Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). IntroductionA cardinal clinical feature of multiple myeloma (MM) is the presence of osteolytic bone lesions. Myeloma cells disrupt the delicate balance between bone formation and bone resorption. 1,2 Various clinical observations 3 and experimental studies 4,5 have linked the level of MM bone disease with disease burden. Increased osteoclastic activity and its molecular basis have long been considered a primary pathogenic event in MM bone disease. However, a molecular basis for the well recognized lack of osteoblast (OB) function, specifically DKK-1, in the MM bone disease has only recently been described. 6,7 Canonical Wnt pathway plays an important role in controlling proliferation, differentiation, and survival of OB. [8][9][10][11] Previous studies have reported high expression levels of the canonical Wnt inhibitor DKK1 and osteolytic bone lesions in various tumor types including breast, 12,13 neuroblastoma, 14 esophageal, and lung cancer, 15 and conversely enhanced OB activity and osteoblastic bone lesions associated with decreased DKK1 levels in prostate and colon cancers. [16][17][18] In MM, high serum DKK1 levels were correlated with focal bone lesions. 19 The DKK1 produced by MM cells can inhibit the differentiation of OB precursor cells 19 and bone formation in vitro 20 through a DKK1-mediated attenuation of Wnt3a-induced stabilization of ␤-catenin. 21 These findings confirm DKK1 as an important regulator of bone formation in the bone microenvironment. The importance of DKK1 secretion in diseases associated with bone destruction is reinforced by a recent study showing that DKK1 mediates the bone destructive effects of rheumatoid arthritis and that a neutralizing antibody to DKK1 could inhibit the bone destructive process in that disease. 22 There is also emerging evidence that the cellular bone compartment affects MM cell growth and progression. This is supported by the observation that osteoclasts can support long-term survival and proliferation of primary MM cells, 23,24 and OB may impede MM cell growth. 7,25 Thus, targeting these cellular elements may also favorably affect disease control. Therefore, we have evaluated DKK1 as a therapeutic target in MM in the context of the bone marrow (BM) microenvironment, analyzing the effect of a human DKK1 neutralizing antibody (BHQ880). We show that this clinically applicable antibody increases OB function and number and also has anti-MM effect when evaluated in the presence of the BM milieu. Methods ReagentsBHQ880 is a phage-derived DKK1 neutralizing human immunoglobulin G1 (IgG1) antibody (provided by Novartis, Cambridge, MA). BHQ880 has a high affinity for and can neutralize both human DKK1 and murine DKK1. IgG1 isotype antibody was used as control. CellsBone marrow mononuclear cells (BMMNCs) and primary MM cells were isolated using Ficoll-Hypaque density gradient sedimentation from BM Submitted November 25, 2008; accepted April 20, 2009. Prepublished online ...

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Purpose: We investigated the in vitro and in vivo anti-multiple myeloma activity of monoclonal antibody (mAb) 1339, a high-affinity fully humanized anti-interleukin 6 mAb (immunoglobulin G1), alone and in combination with conventional and novel anti-multiple myeloma agents, as well as its effect on bone turnover. Experimental Design: We examined the growth inhibitory effect of 1339 against multiple myeloma cell lines in the absence and in the presence of bone marrow stromal cells, alone or in combination with dexamethasone, bortezomib, perifosine, and Revlimid. Using the severe combined immunodeficient (SCID)-hu murine model of multiple myeloma, we also examined the effect of 1339 on multiple myeloma cell growth and multiple myeloma bone disease. Results: mAb 1339 significantly inhibited growth of multiple myeloma cell in the presence of bone marrow stromal cell in vitro, associated with inhibition of phosphorylation of signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, and Akt. In addition, mAb 1339 enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly mAb 1339 significantly enhanced growth inhibitory effects of dexamethasone in vivo in SCID-hu mouse model of multiple myeloma. mAb 1339 treatment also resulted in inhibition of osteoclastogenesis in vitro and bone remodeling in SCID-hu model. Conclusions: Our data confirm in vitro and in vivo anti-multiple myeloma activity of, as well as inhibition of bone turnover by, fully humanized mAb 1339, as a single agent and in combination with conventional and novel agents, providing a rationale for its clinical evaluation in multiple myeloma. (Clin Cancer Res 2009;15(23):7144-52)

Large segmental defects in bone fail to heal and remain a clinical problem. Muscle is highly osteogenic, and preliminary data suggest that autologous muscle tissue expressing bone morphogenetic protein-2 (BMP-2) efficiently heals critical size defects in rats. Translation into possible human clinical trials requires, inter alia, demonstration of efficacy in a large animal, such as the sheep. Scale-up is fraught with numerous biological, anatomical, mechanical and structural variables, which cannot be addressed systematically because of cost and other practical issues. For this reason, we developed a translational model enabling us to isolate the biological question of whether sheep muscle, transduced with adenovirus expressing BMP-2, could heal critical size defects in vivo. Initial experiments in athymic rats noted strong healing in only about one-third of animals because of unexpected immune responses to sheep antigens. For this reason, subsequent experiments were performed with Fischer rats under transient immunosuppression. Such experiments confirmed remarkably rapid and reliable healing of the defects in all rats, with bridging by 2 weeks and remodelling as early as 3-4 weeks, despite BMP-2 production only in nanogram quantities and persisting for only 1-3 weeks. By 8 weeks the healed defects contained well-organised new bone with advanced neo-cortication and abundant marrow. Bone mineral content and mechanical strength were close to normal values. These data demonstrate the utility of this model when adapting this technology for bone healing in sheep, as a prelude to human clinical trials.