CNTO 530 functions as a potent EPO mimetic via unique sustained effects on bone marrow proerythroblast pools.

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 PODXL(high)CD71(high) (pro)erythroblasts at frequencies multifold above epoetin-alfa or darbepoietin-alfa. CNTO 530 moreover supported the sustained expansion of a bone marrow-resident Kit(neg)CD71(high)Ter119(neg) progenitor pool. Based on these distinct erythropoietic and EPOR signaling properties, CNTO 530 holds excellent promise as a new EPO mimetic.

Effects of CNTO 530, an erythropoietin mimetic-IgG4 fusion protein, on embryofetal development in rats and rabbits.

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, 0.9/1, 6, or 60 mg/kg subcutaneously (SC) on gestation day (GD)7 followed by maintenance doses of 0, 0.3, 2, or 20 mg/kg 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 high-dose rabbits only. Rat and rabbit fetuses were exposed to CNTO 530 in all dose groups. CONCLUSIONS: These studies show that the embryo/fetal 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 FcRn-mediated transcytosis.

Critical review of preclinical approaches to evaluate the potential of immunosuppressive drugs to influence human neoplasia.

Many immunosuppressive drugs are associated with an increased risk of B-cell lymphoma, squamous cell carcinoma, and Kaposi sarcoma. Thirteen immunosuppressive drugs have been tested in 2-year carcinogenicity studies (abatacept; azathioprine; busulfan; cyclophosphamide; cyclosporine; dexamethasone; everolimus; leflunomide; methotrexate; mycophenolate mofetil; prednisone; sirolimus; and tacrolimus) and in additional models including neonatal and genetically modified mice; chemical, viral, ultraviolet, and ionizing radiation co-carcinogenesis, and in models with transplanted tumor cells. The purpose of this review is to outline the mechanisms by which immunosuppressive drugs can influence neoplasia, to summarize the available preclinical data on the 13 drugs, and to critically review the performance of the models. A combination of primary tumor and metastasis assays conducted with transplanted cells may provide the highest value for hazard identification and can be applied on a case-by-case basis. However, for both small molecules and therapeutic proteins, determining the relative risk to patients from preclinical data remains problematic. Classifying immunosuppressive drugs based on their mechanism of action and hazard identification from preclinical studies and a prospective pharmacovigilance program to monitor carcinogenic risk may be a feasible way to manage patient safety during the clinical development program and postmarketing.

Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice.

BACKGROUND: The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases. METHODS: TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C). RESULTS: There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFalpha were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy. CONCLUSION: These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

Sequential univariate gating approach to study the effects of erythropoietin in murine bone marrow.

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 Kolmogorov-Smirnov distance (D) statistic. The performance of the algorithm is evaluated by applying it to a known three-color data set looking at activation of CD4+ and CD8+ lymphocytes with anti-CD3 antibody treatment and comparing the results to the expert analysis. The algorithm is then applied to a four-color 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 state-of-the-art 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 anti-CD3 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 poly/orthochromic 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.com/acidrap18.

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor-alpha transgenic mice.

TNF-alpha is a pleitropic cytokine that expresses both pro- and anti-inflammatory activity and transgenic mice expressing human tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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-alpha 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-alpha were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL-beta, IL-5, IL-6, IL-10, IL-12(p70), IL-17, TNF-alpha, IFNgamma, GM-CSF, MIP-1alphaJE, 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-alpha may contribute to myelodysplasia in ACD. Moreover, since human TNF-alpha can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD.

Genetic aspects of immune-mediated adverse drug effects.

Adverse drug effects (ADEs) are of great importance in medicine and account for up to 5% of all hospital admissions. ADEs can arise from several mechanisms and a wide range of drugs can cause immune-mediated ADEs (IMADEs). For a drug to elicit an IMADE, it must be both immunogenic (that is, able to sensitize the immune system) and antigenic (that is, able to evoke a response from a sensitized immune system). Unlike protein therapeutics, small-molecule drugs (or xenobiotics) are usually neither immunogenic nor antigenic. IMADEs are therefore the result of complex interactions between drug-metabolizing enzymes, immune sensitization and immune effectors. The genetic aspects of this interplay are discussed in this review.

Regulation of vascular endothelial growth factor expression by EMMPRIN via the PI3K-Akt signaling pathway.

Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) is a cell surface glycoprotein overexpressed in many solid tumors. In addition to its ability to stimulate stromal MMP expression, tumor-associated EMMPRIN also induces vascular endothelial growth factor (VEGF) expression. To explore the underlying signaling pathways used by EMMPRIN, we studied the involvement of phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), JUN, and p38 kinases in EMMPRIN-mediated VEGF regulation. Overexpression of EMMPRIN in MDA-MB-231 breast cancer cells stimulated the phosphorylation of only Akt and MAPKs but not that of JUN and p38 kinases. Conversely, inhibition of EMMPRIN expression resulted in suppressed Akt and MAPK phosphorylation. Furthermore, the PI3K-specific inhibitor LY294002 inhibited VEGF production by EMMPRIN-overexpressing cells in a dose- and time-dependent manner. On the other hand, the MAPK inhibitor U0126 did not affect VEGF production. In vivo, EMMPRIN-overexpressing tumors with elevated VEGF expression had a high level of phosphorylation of Akt and MAPK. Finally, when fibroblast cells were treated with recombinant EMMPRIN, Akt kinase but not MAPK was phosphorylated concomitant with an increase in VEGF production. Both the activation of Akt kinase and the induction of VEGF were specifically inhibited with a neutralizing antibody to EMMPRIN. Our results show that in both tumor and fibroblast cells EMMPRIN regulates VEGF production via the PI3K-Akt pathway but not via the MAPK, JUN, or p38 kinase pathways.

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.

BACKGROUND: 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. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.

Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are endopeptidases that play pivotal roles in promoting tumor disease progression, including tumor angiogenesis. In many solid tumors, MMP expression could be attributed to tumor stromal cells and is partially regulated by tumor-stroma interactions via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). The role of EMMPRIN during tumor angiogenesis and growth was explored by modulating EMMPRIN expression and activity using recombinant DNA engineering and neutralizing antibodies. In human breast cancer cells, changes in EMMPRIN expression influenced vascular endothelial growth factor (VEGF) production at both RNA and protein levels. In coculture of tumor cells and fibroblasts mimicking tumor-stroma interactions, VEGF expression was induced in an EMMPRIN- and MMP-dependent fashion, and was further enhanced by overexpressing EMMPRIN. Conversely, VEGF expression was inhibited by suppressing EMMPRIN expression in tumor cells, by neutralizing EMMPRIN activity, or by inhibiting MMPs. In vivo, EMMPRIN overexpression stimulated tumor angiogenesis and growth; both were significantly inhibited by antisense suppression of EMMPRIN. Expression of both human and mouse VEGF and MMP, derived from tumor and host cells, respectively, was regulated by EMMPRIN. These results suggest a novel tumor angiogenesis mechanism in which tumor-associated EMMPRIN functionally mediates tumor-stroma interactions and directly contributes to tumor angiogenesis and growth by stimulating VEGF and MMP expression.

Is murine gammaherpesvirus-68 (MHV-68) a suitable immunotoxicological model for examining immunomodulatory drug-associated viral recrudescence?

Immunosuppressive agents are used for treatment of a variety of autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosis (SLE), and psoriasis, as well as for prevention of tissue rejection after organ transplantation. Recrudescence of herpesvirus infections, and increased risk of carcinogenesis from herpesvirus-associated tumors are related with immunosuppressive therapy in humans. Post-transplant lymphoproliferative disorder (PTLD), a condition characterized by development of Epstein Barr Virus (EBV)-associated B-lymphocyte lymphoma, and Kaposi's Sarcoma (KS), a dermal tumor associated with Kaposi Sarcoma-associated virus (KSHV), may develop in solid organ transplant patients. KS also occurs in immunosuppressed Acquired Immunodeficiency (AIDS) patients. Kaposi Sarcoma-associated virus (KSHV) is a herpes virus genetically related to EBV. Murine gammaherpes-virus-68 (MHV-68) is proposed as a mouse model of gammaherpesvirus infection and recrudescence and may potentially have relevance for herpesvirus-associated neoplasia. The pathogenesis of MHV-68 infection in mice mimics EBV/KSHV infection in humans with acute lytic viral replication followed by dissemination and establishment of persistent latency. MHV-68-infected mice may develop lymphoproliferative disease that is accelerated by disruption of the immune system. This manuscript first presents an overview of gammaherpesvirus pathogenesis and immunology as well as factors involved in viral recrudescence. A description of different types of immunodeficiency then follows, with particular focus on viral association with lymphomagenesis after immunosuppression. Finally, this review discusses different gammaherpesvirus animal models and describes a proposed MHV-68 model to further examine the interplay of immunomodulatory agents and gammaherpesvirus-associated neoplasia.

Gene expression profiling for pharmaceutical toxicology screening.

Advances in medicinal chemistry and high-throughput pharmacological screening are creating a multitude of potential lead compounds. There is also heightened concern about drug-induced toxicity, which is all too often uncovered late in development or at the post marketing stage. Together, these factors have created a need for novel approaches to screen for toxicity. There have been technological advances that enable study of changes in the gene expression profile caused by toxic insults and important steps made toward unraveling target organ toxicity at the molecular level. Thus, gene expression profile-based screens hold the promise to revolutionize the way in which compounds are selected for development. For screens focused on specific mechanisms of toxicity, reporter gene systems have proven utility, albeit modest because of our limited knowledge of which genes are true surrogate markers for toxicity. For broader forecasts of toxicity, DNA microarrays hold great promise for delivering practical gene expression profile screens (GEPS). For this promise to be realized, however, a number of technological hurdles must be cleared: (i) cost; (ii) reproducibility; (iii) throughput; and (iv) data analysis. Of equal if not greater importance, issues relating to the test systems used, the requisite number of genes to be studied and the size and scope of the database upon which forecasts will be based must be addressed. At present, the proof-of-concept for GEPS for toxicity is in hand, and we are poised to realize the goal of creating practical GEPS for application in compound prioritization.

Predictive power of preclinical studies in animals for the immunogenicity of recombinant therapeutic proteins in humans.

Despite advances at the level of gene sequence (eg, humanized versus murine antibodies), expression systems (eg, mammalian versus prokaryotic) and post-expression modification (eg, 'PEGylation'), clinical immunogenicity of therapeutic proteins remains a concern. Although animals are routinely and effectively used to evaluate pharmacological activity and to support a claim of safety for recombinant proteins, their usefulness for predicting clinical immunogenicity is more questionable. This review argues that recombinant proteins can be grouped into immunogenic classes; for some of these classes, for example, bacterial proteins, immunogenicity in animals is often predictive for humans, but for others, for example, fully human proteins, even data from non-human primates can have little predictive value. We will attempt to make the case that for a variety of immunological and practical constraints, animal studies, even those conducted in non-human primates, have limited predictive power for immunogenicity in humans, and tend to over-predict clinical immunogenicity.

Engineering peptide therapeutics using MIMETIBODY™ technology.

The MIMETIBODY™ platform was developed to expand the opportunities for application of biotherapeutics. While the utility of antibodies as antagonists has been well demonstrated, their application as agonists has been more challenging. For steric reasons, antibodies may be less well suited to perform as agonists or as inhibitors of GPCRs. In contrast, many bioactive peptides function as agonists or by interaction with GPCRs but their development as therapeutics has been challenging due to their small size and metabolic lability. The MIMETIBODY™ platform has been used to develop a variety of stable, long-lived molecules with intrinsic activities similar to that of their parent peptides. This chapter describes methods for construction of expression plasmids, expression and purification strategies, and methods for characterizing the activity of these novel proteins.

CNTO 530 increases expression of HbA and HbF in murine models of ß-thalassemia and sickle cell anemia.

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 ß-thalassemia and sickle cell anemia (Berkeley mice). ß- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human ß-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-α (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 ß- 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 ß-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Concordance of preclinical and clinical pharmacology and toxicology of therapeutic monoclonal antibodies and fusion proteins: cell surface targets.

Monoclonal antibodies (mAbs) and fusion proteins directed towards cell surface targets make an important contribution to the treatment of disease. The purpose of this review was to correlate the clinical and preclinical data on the 15 currently approved mAbs and fusion proteins targeted to the cell surface. The principal sources used to gather data were: the peer reviewed Literature; European Medicines Agency 'Scientific Discussions'; and the US Food and Drug Administration 'Pharmacology/Toxicology Reviews' and package inserts (United States Prescribing Information). Data on the 15 approved biopharmaceuticals were included: abatacept; abciximab; alefacept; alemtuzumab; basiliximab; cetuximab; daclizumab; efalizumab; ipilimumab; muromonab; natalizumab; panitumumab; rituximab; tocilizumab; and trastuzumab. For statistical analysis of concordance, data from these 15 were combined with data on the approved mAbs and fusion proteins directed towards soluble targets. Good concordance with human pharmacodynamics was found for mice receiving surrogates or non-human primates (NHPs) receiving the human pharmaceutical. In contrast, there was poor concordance for human pharmacodynamics in genetically deficient mice and for human adverse effects in all three test systems. No evidence that NHPs have superior predictive value was found.

Concordance of preclinical and clinical pharmacology and toxicology of monoclonal antibodies and fusion proteins: soluble targets.

Monoclonal antibodies (mAbs) and fusion proteins directed towards soluble targets make an important contribution to the treatment of disease. The purpose of this review was to correlate the clinical and preclinical data on the 14 currently approved mAbs and fusion proteins targeted to soluble targets. The principal sources used to gather data were: the peer reviewed Literature; European Medicines Agency 'Scientific Discussions' and United States Food and Drug Administration 'Pharmacology/Toxicology Reviews' and package inserts (United States Prescribing Information). Data on the following approved biopharmaceuticals were included: adalimumab, anakinra, bevacizumab, canakinumab, certolizumab pegol, denosumab, eculizumab, etanercept, golimumab, infliximab, omalizumab, ranibizumab, rilonacept and ustekinumab. Some related biopharmaceuticals in late-stage development were also included for comparison. Good concordance with human pharmacodynamics was found for both non-human primates (NHPs) receiving the human biopharmaceutical and mice receiving rodent homologues (surrogates). In contrast, there was limited concordance for human adverse effects in genetically deficient mice, mice receiving surrogates or NHPs receiving the human pharmaceutical. In summary, the results of this survey show that although both mice and NHPs have good predictive value for human pharmacodynamics, neither species have good predictive value for human adverse effects. No evidence that NHPs have superior predictive value was found.

Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice.

Many immunosuppressive drugs are associated with an increased risk of neoplasia, principally non-melanoma skin cancers and B-cell lymphomas. However, only 6 of the 13 immunosuppressive drugs tested in 2 year bioassays increased the incidence of neoplasia. For example, the 2-year bioassays conducted with cyclosporine (CSA), an International Agency for Research on Cancer (IARC) Group 1 human carcinogen, were negative. The purpose of these investigations was to use transplanted tumor models in immunocompetent, syngeneic mice to gain insight into the failure of the 2-year bioassay to show an increased incidence of neoplasia with CSA. C3H HeN mice were used in a battery of assays with a transplanted squamous cell carcinoma (SCC VII cells) or a B-cell, lymphoma (38C13 cells) cells to study effects of CSA on local growth and metastases, experimental metastases, and progression of established metastases. Mice received CSA twice weekly by subcutaneous (SC) injection at doses of 0.5, 5, or 50 mg/kg; controls received the CSA vehicle. CSA had a modest inhibitory effect on SC tumors initiated by 38C13 cells and on intramuscular tumors initiated by SCC VII cells. CSA also decreased the number of lung colonies and decreased the size, growth fraction and vascularity of established lung metastases initiated by SCC VII cells. In contrast, CSA increased progressive growth of metastases to the sentinel lymph node from an intramuscular SCC VII tumor, but had no effect cellular traffic to the node. In conclusion, CSA at doses up to 50 mg/kg did not facilitate tumor progression and it partially inhibited tumor growth, suggesting that suppression of tumor progression may partially explain the failure of CSA to act as a carcinogen in 2 year bioassays.

Loss of toll-like receptor 3 function improves glucose tolerance and reduces liver steatosis in obese mice.

OBJECTIVE: Emerging evidence suggests a link between innate immunity and development of type 2 diabetes mellitus (T2D); however, the molecular mechanisms linking them are not fully understood. Toll-like Receptor 3 (TLR3) is a pathogen pattern recognition receptor that recognizes the double-stranded RNA of microbial or mammalian origin and contributes to immune responses in the context of infections and chronic inflammation. The objective of this study was to determine whether TLR3 activity impacts insulin sensitivity and lipid metabolism. MATERIALS AND METHODS: Wild type (WT) and TLR3 knock-out (TLR3(-/-)) mice were fed a high fat diet (HFD) and submitted to glucose tolerance tests (GTTs) over a period of 33 weeks. In another study, the same group of mice was treated with a neutralizing monoclonal antibody (mAb) against mouse TLR3. RESULTS: TLR3(-/-) mice fed an HFD developed obesity, although they exhibited improved glucose tolerance and lipid profiles compared with WT obese mice. In addition, the increase in liver weight and lipid content normally observed in WT mice on an HFD was significantly ameliorated in TLR3(-/-) mice. These changes were accompanied by up-regulation of genes involved in cholesterol efflux such as PPARδ, LXRα, and LXRα-targeting genes and down-regulation of pro-inflammatory cytokine and chemokine genes in obese TLR3(-/-) mice. Furthermore, global gene expression profiling in liver demonstrated TLR3-specific changes in both lipid biosynthesis and innate immune response pathways. CONCLUSIONS: TLR3 affects glucose and lipid metabolism as well as inflammatory mediators, and findings in this study reveal a new role for TLR3 in metabolic homeostasis. This suggests antagonizing TLR3 may be a beneficial therapeutic approach for the treatment of metabolic diseases.

Development of a human whole blood assay for prediction of cytokine release similar to anti-CD28 superagonists using multiplex cytokine and hierarchical cluster analysis.

Anti-CD28 superagonist (SA) mediated cytokine release syndrome (CRS), an adverse event resulting in systemic release of cytokines, is an emergent issue in drug development. CRS is of potential concern for all monoclonal antibodies (mAbs) particularly those directed against cell surface targets on lymphocytes. Concern regarding patient safety requires development of novel methods to predict these adverse reactions. Due to the inability of animal studies to predict CRS, we have developed a whole blood in vitro screen to support First in Human studies and assess the potential for mAbs to cause anti-CD28 SA-like CRS. For this purpose we have immobilized marketed mAbs, whose potential for causing CRS and milder infusion reactions is known, on Protein A beads and used these beads to stimulate cytokine release. After culture, supernatants are harvested and frozen for later multiplex analysis of cytokines using Searchlight™ technology. We have employed hierarchicalluster analysis (HCA) to allow comparison of 12 different cytokine levels across numerous donors, treatments, and experiments. Results conclusively distinguish test mAb responses from an anti-CD28 superagonist mAb response. As part of a global analysis of preclinical data, the results of this assay can facilitate entry into First in Human clinical trials, help with selection of starting doses and may allow more rapid dose escalation using smaller cohorts.