Recombinant humanized anti-CD40 monoclonal antibody triggers autologous antibody-dependent cell-mediated cytotoxicity against multiple myeloma cells

Multiple myeloma (MM) is currently incurable, and novel therapies are needed. In this study, we examined a novel recombinant humanized monoclonal antibody against CD40 (rhuCD40 mAb) and demonstrate for the first time that rhuCD40 mAb induces antibody-dependent cell-mediated cytotoxicity (ADCC) against CD40-positive MM cells. Importantly, we show that rhuCD40 mAb induces autologous ADCC against primary patient MM cells, without triggering ADCC against normal B cells. This study, therefore, both demonstrates that rhuCD40 mAb triggers autologous ADCC against patient MM cells and provides the framework for the clinical evaluation of rhuCD40 mAb immunotherapy to improve patient outcome in MM.     

Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.     

Targeting MEK induces myeloma-cell cytotoxicity and inhibits osteoclastogenesis

Activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade mediates human multiple myeloma (MM) growth and survival triggered by cytokines and adhesion to bone marrow stromal cells (BMSCs). Here, we examined the effect of AZD6244 (ARRY-142886), a novel and specific MEK1/2 inhibitor, on human MM cell growth in the bone marrow (BM) milieu. AZD6244 blocks constitutive and cytokine-stimulated ERK1/2 phosphorylation and inhibits proliferation and survival of human MM cell lines and patient MM cells, regardless of sensitivity to conventional chemotherapy. Importantly, AZD6244 (200 nM) induces apoptosis in patient MM cells, even in the presence of exogenous interleukin-6 or BMSCs associated with triggering of caspase 3 activity. AZD6244 sensitizes MM cells to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. AZD6244 down-regulates the expression/secretion of osteoclast (OC)-activating factors from MM cells and inhibits in vitro differentiation of MM patient PBMCs to OCs induced by ligand for receptor activator of NF-kappaB (RANKL) and macrophage-colony stimulating factor (M-CSF). Finally, AZD6244 inhibits tumor growth and prolongs survival in vivo in a human plasmacytoma xenograft model. Taken together, these results show that AZD6244 targets both MM cells and OCs in the BM microenvironment, providing the preclinical framework for clinical trials to improve patient outcome in MM.     

Patupilone (epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo

In this study, we investigated the in vitro and in vivo efficacy of patupilone (epothilone B, EPO906), a novel nontaxane microtubule stabilizing agent, in treatment of multiple myeloma (MM). Patupilone directly inhibited growth and survival of MM cells, including those resistant to conventional chemotherapies, such as the taxane paclitaxel. Patupilone induced G2M arrest of MM cells, with subsequent apoptosis. Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), 2 known growth and survival factors for MM, did not protect MM.1S cells against patupilone-induced cell death. Proliferation of MM cells induced by adherence to bone marrow stromal cells (BMSCs) was also inhibited by patupilone and was paralleled by down-regulation of vascular endothelial growth factor (VEGF) secretion. Importantly, stimulation of cells from patients with MM, either with IL-6 or by adherence to BMSCs, enhanced the anti-proliferative and proapoptotic effects of patupilone. Moreover, patupilone was effective against MM cell lines that overexpress the MDR1/P-glycoprotein multidrug efflux pump. In addition, patupilone was effective in slowing tumor growth and prolonging median survival of mice that received orthotopical transplants with MM tumor cells. Taken together, these preclinical findings suggest that patupilone may be a safe and effective drug in the treatment of MM, providing the framework for clinical studies to improve patient outcome in MM.     

Targeting mitochondrial factor Smac/DIABLO as therapy for multiple myeloma (MM)

Second mitochondria-derived activator of caspases (Smac) promotes apoptosis via activation of caspases. Here we show that a low-molecular-weight Smac mimetic LBW242 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Examination of purified patient MM cells demonstrated similar results, without significant cytotoxicity against normal lymphocytes and bone marrow stromal cells (BMSCs). Importantly, LBW242 abrogates paracrine MM cell growth triggered by their adherence to BMSCs and overcomes MM cell growth and drug-resistance conferred by interleukin-6 or insulinlike growth factor-1. Overexpression of Bcl-2 similarly does not affect LBW242-induced cytotoxicity. Mechanistic studies show that LBW242-induced apoptosis in MM cells is associated with activation of caspase-8, caspase-9, and caspase-3, followed by PARP cleavage. In human MM xenograft mouse models, LBW242 is well tolerated, inhibits tumor growth, and prolongs survival. Importantly, combining LBW242 with novel agents, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the proteasome inhibitors bortezomib and NPI-0052, as well as with the conventional anti-MM agent melphalan, induces additive/synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating LBW242, alone and together with other anti-MM agents, to improve patient outcome in MM.     

Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor,induces pleiotropic anti‐myeloma activity in vitro and in vivo

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine‐induced osteoclast differentiation more potently (9‐ to 10‐fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G₀‐G₁ cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti‐MM therapies. Significant tumour growth reduction in AS703026‐ vs. vehicle‐treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC‐induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti‐MM agents, to improve patient outcome.     

Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)

In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester-induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCmu. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.     

CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies

Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.     

Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells

Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF-beta1) is also produced by BMSCs and can regulate IL- 6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF- beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N-BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N- BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM-BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF- beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion.     

CD48 as a novel molecular target for antibody therapy in multiple myeloma

Monoclonal antibody (mAb) drugs are desirable for the improvement of multiple myeloma (MM) treatment. In this study, we found for the first time that CD48 was highly expressed on MM plasma cells. In 22 out of 24 MM patients, CD48 was expressed on more than 90% of MM plasma cells at significantly higher levels than it was on normal lymphocytes and monocytes. CD48 was only weakly expressed on some CD34(+) haematopoietic stem/progenitor cells, and not expressed on erythrocytes or platelets. We next examined whether CD48 could serve as a target antigen for mAb therapy against MM. A newly generated in-house anti-CD48 mAb induced mild antibody-dependent cell-mediated cytotoxicity and marked complement-dependent cytotoxicity against not only MM cell lines but also primary MM plasma cells in vitro. Administration of the anti-CD48 mAb significantly inhibited tumour growth in severe combined immunodeficient mice inoculated subcutaneously with MM cells. Furthermore, anti-CD48 mAb treatment inhibited growth of MM cells transplanted directly into murine bone marrow. Finally and importantly, we demonstrated that the anti-CD48 mAb did not damage normal CD34(+) haematopoietic stem/progenitor cells. These results suggest that the anti-CD48 mAb has the potential to become an effective therapeutic mAb against MM.     

多发性骨髓瘤血管内皮生长因子和白细胞介素-6相互作用的研究

目的　研究人类多发性骨髓瘤(MM)细胞系和MM患者骨髓基质细胞(BMSCs)之间相互作用对血管内皮生长因子(VEGF)和IL6分泌的调控作用,分析VEGF和IL6的相互作用在MM发病机制中的意义。方法　建立MMBMSCs和正常人BMSCs(NBMSCs)的培养体系,用IL6、抗IL6抗体、VEGF、抗VEGF抗体作用于BMSCs和(或)MM细胞系U266后,ELISA方法检测其VEGF和IL6的分泌量。结果　U266分泌VEGF,但不分泌IL6,而MMBMSCs和NBMSCs既分泌VEGF又分泌IL6。重组人VEGF刺激BMSCs后,以时间和剂量依赖性的方式诱导IL6的分泌,此效应可被抗VEGF抗体抑制。外源性IL6促进BMSCs分泌VEGF。当U266与BMSCs黏附后,VEGF分泌增加25~50倍,IL6增加55~90倍,两者差异有统计学意义(P<005);分别加入抗VEGF或抗IL6抗体,IL6或VEGF的分泌受抑。重组人IL6作用于U266,可诱导剂量依赖性的VEGF分泌的增加,此反应可被抗IL6抗体抑制。结论　在MM中,MM细胞和BMSCs之间的相互作用调节VEGF和IL6的分泌,促进MM细胞的生长和血管新生,在MM的发病机制中发挥重要作用,为针对骨髓微环境的靶位治疗提供了理论依据。     

SDX-101, the R-enantiomer of etodolac, induces cytotoxicity, overcomes drug resistance, and enhances the activity of dexamethasone in multiple myeloma

In this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex.     

Inhibition of Akt induces significant downregulation of survivin and cytotoxicity in human multiple myeloma cells

Akt mediates growth and drug resistance in multiple myeloma (MM) cells in the bone marrow (BM) microenvironment. We have shown that a novel Akt inhibitor Perifosine induces significant cytotoxicity in MM cells in the BM milieu. This study further delineated molecular mechanisms whereby Perifosine triggered cytotoxicity in MM cells. Neither the intensity of Jun NH(2)-terminal kinase phosphorylation nor caspase/poly (ADP-ribose) polymerase cleavage correlated with Perifosine-induced cytotoxicity in MM.1S, INA6, OPM1 and OPM2 MM cells. However, survivin, which regulates caspase-3 activity, was markedly downregulated by Perifosine treatment, without changes in other anti-apoptotic proteins. Downregulation of survivin by siRNA significantly inhibited OPM1 MM cell growth, confirming that survivin mediates MM cell survival. Perifosine significantly downregulated both function and protein expression of beta-catenin. Co-culture with BM stromal cells (BMSCs) upregulated both beta-catenin and survivin expression in MM cells, which was blocked by Perifosine. Importantly, Perifosine treatment also downregulated survivin expression in human MM cells grown in vivo in a severe combined immunodeficient mouse xenograft model. Finally, Perifosine inhibited bortezomib-induced upregulation of survivin, associated with enhanced cytotoxicity of combined bortezomib and Perifosine treatment. These preclinical studies provide the framework for clinical trials of bortezomib with Perifosine to improve patient outcome in MM.     

Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents

Multiple Myeloma (MM), a malignancy of plasma cells, remains incurable despite the use of conventional and novel therapies. Halofuginone (HF), a synthetic derivative of quinazolinone alkaloid, has recently been shown to have anti-cancer activity in various preclinical settings. This study demonstrated the anti-tumour activity of HF against a panel of human MM cell lines and primary patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. HF showed anti-MM activity in vivo using a myeloma xenograft mouse model. HF suppressed proliferation of myeloma cells alone and when co-cultured with bone marrow stromal cells. Similarly, HF induced apoptosis in MM cells even in the presence of insulin-like growth factor 1 or interleukin 6. Importantly, HF, even at high doses, did not induce cytotoxicity against CD40 activated peripheral blood mononuclear cells from normal donors. HF treatment induced accumulation of cells in the G(0) /G(1) cell cycle and induction of apoptotic cell death associated with depletion of mitochondrial membrane potential; cleavage of poly (ADP-ribose) polymerase and caspases-3, 8 and 9 as well as down-regulation of anti-apoptotic proteins including Mcl-1 and X-IAP. Multiplex analysis of phosphorylation of diverse components of signalling cascades revealed that HF induced changes in P38MAPK activation; increased phosphorylation of c-jun, c-jun NH(2)-terminal kinase (JNK), p53 and Hsp-27. Importantly, HF triggered synergistic cytotoxicity in combination with lenalidomide, melphalan, dexamethasone, and doxorubicin. Taken together, these preclinical studies provide the preclinical framework for future clinical studies of HF in MM.     

A novel immunogenic CS1-specific peptide inducing antigen-specific cytotoxic T lymphocytes targeting multiple myeloma

The CS1 antigen provides a unique target for the development of an immunotherapeutic strategy to treat patients with multiple myeloma (MM). This study aimed to identify HLA‐A2⁺ immunogenic peptides from the CS1 antigen, which induce peptide‐specific cytotoxic T lymphocytes (CTL) against HLA‐A2⁺ MM cells. We identified a novel immunogenic HLA‐A2‐specific CS1_239‐247 (SLFVLGLFL) peptide, which induced CS1‐specific CTL (CS1‐CTL) to MM cells. The CS1‐CTL showed a distinct phenotype, with an increased percentage of effector memory and activated CTL and a decreased percentage of naïve CTL. CS1_239‐247 peptide‐specific CD8⁺ T cells were detected by DimerX analyses and demonstrated functional activities specific to the peptide. The CTL displayed HLA‐A2‐restricted and antigen‐specific cytotoxicity, proliferation, degranulation and γ‐interferon (IFN‐γ) production against both primary MM cells and MM cell lines. In addition, the effector memory cells subset (CD45RO⁺CCR7^?/CD3⁺CD8⁺) within CS1‐CTL showed a higher level of CD107a degranulation and IFN‐γ production as compared to effector cells (CD45RO^?CCR7^?/CD3⁺CD8⁺) against HLA‐A2⁺ primary MM cells or MM cell lines. In conclusion, this study introduced a novel immunogenic HLA‐A2‐specific CS1_239‐247 peptide capable of inducing antigen‐specific CTL against MM cells that will provide a framework for its application as a novel MM immunotherapy.     

Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P <.001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold; P =.02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P =.002). This effect was inhibited by anti-IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.