Endosialin/TEM1 was originally discovered as a human embryonic fibroblast-specific antigen and was later found to be differentially expressed in tumor stroma and endothelium. Endosialin/TEM1 overexpression has been observed in many cancers of various tissue origin, including colon, breast, pancreatic, and lung. The knockout (KO) mouse model showed the absence of endosialin/TEM1 expression reduced growth, invasion, and metastasis of human tumor xenografts. In addition, lack of endosialin/TEM1 led to an increase in small immature blood vessels and decreased numbers of medium and large tumor vessels. This abnormal angiogenic response could be responsible for the reduced tumor growth and invasion observed in endosialin/TEM1 KO mice, suggesting a role for endosialin/TEM1 in controlling the interaction among tumor cells, endothelia, and stromal matrix. Here we report the identification of fibronectin (FN) and collagen types I and IV as specific ligands for endosialin/TEM1. More importantly, cells expressing endosialin/TEM1 exhibit enhanced adhesion to FN as well as enhanced migration through matrigel, although these properties could be blocked by a humanized antibody directed against human endosialin/TEM1. Our results pinpoint to a molecular mechanism by which expression of endosialin/TEM1 in the tumor stroma and endothelium may support tumor progression and invasion.CD248 ͉ fibronectin ͉ stroma ͉ collagen T he process of angiogenesis, which is critical for physiological tissue growth, wound healing, and embryonic development, also is required for the formation of large solid tumors (1). Within the developing capillary, extracellular matrix (ECM) proteins serve as a structural scaffold for proliferating endothelial and tumor tissues and, more important, provide support for the growth of tumor cells. Therefore, anticancer strategies aimed at disrupting these processes could result in effective therapies.Endosialin/TEM1 (CD248) originally discovered as a human embryonic fibroblast-specific antigen was later found to be differentially expressed in tumor stroma and endothelium. The monoclonal antibody FB5, generated through immunization of mice with human embryonic fibroblasts, was found to recognize an antigen, named endosialin, present on tumor stromal cells (2). Subsequently, examination of gene expression patterns in normal and neoplastic tissue indicated up-regulation of endosialin/TEM1 mRNA expression in tumor neovessels (3). Similar endosialin/TEM1 expression levels were noted in human colorectal cancer (4), breast cancer tissues (5), and histiocytomas (6). Moreover, human endosialin expression has been observed in highly invasive glioblastoma, anaplastic astrocytomas, and metastatic carcinomas, including melanomas (7,8).Tem1 knockout (KO) mice develop normally and exhibit normal wound healing, suggesting that endosialin/TEM1 is not required for neovascularization during fetal development or wound repair (9). However, when colorectal cancer cells were implanted in the abdominal sites of Tem1 KO mice, the loss of en...

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon ؉ cells, increased 38-fold in lon ؉ cells carrying a Tn10::⌬kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::⌬kan insertion. Sequencing of both sides of the Tn10::⌬kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.Regulation of colanic acid capsular polysaccharide gene (cps) expression in Escherichia coli is multilayered, as evidenced by the numerous direct and indirect regulatory mechanisms that have been identified to date (for reviews, see references 6 and 8). The current model proposes two pathways for activating cps expression (6). One pathway involves a twocomponent regulatory system (24). RcsC (regulator of capsule synthesis), which has been described as a membrane-bound sensor protein based on homology with the sensor component of two-component sensor regulator pairs (24), appears to be activated by environmental stimuli, such as desiccation (18) or osmotic shock (22). Presumably, activated RcsC either directly or indirectly modifies RcsB, the proposed effector of the twocomponent system, which in turn activates cps expression (6, 7). In the alternate pathway, the other positive effector of cps expression, RcsA, presumably forms a complex with RcsB, resulting in the activation of cps expression (2, 6, 25). RcsA is highly unstable and appears to be degraded in a Lon-dependent fashion (26; for a review, see reference 6). In lon ϩ cells, RcsA levels are low, and these cells produce little colanic acid (26; for a review, see reference 6). Conversely, in ⌬lon cells, RcsA levels are high, leading to increased colanic acid production and mucoid colonies (26; for a review, see reference 6). In this model, both pathways require RcsB for high-level expression of cps (6).Current evidence suggests that additional regulators of cps expression exist: mutations in hns (6 min) (21, 29), capS (22.5 min) (14), opsX (62 min) (30), or capT (unknown location) (14) lead to an increase in colanic acid production. Furthermore, mutations within the rfa locus (82 min) both alter lipopolysaccharide structure and synthesis and increase colanic acid production (19). Given the complexity of the cps system, identification of additional cps regulators seems probable. (SG20780 [2]) strains carrying the zce-23::⌬kan insertion were assayed for ␤-galactosidase activity (Table 1). A 38-fold increase in the level of cps expression was observed in either Luria-Bertani (LB) or minimal (M63 salts, 0.4% glucose, 0.1% Casamino Acids) medium with the introduction of the zce-23:: ⌬kan insertion into a lon ϩ strain. The zce-23::⌬kan insertion had no effect on cps expression in ⌬lon cells.The increase in cpsB10::lacZ expression is abolished in lon ؉ s...

Purpose Endosialin (TEM-1, CD248) is a protein expressed on the surface of activated mesenchymal cells, including certain subsets of tumors. Preclinical models suppressing endosialin function have shown antitumor activity. A humanized monoclonal antibody, MORAb-004, was engineered to target endosialin and is the first agent in clinical development for this mesenchymal cell target. Experimental Design This first-in-human, open-label, phase I study recruited patients with treatment-refractory solid tumors. MORAb-004 was administered intravenously once weekly in 4-week cycles. Objectives included determination of the safety of multiple infusions of MORAb-004, identification of the maximum tolerated dose (MTD), pharmacokinetic modeling, detection of any anti-human antibody response, and assessment of objective radiographic response to therapy. Results Thirty-six patients were treated at 10 dose levels of MORAb-004, ranging from 0.0625 to 16 mg/kg. Drug-related adverse events were primarily grade 1–2 infusion toxicities. Dose-limiting toxicity of grade 3 vomiting was observed at 16 mg/kg. Eighteen of 32 evaluable patients across all doses achieved disease stability, with minor radiographic responses observed in 4 patients (pancreatic neuroendocrine, hepatocellular, and sarcoma tumor types). Pharmacokinetics showed MORAb-004 accumulation beginning at 4 mg/kg and saturable elimination beginning at 0.25 mg/kg. Exposure increased in a greater-than-dose-proportional manner with terminal half-life increasing proportionally with dose. The MTD was identified as 12 mg/kg. Conclusions Preliminary antitumor activity was observed. Safety profile, pharmacokinetics, and early antitumor activity suggest that MORAb-004 is safe at doses up to 12 mg/kg and should be studied further for efficacy.

Capsule (cps) gene expression in Escherichia coli is controlled by a complex network of regulators. Transcription of the cps operon is controlled by at least two positive regulators, RcsA and RcsB. We show here that RcsA functions to activate its own expression, as seen by the 100-fold-increased expression of arcsA::lacZ transcriptional fusion in strains with high levels of RcsA protein, either due to a mutation inlon or due to overexpression of RcsA from a multicopy plasmid. Expression of the rcsA::lacZfusion is increased by but not dependent on the presence of RcsB. In addition, the effects of H-NS and RcsB on the expression ofrcsA are independent of each other. A sequence motif, conserved between the E. coli cps promoter and theErwinia amylovora ams promoter and previously shown to be the RcsA-RcsB binding site, was identified in the rcsApromoter region and shown to be required for high-level expression ofrcsA.

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes, humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however, this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g., affinity and titers) of mAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigenspecific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing.