Supplementary Figure 1 from Extracellular Matrix Metalloproteinase Inducer Stimulates Tumor Angiogenesis by Elevating Vascular Endothelial Cell Growth Factor and Matrix Metalloproteinases

Human immunodeficiency virus (HIV) in- fects cells after binding of the viral envelope glycoprotein gpl20 to the cell surface recognition marker CD4. gpl20 is nonco- valently associated with the HIV transmembrane envelope glycoprotein gp4l, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gpl20 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 pro- teins sT4, V1V2,DT, and V1(106) (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gpl20 from HIV-1 and HIV-1-infected cells. gpl20 release was con- centration-, time-, and temperature-dependent. The reaction was biphasic at 37?C and did not take place at 4?C, indicating that binding of soluble CD4 was not sufficient to release gpl20. The appearance of free gpl20 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp4l on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gpl20 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inacti- vate HIV by inducing the release of gpl20. We propose that HIV envelope-mediated fusion is initiated following rearrange-

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.

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

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.

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.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the involvement of multiple organ systems with alternating clinical exacerbations and remissions. Circulating immune complexes and autoantibodies can cause tissue damage and organ dysfunction with manifestations involving the skin, serosal surfaces, central nervous system, and kidneys (Rahman & Isenberg, 2008). B cells are believed to play an important role in SLE. B cells can function as APCs, produce cytokines and chemokines contributing to lymphoid regulation, and can respond to stimuli in the microenvironment at local tissues (Ramanujam & Davidson, 2008). Pathogenic autoantibodies produced by autoreactive B cells are believed to play an important role in the pathogenesis of SLE. CXCL13 has been shown to be a key mediator of organization of lymphoid tissues. CXCL13 is a B cell chemoattractant that is expressed by peritoneal macrophages and follicular dendritic cells in secondary lymphoid organs, such as the follicles of Peyer’s patches, the spleen and lymph nodes. Through interaction with CXCR5, a G-protein coupled receptor, CXCL13 attracts B lymphocytes and promotes migration of small numbers of T helper follicular cells and macrophages (Gunn et al., 1998). CXCL13 is critical for B cell homing and follicle formation in lymph node and spleen, and it is required for the development of lymph nodes and Peyer’s patches (Ansel et al., 2000). CXCL13 protein level is elevated in ectopic B cell follicles formed in the inflamed tissues of multiple chronic diseases, and plays an important role in maintaining inflammation by actively recruiting B cells (Carlsen et al., 2004; Magliozzi et al., 2004; Salolonsson et al., 2002; Shi et al., 2001;). CXCL13 has been shown to have increased expression in the thymus and kidney of aged NZB/W F1 mice, and may play a role in breaking immune tolerance in the thymus of autoimmune prone mice (Ishikawa et al., 2001). Treatment with anti-CXCL13 has shown efficacy in animal models of RA and EAE (Bagaeva et al. 2006; Zheng et al., 2005). Because of its function and presence in various pathological conditions, CXCL13 and CXCL13 dependent pathways are thought to be instrumental in the pathogenesis of a variety of diseases where B cells may play a significant role, including RA, OA, UC, and SLE, and could be potential targets for autoimmune therapy (Table 1).

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.

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 PPAR delta, LXR alpha, and LXR alpha-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. (C) 2012 Elsevier Inc. All rights reserved.

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.

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. Here, we report on experiments designed to study how the pharmacokinetic profiles of EPO receptor agonists, ranging from the short-lived epoetin-? to the long-lived EPO-MIMETIBODYTM constructs CNTO 530 and CNTO 531, influence the pharmacodynamic response in rats. Rats received a single dose of an EPO-R agonist and the effects on reticulocytes, red blood cells and hemoglobin were measured over time. The increase in red blood cells and hemoglobin were negatively correlated with clearance. At doses that cause a similar effect on reticulocytes, very long-lived EPO-R agonists caused prolonged production of red blood cells. In conclusion, we have shown that very long-lived EPO-R agonists cause prolonged production of red blood cells and increase in hemoglobin that is independent of their in vitro potency or the peak release of reticulocytes. These data suggest that EPO may be a survival factor for reticulocytes.

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. Here, we report on experiments designed to study how the pharmacokinetic profiles of EPO receptor agonists, ranging from the short-lived epoetin-? to the long-lived EPO-MIMETIBODYTM constructs CNTO 530 and CNTO 531, influence the pharmacodynamic response in rats. Rats received a single dose of an EPO-R agonist and the effects on reticulocytes, red blood cells and hemoglobin were measured over time. The increase in red blood cells and hemoglobin were negatively correlated with clearance. At doses that cause a similar effect on reticulocytes, very long-lived EPO-R agonists caused prolonged production of red blood cells. In conclusion, we have shown that very long-lived EPO-R agonists cause prolonged production of red blood cells and increase in hemoglobin that is independent of their in vitro potency or the peak release of reticulocytes. These data suggest that EPO may be a survival factor for reticulocytes.

CNTO 530 and darbepoetin- are long lived erythropoietin receptor agonists (ERAs).Clinically, anemia of chronic disease (ACD) is associated with increased expression of tumor necrosis factor- (TNF-) and mice transgenic for human TNF- develop ACD.The purpose of this investigation was to compare the effects of these agents in a murine model of ACD.Human TNF- expressing (Tg197) mice were administered a single subcutaneous dose of CNTO 530 or darbepoetin- and the pharmacodynamic response in bone marrow spleen and peripheral blood evaluated.RBC life span and reticulocyte age distribution were also evaluated.CNTO 530 induced a dose responsive increase in reticulocytes, RBCs and Hgb in both wild type and Tg197 mice.Although the reticulocyte response was similar to wild types, the RBC and Hgb response to CNTO 530 in Tg197 mice was blunted.There was no statistically significant difference in RBC life span with either compound.Darbepoetin-α caused a greater peak in % dead Pro/basophilic erythroblasts, greater peak EMH in the spleen and a greater increase in reticulocyte maturation time.In contrast, despite a similar peak increase, CNTO 530 caused a more sustained response of reticulocyte, EMH, RBC and Hgb, consistent with increased exposure.In conclusion, CNTO 530 and darbepoetin- increased RBC and hemoglobin in a murine model of ACD.Compared to darbepoetin-, CNTO 530 had a more sustained effect, consistent with increased exposure.

Non‐selective immunosuppressive drugs are associated with an increased risk of neoplasia. Although selective immunosuppressants, e.g., monoclonal antibodies, will likely pose less of a hazard, concerns remain about an increased risk of neoplasia, particularly squamous cell carcinoma. The 2 year bioassay has been the industry standard for evaluating carcinogenicity. However, it has poor predictive value for immunosuppressive drugs, accurately identifying a hazard with only 5 of the 13 drugs tested. Thus, there is need for an alternative testing strategy. We have developed a novel 3‐stage model to study tumor promotion and progression in vivo. We chose a murine squamous skin cell carcinoma (SCC VII) cell line to study local invasion and dissemination, colonization, and tumor progression in C3H‐HeN, SCID and SCID/bg mice in the presence and absence of cyclosporin A. Local invasion and dissemination were evaluated by following emigration of quantum dot labeled tumor cells to the draining lymph node. Lung colony counts were made after iv injection of tumor cells and angiogenesis and growth fraction were evaluated morphometrically. Immunosuppression, was associated with increased local growth and lung colonization but with decreased angiogenesis and growth rates. These results suggest that a series of models described here may offer a viable alternative to the 2 year bioassay.

INTRODUCTION:Immunosuppressive drugs are associated with an increased risk of infections and in some cases neoplasia, particularly non-melanoma skin cancers. This paper describes the development of a model to test the effects of immunosuppressive drugs on local invasion and metastases of a squamous cell carcinoma in syngeneic, immunocompetent mice. METHODS:SCC VII cells were labeled with 655 quantum dots (QDs), injected intramuscularly into C3H HEN mice and traffic and progressive growth in the draining popliteal lymph node were evaluated. RESULTS:SCC VII cells express RAE-1, an NKG2D ligand, and were sensitive to natural killer (NK) cells in vitro. QDs were stable in SCC VII cells and showed no evidence of toxicity to the cells. In vivo, confocal microscopy showed that QD-labeled SCC VII cells could migrate to the draining node and microfluorimetry showed progressive traffic of QDs to the node. There was no evidence of systemic toxicity of QDs. Primary immunosuppression in SCID and SCID-beige mice and treatment of normal mice with immunosuppressive agents (anti-asialoGM1 and cyclophosphamide) can enhance traffic of QDs and/or metastases to the draining lymph node. In contrast, cyclosporine had no effect on traffic or metastases. CONCLUSION:This model of local invasion and metastases may be useful in immunotoxicology for identifying and characterizing the hazard posed by selective immunosuppressive drugs.

BACKGROUNDCNTO 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.METHODSFor 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.RESULTSAdministration 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.CONCLUSIONSThese 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.

Infusion reactions and cytokine release syndrome (CRS) are an emerging issue in drug development and are of particular importance with the development of new therapeutic proteins. Increasing concerns regarding patient safety require a better understanding of the mechanism involved and the development of novel methods for preventing and predicting such reactions and CRS. This review discusses developments during the past few years in understanding the mechanisms that cause infusion reactions and CRS, advances in approaches to prevent CRS, the reason why preclinical animal models are unreliable predictors of CRS, and new developments in the design and analysis of in vitro screening systems for the prediction of CRS.

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.