• Selective myeloma cell killing and enhanced effector function of a novel anti-BCMA antibody conjugated with MMAF via noncleavable linker.• Specific multiple myeloma antigen for monoclonal antibody-based immunotherapy.B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G 2 /M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer. (Blood. 2014;123(20):3128-3138) IntroductionAlthough there is no monoclonal antibody (mAb)-based targeted therapy approved to treat patients with multiple myeloma (MM), many mAbs targeting different antigens have been preclinically and clinically evaluated. ) were either moved toward or remain in clinical development based on encouraging results from preclinical studies. However, these antigens still lack specificity and are also expressed in other normal tissues including natural killer (NK) or other effectors, which could limit their clinical utility. Therefore, novel therapeutic mAbs to achieve improved MM selectivity, simultaneously targeting cytotoxic drugs to MM cells, are urgently needed.B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily (TNFRSF17), is selectively induced during plasma cell differentiation and is nearly absent on naive and memory B cells. 13,14 Upon binding to its ligands B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), the survival of bone marrow (BM) plasma cells and plasmablasts is promoted.15,16 BCMA does not maintain normal B-cell homeostasis, but is required for the survival of long-lived plasma cells. 17 In MM, BCMA messenger RNA (mRNA) is commonly expressed at high levels in malignant plasma...

The neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution.

In vitro studies indicate the therapeutic potential of mTOR inhibitors in treating multiple myeloma. To provide further support for this potential, we used the rapamycin analog CCI-779 in a myeloma xenograft model. CCI-779, given as 10 intraperitoneal injections, induced significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 cell lines. Effective doses of CCI-779 were associated with modest toxicity, inducing only transient thrombocytopenia and leukopenia. Immunohistochemical studies demonstrated the antitumor responses were associated with inhibited proliferation and angiogenesis, induction of apoptosis, and reduction in tumor cell size. Although CCI-779-mediated inhibition of the p70 mTOR substrate was equal in 8226 and OPM-2 tumor nodules, OPM-2 tumor growth was considerably more sensitive to inhibition of proliferation, angiogenesis, and induction of apoptosis. Furthermore, the OPM-2 tumors from treated mice were more likely to show downregulated expression of cyclin D1 and c-myc and up-regulated p27 expression.Because earlier work suggested heightened AKT activity in OPM-2 tumors might induce hypersensitivity to mTOR inhibition, we directly tested this by stably transfecting a constitutively active AKT allele into U266 cells. The in vivo growth of the latter cells was remarkably more sensitive to CCI-779 than the growth of control U266 cells. IntroductionThe phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) signaling pathway is important for the survival and growth of multiple myeloma (MM) cells and is an attractive target for antitumor therapy. [1][2][3] An important downstream target of PI3-K/AKT is the mammalian target of rapamycin (mTOR), which mediates phosphorylation of p70S6 kinase (p70) and 4E-BP1, 4 proteins responsible for the translation and expression of D-type cyclins and c-myc. 5,6 By preventing these phosphorylation events, mTOR inhibitors down-regulate such expression and induce G 1 cell cycle arrest. 7 In addition, these drugs up-regulate expression of the p27 CDK inhibitor, which may also contribute to G 1 arrest. 8 The in vitro sensitivity of MM cells to the antitumor effects to mTOR inhibitors frequently correlates with heightened AKT activity. [9][10][11] Rapamycin is a classical mTOR inhibitor. The poor solubility that compromised rapamycin as an intravenous agent led to the development of a more soluble ester analog of rapamycin, CCI-779. 12 We have shown in vitro anti-MM activity of rapamycin and CCI-779. 9,11,13 Exposure to these mTOR inhibitors prevents the proliferation of PTEN-and RAS-mutated myeloma cell lines and of interleukin-6 (IL-6)-stimulated proliferation of nonmutated myeloma clones. To provide a further preclinical rationale for the development of mTOR inhibitors in patients, we initiated the current study testing the effects of CCI-779 in vivo against human MM tumor growth in a murine xenograft model. Our results confirm that CCI-779 is effective in vivo against myeloma cells and demonstrate inhibited proliferation, angiogenes...

Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress-inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib.