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CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of -thalassemia and sickle cell anemia (Berkeley mice). - thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human -thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin- (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in - thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of -thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Renal insufficiency is commonly associated with an erythropoietin (EPO) dependent anemia. CNTO 530 is an EPO-mimetic antibody fusion protein with EPO-receptor agonist activity. The pharmacokinetics and pharmacodynamic activity of CNTO 530 were evaluated in a rat model of chronic renal insufficiency (CRI) induced anemia. Following 5/6 nephrectomy and 13 -17 weeks of stabilization, rats received a single subcutaneous (SC) dose of saline or CNTO 530 (0.03, 0.1, or 0.3 mg/kg). CNTO 530 caused dose-dependent reticulocytosis and long-lived increases in red blood cells, hemoglobin, and hematocrit. After receiving a dose of 0.1 mg/kg, red cell indices increased and remained within normal range for 44 days. Pharmacokinetic analysis showed that clearance of CNTO 530 increased 1.4 fold and terminal t1/2 and AUC decreased 1.6 fold in CRI rats compared to controls. CNTO 530 is active in a CRI rat model of anemia and widely spaced treatments with CNTO 530 may be sufficient to combat anemia.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

BACKGROUND:CNTO 530is a biopharmaceutical consisting of a novel peptide that mimics the actions of erythropoietin, fused to the Fc fragment of human IgG4. Pharmacokinetic and pharmacodynamic studies showed that CNTO 530 produced sustained increases in red blood cell parameters in rats and rabbits and that the serum half life of CNTO 530 was 2 days in rabbits and 3 days in rats. METHODS:For the evaluation of embryofetal development, CNTO 530 was injected at loading doses of 0, 0.91, 6, or 60 mgkg subcutaneously SC on gestation day GD7 followed by maintenance doses of 0, 0.3, 2, or 20 mgkg SC every 3 days through GD16 in rats and every 2 days through GD19 in rabbits GD0 was the day of mating. Rats were Caesarean sectioned on GD21, rabbits on GD29. RESULTS:Administration of CNTO 530 was associated with an increase in hematocrit at all dose levels and a decrease in maternal body weight gains. Fetuses exhibited reduced body weight and delayed ossification. Soft tissue changes were limited to cardiovascular alterations in the highdose rabbits only. Rat and rabbit fetuses were exposed to CNTO 530 in all dose groups. CONCLUSIONS:These studies show that the embryofetal development effects observed following CNTO 530 treatment during organogenesis are qualitatively similar to those seen with other erythropoietin agonists and are likely a secondary consequence of increased hematocrit in the dams. Unlike other erythropoietin receptor agonists, CNTO 530 was able to cross the placental barrier, which was considered likely the result of FcRnmediated transcytosis. Birth Defects Res Part B89:87–96, 2010. © 2010 WileyLiss, Inc.

Anemia as associated with numerous clinical conditions can be debilitating, but frequently can be treated via administration of epoetin-alfa, darbepoietin-alfa, or methoxy-PEG epoetin-beta. Despite the complexity of EPO-EPO receptor interactions, the development of interesting EPO mimetic peptides (EMPs) also has been possible. CNTO 530 is one such novel MIMETIBODY Fc-domain dimeric EMP fusion protein. In a mouse model, single-dose CNTO 530 (unlike epoetin-alfa or darbepoietin-alfa) bolstered red cell production for up to 1 month. In 5-fluorouracil and carboplatin-paclitaxel models, CNTO 530 also protected against anemia with unique efficiency. These actions were not fully accounted for by half-life estimates, and CNTO 530 signaling events therefore were studied. Within primary bone marrow erythroblasts, kinetics of STAT5, ERK, and AKT activation were similar for CNTO 530 and epoetin-alfa. p70S6K activation by CNTO 530, however, was selectively sustained. In vivo, CNTO 530 uniquely stimulated the enhanced formation of PODXLhighCD71high (pro)erythroblasts at frequencies multifold above epoetin-alfa or darbepoietin-alfa. CNTO 530 moreover supported the sustained expansion of a bone marrow–resident KitnegCD71highTer119neg progenitor pool. Based on these distinct erythropoietic and EPOR signaling properties, CNTO 530 holds excellent promise as a new EPO mimetic.

Analysis of multicolor flow cytometric data is traditionally based on the judgment of an expert, generally time consuming, sometimes incomplete and often subjective in nature. In this article, we investigate another statistical method using a Sequential Univariate Gating SUG algorithm to identify regions of interest between two groups of multivariate flow cytometric data. The metric used to differentiate between the groups of univariate distributions in SUG is the KolmogorovSmirnov distance D statistic. The performance of the algorithm is evaluated by applying it to a known threecolor data set looking at activation of CD4 and CD8 lymphocytes with antiCD3 antibody treatment and comparing the results to the expert analysis. The algorithm is then applied to a fourcolor data set used to study the effects of recombinant human erythropoietin rHuEPO on several murine bone marrow populations. SUG was used to identify regions of interest in the data and results compared to expert analysis and the current stateoftheart statistical method, Frequency Difference Gating FDG. Cluster analysis was then performed to identify subpopulations responding differently to rHuEPO. Expert analysis, SUG and FDG identified regions in the data that showed activation of CD4 and CD8 lymphocytes with antiCD3 treatment. In the rHuEPO treated data sets, the expert and SUG identified a dose responsive expansion of only the erythroid precursor population. In contrast, FDG resulted in identification of regions of interest both in the erythroid precursors as well as in other bone marrow populations. Clustering within the regions of interest defined by SUG resulted in identification of four subpopulations of erythroid precursors that are morphologically distinct and show a differential response to rHuEPO treatment. Greatest expansion is seen in the basophilic and polyorthochromic erythroblast populations with treatment. Identification of populations of interest can be performed using SUG in less subjective, time efficient, biologically interpretable manner that corroborates with the expert analysis. The results suggest that basophilic erythroblasts cells or their immediate precursors are an important target for the effects of rHuEPO in murine bone marrow. The MATLAB implementation of the method described in the article, both experimental data and other supplemental materials are freely available at http:web.mac.comacidrap18. © 2008 International Society for Advancement of Cytometry

TNF‐α is a pleitropic cytokine that expresses both pro‐ and anti‐inflammatory activity and transgenic mice expressing human tumor necrosis factor‐α (TNF‐α) exhibit a progressive polyarthritis that models rheumatoid arthritis (RA). One of the common comorbidities of RA is anemia of chronic disease (ACD). The purpose of these experiments was to study the changes in the bone marrow and peripheral blood that accompany polyarthritis in TNF‐α transgenic mice in an effort to better understand the pathogenesis of myelodysplasia and ACD. Polychromatic cytometry, hematology and serum cytokine analysis were used to study the pathogenesis of ACD in human TNF‐α transgenic mice. Our hematological evaluation revealed a mild, compensated, microcytic hypochromic anemia, and monocytosis. In the bone marrow, we observed alterations in cell kinetics, decreased relative expression of transferrin receptor and increased apoptosis and cell death in several late precursor cell populations. Although significant levels of human TNF‐α were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL‐β, IL‐5, IL‐6, IL‐10, IL‐12(p70), IL‐17, TNF‐α, IFNγ, GM‐CSF, MIP‐1αJE, MCP‐5 was observed. Tg197 mice develop a compensated, microcytic, hypochromic anemia, and a functional iron deficiency by 9 weeks of age. Changes in peripheral blood are reflected in alterations in cell kinetics, transferrin receptor expression and markedly increased apoptosis and cell death in the bone marrow indicating that TNF‐α may contribute to myelodysplasia in ACD. Moreover, since human TNF‐α can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD © 2008 International Society for Analytical Cytology

Classically, B-lymphocytes (B cells) are considered to be the mediators of humoral immunity and their role in inflammatory disease largely confined to the down-stream function of antigen-antibody complexes, e.g., in fixing complement and mediating antibody dependent cellular cytotoxicity. More recently, and with the growing acceptance of the view that the immune system operates as an interconnected web of cells and cytokines, a larger role for B cells has been proposed. In this review, with a focus on how B cells and their cytokine products may present novel therapeutic targets, we will briefly discuss B cell ontogeny and discuss the evidence supporting a larger role for B cells in a variety of inflammatory diseases. Special emphasis will be placed on autoimmune diseases. These discussions are intended to provide the reader with the basis for viewing B cells as players of a broader role in inflammatory disease and thus suggest avenues for exploiting B cell directed therapy in novel ways.

Cellular binding of annexin V and membrane permeability to 7‐aminoactinomycin D (7AAD) are important tools for studying apoptosis and cell death by flow cytometry. Combining viability markers with cell surface marker expression is routinely used to study various cell lineages. Current classification methods using strict thresholds, or “gates,” on the fluorescent intensity of these markers are subjective in nature and may not fully describe the phenotypes of interest. We have developed objective criteria for phenotypic boundary recognition through the application of statistical pattern recognition. This task was achieved using artificial neural networks (ANNs) that were trained to recognize subsets of cells with known phenotypes, and then used to determine decision boundaries based on statistical measures of similarity. This approach was then used to test the hypothesis that erythropoietin (EPO) inhibits apoptosis and cell death in erythroid precursor cells in murine bone marrow.Our method was developed for classification of viability using an in vitro cell system and then applied to an ex vivo analysis of murine late‐stage erythroid progenitors. To induce apoptosis and cell death in vitro, an EPO‐dependent human leukemic cell line, UT‐7EPO cells were incubated without recombinant human erythropoietin (rhEPO) for 72 h. Five different ANNs were trained to recognize live, apoptotic, and dead cells using a “known” subset of the data for training, and a K‐fold cross validation procedure for error estimation. The ANNs developed with the in vitro system were then applied to classify cells from an ex vivo study of rhEPO treated mice. Tg197 (human tumor necrosis‐α transgenic mice, a model of anemia of chronic disease) received a single s.c. dose of 10,000 U/kg rhEPO and femoral bone marrow was collected 1, 2, 4, and 8 days after dosing. Femoral bone marrow cells were stained with TER‐119 PE, CD71 APC enable identification of erythroid precursors, and annexin V FITC and 7AAD to identify the apoptotic and dead cells. During classification forward and side angle light scatter were also input to all pattern recognition systems.Similar decision boundaries between live, apoptotic, and dead cells were consistently identified by the neural networks. The best performing network was a radial basis function multi‐perceptron that produced an estimated average error rate of 4.5% ± 0.9%. Using these boundaries, the following results were reached: depriving UT‐7EPO cells of rhEPO induced apoptosis and cell death while the addition of rhEPO rescued the cells in a dose‐dependent manner. In vivo, treatment with rhEPO resulted in an increase of live erythroid cells in the bone marrow to 119.8% ± 9.8% of control at the 8 day time point. However, a statistically significant transient increase in TER‐119+ CD71+ 7AAD+ dead erythroid precursors was observed at the 1 and 2 day time points with a corresponding decrease in TER‐119+ CD71+ 7AAD− Annexin V− live erythroid precursors, and no change in the number of TER‐119+ CD71+ annexin V+ 7AAD− apoptotic erythroid precursors in the bone marrow.A statistical pattern recognition approach to viability classification provides an objective rationale for setting decision boundaries between “positive” and “negative” intensity measures in cytometric data. Using this approach we have confirmed that rhEPO inhibits apoptosis and cell death in an EPO dependent cell line in vitro, but failed to do so in vivo, suggesting EPO may not act as a simple antiapoptotic agent in the bone marrow. Rather, homeostatic mechanisms may regulate the pharmacodynamic response to rhEPO. © 2007 International Society for Analytical Cytology

Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model.

In vivo models have demonstrated that interleukin-13 (IL-13) plays an important role in asthma; however, few studies have evaluated the effect of inhibition of IL-13 on established and persistent disease. In the present study, we have investigated the effect of a therapeutic dosing regimen with an anti-IL-13 monoclonal antibody (mAb) in a chronic mouse model of persistent asthma. BALB/c mice were sensitized to allergen [ovalbumin (OVA); on days 1 and 8] and challenged with OVA weekly from day 22. Anti-IL-13 mAb or vehicle dosing was initiated following two OVA challenges when disease was established. At this time, mice exhibited airway hyperresponsiveness (AHR), increased mucus production, inflammation, and initiation of subepithelial fibrosis compared with saline-challenged mice. Mice received four additional OVA challenges. Treatment with anti-IL-13 mAb inhibited AHR and prevented the further development of subepithelial fibrosis and progression of inflammation. Furthermore, mAb treatment reversed the mucus hyperplasia to basal levels. These effects were associated with an inhibition of cytokines, chemokines, and matrix metalloproteinase-9. These data demonstrate that neutralization of IL-13 can inhibit the progression of established disease in the presence of repeated allergen exposures.

Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO-induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress. Novikoff rat hepatoma (N1S1) cells were incubated with TRO, troglitazone quinone (TQ), thiazolidinedione-phenoxyacetic acid (TD-PAA) or rosiglitazone (RSG). Membrane peroxidation, intracellular glutathione (GSH) content, and cellular viability were monitored simultaneously by multiparameter flow cytometry. TRO and TQ increased membrane peroxidation, decreased intracellular GSH, and decreased cell viability in a concentration-dependent manner. In contrast, TD-PAA and RSG neither increased membrane peroxidation nor induced loss of cell viability. In addition, TRO caused a concentration-dependent increase in intracellular superoxide generation accompanied by a collapse in mitochondrial membrane potential. Multiparameter flow cytometric evaluation of N1S1 cells indicated that the chromane ring of TRO, rather than the TZD moiety, may be responsible for oxidative stress and suggested that a direct effect on mitochondrial physiology may play a role in TRO-mediated hepatotoxicity. Cytometry Part A 52A:28–35, 2003. © 2003 Wiley-Liss, Inc.