A multicenter, randomized, rater-blinded, parallel-group, phase 3 study to compare the efficacy, safety, and immunogenicity of biosimilar RGB-10 and reference once-daily teriparatide in patients with osteoporosis.

The efficacy and safety of RGB-10 and reference teriparatide were evaluated in a randomized 52-week study in 250 patients with osteoporosis at high risk of fracture. RGB-10 was equivalent to reference teriparatide in efficacy and had a comparable safety profile. INTRODUCTION: RGB-10 is the first biosimilar teriparatide authorized in the European Union. This multicenter, randomized, rater-blinded, parallel-group phase 3 study evaluated equivalence in efficacy and compared safety between RGB-10 and reference teriparatide in patients with osteoporosis at high risk of fracture for registration in Japan. METHODS: Ambulatory postmenopausal women and men (≥ 55 years of age) with osteoporosis at high risk of fracture were randomized 1:1 to receive either RGB-10 or reference teriparatide 20 µg once daily via subcutaneous self-injection for 52 weeks. The primary efficacy endpoint was the percent change from baseline to 52 weeks in lumbar spine (L2-L4) bone mineral density (BMD). Safety outcomes and immunogenicity were also assessed. RESULTS: In total, 250 patients (125 in each group) were randomized. The percent change from baseline to 52 weeks in lumbar spine (L2-L4) BMD (mean ± standard deviation) was 8.94% ± 6.19% in the RGB-10 group and 9.65% ± 6.22% in the reference teriparatide group. The estimated between-group difference (95% confidence interval) was - 0.65% (- 2.17% to - 0.87%) within the pre-specified equivalence margin (± 2.8%), which indicates equivalence in efficacy between the two groups. Changes in BMD at lumbar spine (L1-L4), femoral neck, and total hip and serum procollagen type I amino-terminal propeptide were also similar between the groups. Safety profiles, including immunogenicity, were comparable. CONCLUSIONS: The therapeutic equivalence of RGB-10 to reference teriparatide was demonstrated. RGB-10 had comparable safety profile to that of reference teriparatide.

The Therapeutic Antibody LM609 Selectively Inhibits Ligand Binding to Human αVß3 Integrin via Steric Hindrance.

The LM609 antibody specifically recognizes αVß3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate-independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVß3-targeted radioimmunotherapy. To elucidate the mechanisms of recognition and inhibition of αVß3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVß3. Using single-particle electron microscopy, we show that LM609 binds at the interface between the ß-propeller domain of the αV chain and the ßI domain of the ß3 chain, near the RGD-binding site, of all observed integrin conformational states. Integrating these data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD-binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.

Pathogenesis of Osteosclerotic Change Following Treatment with an Antibody Against RANKL for Giant Cell Tumour of the Bone.

BACKGROUND: Giant cell tumours (GCTs) of the bone are intermediate tumours that are locally aggressive. Denosumab, an antibody against receptor activator of nuclear factor kappa-B ligand (RANKL), was recently developed; however, it induces osteosclerotic change through an unknown mechanism. We determined whether osteosclerotic change could be induced by neoplastic stromal cells of giant cell tumours (GCTs). PATIENTS AND METHODS: Participants included four patients with GCT of the bone who were treated with neoadjuvant denosumab. Expression of alkaline phosphatase (ALP), osteocalcin (OCN), RANKL and histone H3K36 trimethylation were assessed through immunohistochemistry of biopsy and surgical specimens. RESULTS: OCN expression was significantly elevated after denosumab treatment, whereas ALP and RANKL expressions were not significantly elevated. Immunofluorescence staining revealed OCN expression and H3K36 trimethylation while their co-localisation was confirmed in the surgical specimens. CONCLUSION: Denosumab promoted OCN expression and might induce the osteogenic differentiation of GCT stromal cells.

Current status in vitamin D and regulatory T cells--immunological implications.

There has been a continuous effort to understand possible non-Ca metabolism roles of vitamin D, including its role in the immune system and, in particular, in T cell-medicated immunity. Vitamin D receptor is found in significant concentrations in the T lymphocyte and macrophage populations, when we refer to immune system, and pretty much in any human tissue and cells. Until the eighties, no one had imagined that vitamin D might play a role in the functioning of the immune system. Today we accepted that the normal immune system harbors a regulatory T cell (Treg) population specialized for immune suppression. Currently, the most commonly known regulatory T-cell lineage is called CD4+ CD25high FoxP3+ regulatory T cells. Several autoimmune disorders have been linked to a deficiency in vitamin D3. In some autoimmune diseases, including multiple sclerosis (MS), a compromised Treg function is believed to be critically involved in the disease process. Vitamin D insufficiency has ramifications not only for bone health, but also in other non-skeletal areas of vitamin D function, such as immune cells, muscle cells and, perhaps, adipocytes. As a final conclusion, further researches in the field of vitamin D, Tregs, immunity (inflammatory processes, rejection, autoimmune diseases, etc.), either in vitro on cell cultures or in vivo using lab animals or volunteers are still necessary.

Inhibition of Behcet's disease by calcitonin.

Behcet's disease (BD) is a chronic, multisystem inflammatory disorder characterized by relapsing oral aphthous and genital ulcers, ocular inflammation, erythemanodosum and folliculitis-like lesions of the skin, arthritis, and central nervous system involvement. Its pathogenesis has not been fully elucidated but the etiology is accepted to be multifactorial, therefore the treatment of Behcet's disease continues to be a major therapeutic challenge. The identification of novel therapeutic agents for the treatment of these disorders is important. Calcitonin (CT), a peptide hormone secreted in response to hypercalcemia, has the dual effect of inhibiting osteoclast recruitment as well as their resorptive activity. A number of reviews have concluded that salmon calcitonin is safe and effective in the treatment of osteoporosis. Calcitonin abrogated the stimulating effect of RANKL or prednisolone; similar results were obtained with OPG. Additionally, the analgesic activity of salmon calcitonin has been shown in several controlled prospective double-blind studies to improve pain. Exogenous calcitonin is thought to cross the blood-brain barrier and to accumulate slowly in the brain, inducing analgesia once sufficient receptors are occupied. Since CT could antagonize resorptive and analgesic activity by competitively binding to CTR and has been considered as a specific antagonist, we postulate that the CT could function as a novel agent to inhibit BD. In our opinion, if the hypothesis proved to be practical, CT could be widely used in clinical settings to treat BD.

Two-year treatment with denosumab (AMG 162) in a randomized phase 2 study of postmenopausal women with low BMD.

UNLABELLED: Denosumab is a monoclonal antibody to RANKL. In this randomized, placebo-controlled study of 412 postmenopausal women with low BMD, subcutaneous denosumab given every 3 or 6 mo was well tolerated, increased BMD, and decreased bone resorption markers for up to 24 mo. Continued study of denosumab is warranted in the treatment of low BMD in postmenopausal women. INTRODUCTION: Denosumab is a fully human monoclonal antibody that inhibits RANKL, a key mediator of osteoclastogenesis and bone remodeling. This prespecified exploratory analysis evaluated the efficacy and safety of denosumab through 24 mo in the treatment of postmenopausal women with low BMD. MATERIALS AND METHODS: Four hundred twelve postmenopausal women with lumbar spine BMD T-scores of -1.8 to -4.0 or femoral neck/total hip T-scores of -1.8 to -3.5 were randomly assigned to receive double-blind, subcutaneous injections of placebo; denosumab 6, 14, or 30 mg every 3 mo; denosumab 14, 60, 100, or 210 mg every 6 mo; or open-label oral alendronate 70 mg once weekly. Outcome measures included BMD at the lumbar spine, total hip, distal one-third radius, and total body; bone turnover markers; and safety. RESULTS: Denosumab increased BMD at all measured skeletal sites and decreased concentrations of bone turnover markers compared with placebo at 24 mo. At the lumbar spine, BMD increases with denosumab ranged from 4.13% to 8.89%. BMD changes with denosumab 30 mg every 3 mo and > or =60 mg every 6 mo were similar to, or in some cases greater than, with alendronate. The incidence of adverse events was similar in the placebo, denosumab, and alendronate treatment groups. Exposure-adjusted adverse events over 2 yr of treatment were similar to those reported during the first year of treatment. CONCLUSIONS: In these postmenopausal women with low BMD, treatment with denosumab for 2 yr was associated with sustained increases in BMD and reductions in bone resorption markers compared with placebo.

Human gamma delta T cells and tumor immunotherapy.

Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens derived from pathogenic microbes in a TCR-dependent manner, such as pyrophosphomonoester compounds from mycobacteria and malaria parasite and alkyl amines from Proteus, suggesting that this subset of gamma delta T cells is involved in infectious immunity. The precise recognition mechanism has been delineated using a site-directed mutagenesis strategy based on crystal structure of gamma delta TCR. On the other hand, several lines of evidence indicate that human gamma delta T cells are involved in tumor immunity. Although activated gamma delta T cells exhibit a cytolytic activity against most of tumor cells, only a small fraction of tumor cells, like Burkitt lymphoma cells and multiple myeloid cells, is recognized by human gamma delta T cells in a TCR-dependent manner. This implicates that human gamma delta T cells have two distinct pathways for anti-tumor immunity. One is a natural killer-like pathway and the other is a TCR-dependent pathway. Recently, it was shown that treatment of human tumor cells with nitrogen-containing bisphosphonates, therapeutic drugs for hypercalcemia in malignancy, generated antigenic structure on the surface of tumor cells, which could be recognized by human gamma delta T cells in a TCR-dependent manner. This tumor labeling system may lead to a novel strategy for cancer immunotherapy.

Immune regulation of 25-hydroxyvitamin-D3-1alpha-hydroxylase in human monocytes.

UNLABELLED: Monocytes express 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNgamma and CD14/TLR4 activation. Cross-talk between the JAK-STAT, the NF-kappaB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPbeta to its recognition sites in the promoter of the 1alpha-hydroxylase gene is a prerequisite. INTRODUCTION: The activated form of vitamin D3, 1,25(OH)2D3, known for its action in bone and mineral homeostasis, has important immunomodulatory effects. 1,25(OH)2D3 modulates the immune system through specific nuclear receptors, whereas macrophages produce 1,25(OH)2D3. In monocytes, the expression of 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, is regulated by immune stimuli. The aim of this study was to elucidate the intracellular pathways through which interferon (IFN)gamma and Toll-like receptor (TLR) modulation regulate expression of 1alpha-hydroxylase in monocytes/macrophages. MATERIALS AND METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFNgamma (12.5 U/ml) and/or lipopolysaccharide (LPS; 100 ng/ml) for 48 h. The following inhibitors were used: janus kinase (JAK) inhibitor AG490 (50 microM), NF-kappaB inhibitor sulfasalazine (0.25 mM), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microM). 1alpha-hydroxylase mRNA expression was monitored by qRT-PCR. Phosphorylation of transcription factors was studied by Western blotting. Transfection of mutated or deletion promoter constructs, cloned in the pGL3-luciferase reporter plasmid, were performed in the RAW264.7 cell line. Cells were stimulated with IFNgamma (100 U/ml) and LPS (100 microg/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1alpha, NF-kappaB, and C/EBPbeta to their respective binding sites in the promoter was analyzed by gel shift assays. RESULTS: 1alpha-hydroxylase mRNA expression in monocytes is synergistically induced by IFNgamma and CD14/TLR4 ligation and paralleled by 1,25(OH)2D3 production. This induction requires the JAK-STAT, NF-kappaB, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block 1alpha-hydroxylase expression (JAK inhibitor, 60% inhibition, p < 0.01; NF-kappaB inhibitor, 70% inhibition, p < 0.05; p38 MAPK inhibitor, 95% inhibition, p < 0.005). In addition, we show the involvement of the p38 MAPK pathway in phosphorylation of C/EBPbeta. Direct binding of C/EBPbeta to its recognition sites in the 1alpha-hydroxylase promoter is necessary to enable its immune-stimulated upregulation. CONCLUSION: IFNgamma and CD14/TLR4 binding regulate expression of 1alpha-hydroxylase in monocytes in a synergistic way. Combined activation of the JAK-STAT, p38 MAPK, and NF-kappaB pathways is necessary, with C/EBPbeta most probably being the essential transcription factor controlling immune-mediated transcription.