL-6R by SGN-40, correlating with decreased IL-6 downstream signaling and function. Because IL-6 is the major growth and survival factor for human MM, decreased IL-6R induced by SGN-40 treatment would inhibit MM cell growth and survival in the bone marrow milieu. These results are consistent with recent studies demonstrating that IFN- (42)
or IFN-
(43
, 44) significantly inhibits membrane IL-6 binding IL-6R (gp80) protein and mRNA in human plasma cell lines. Since IFN- up-regulates CD40 expression, it may also result in additional inhibition of IL-6R expression induced by SGN-40. In contrast to its effects on IL-6 signaling, SGN-40 did not alter the proliferative and antiapoptotic effects induced by IGF-I. The mechanism by which SGN-40 treatment induces differential responses of MM cells to IL-6 and IGF-I may, at least in part, be explained by differential changes of their cognate receptors because we could not detect any changes of IGF-I receptor at either protein and mRNA levels induced by SGN-40 treatment (data not shown).
In summary, the humanized anti-CD40 mAb SGN-40 has direct anti-MM effects that are independent of antibody-dependent cell-mediated cytotoxicity as we reported previously (24) . SGN-40 mediates apoptosis in human MM cells in the presence of CHX, and SGN-40 up-regulates cytotoxic ligands belonging to the TNF family. In addition to regulating CD40-mediated cytotoxicity, these ligands and/or their receptors also are important in apoptosis induced by a broad spectrum of stimuli, including chemotherapy and radiation. Importantly, inhibition of IL-6R expression by SGN-40 renders MM cells refractory to IL-6-induced proliferative and protective effects, thereby inhibiting MM cell growth and survival in the bone marrow microenvironment. These results provide the framework for the clinical evaluation of SGN-40 to improve patient outcome in MM.
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FOOTNOTES |
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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.
Requests for reprints: Kenneth C. Anderson, Department of Medical Oncology, Dana-Farber Cancer Institute, M557, 44 Binney Street, Boston, MA 02115. Phone: 617-632-2144; Fax: 617-632-2140; E-mail: kenneth_anderson{at}dfci.harvard.edu
Received 11/19/03. Revised 2/ 2/04. Accepted 2/ 5/04.
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, MM.1S; 
, MM.1R) and two patient MM cells (right, 
50,000) using anti-CD40 antibody.
