Luigi Grasso

Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration

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.

Endosialin/TEM-1/CD248 regulates pericyte proliferation through PDGF receptor signaling.

Recent reports have described several cellular phenotypes that appear to be mediated by Endosialin/TEM-1/CD248 (TEM-1), including tubule formation on matrigel, migration, and proliferation. It has been shown that siRNA knock-down of TEM-1 in primary human fibroblasts resulted in reduced proliferation. However, the downstream signaling events that mediate TEM-1 function(s) currently remain unknown. In this study, we demonstrate that TEM-1 mediates proliferation of primary human pericytes through a PDGF receptor signaling pathway. Normal pericytes expressing high levels of TEM-1 were able to proliferate, respond to PDGF-BB stimulation by phosphorylating both the PDGF receptor and the MAP kinase ERK-1/2, and induce the expression of the immediate early transcription factor c-Fos. However, when TEM-1 expression was knocked-down, PDGF-BB-induced proliferation, ERK-1/2 phosphorylation, and c-Fos expression were significantly impaired. Thus, our results provide evidence for a TEM-1-dependent signal pathway that controls proliferation of human pericytes and suggest targeting this pathway for future strategies aimed at mitigating tumor angiogenesis.

The role of p53, p21WAF1/C1P1, and bcl-2 in radioresistant colorectal carcinoma

Genetic alterations in the p53 tumor suppressor gene are common in human colorectal cancers, occurring in approximately 70% of tumors. In vitro studies have shown that wild-type p53 is involved in controlling cell cycle checkpoint functions and apoptosis involved in the cytotoxic response induced by ionizing radiation and several anticancer chemotherapeutic agents. Wild-type p53 protein can transcriptionally activate the WAF gene, which encodes a cyclin-dependent kinase inhibitory protein, p21WAF1/C1PI protein, and transcriptionally repress the bcl-2 gene, which encodes an inhibitor of apoptosis. To learn more about the in vivo relationship between p53 protein and the expression of p21WAF1/C1PI and bcl-2 proteins in human colorectal cancers treated with radiation therapy, we examined the expression of these proteins by immunohistochemistry in pre-irradiated biopsy specimens and surgical specimens with residual tumor of 27 patients with colorectal carcinoma. Cell proliferation was measured using Ki-67 expression in the tumor cells. The p53 protein was not detected in normal colorectal mucosa, but it was expressed in 21 of 27 (78%) of pre-irradiated tumor samples and in 19 of 27 (70%) of post-irradiated tumors. Expression of the bcl-2 protein in normal colorectal mucosa was confined to the basal epithelial cells of the crypts. Diffuse bcl-2 staining was detected in tumor cells in 13 of 27 (48%) of pre-irradiated samples and in 14 of 27 (52%) of post-irradiated samples. p21WAF1/C1PI expression was detected in 14 of 27 (52%) of pre-irradiated samples but only in 7 of 27 (26%) of post-irradiated samples. No inverse relationship between expression of p53 protein and abnormal bcl-2 expression was apparent. p21WAF1/C1PI was expressed in most nonproliferating Ki-67-negative epithelial cells at the apical tips of the crypts in normal colorectal mucosa, but not in proliferating Ki-67-positive cells of adjacent adenomatous mucosa. An inverse relationship between Ki-67 and p21WAF1/C1PI expression was observed in normal colorectal mucosa and adjacent adenomatous mucosa. After radiation therapy, p53 protein accumulation did not change among residual tumors in 18 cases (three of which were initially negative and remained negative); in four cases there was a significant increase, and five cases had a substantial decrease of p53 expression. Aberrant bcl-2 expression is not correlated with expression of p53 and does not increase significantly in post-irradiated tumor cells. p21WAF1/C1PI expression is markedly reduced in tumor cells that survive radiation therapy.

The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity

Because of its high mortality rate, ovarian cancer is a leading cause of death among women and a highly unmet medical need. New therapeutic agents that are effective and well tolerated are needed and cancer antigen-specific monoclonal antibodies that have direct pharmacologic effects or can stimulate immunological responses represent a promising class of agents for the treatment of this disease. The human folate receptor α (FOLR1), which is overexpressed in ovarian cancer but largely absent in normal tissues, appears to play a role in the transformed phenotype in ovarian cancer, cisplatin sensitivity, and growth in depleted folate conditions and therefore has potential as a target for passive immunotherapy. The anti-FOLR1 monoclonal antibody MORAb-003 (farletuzumab) was previously shown to elicit antibody dependent cellular cytotoxicity (ADCC) and inhibit tumor growth of human tumor xenografts in nude mice. Because of its promising preclinical profile, farletuzumab has been evaluated in clinical trials as a potential therapeutic agent for ovarian cancer. In this report, we demonstrated that farletuzumab’s antitumor effect against an experimental model of ovarian cancer is mediated by its ADCC activity.

Co-Development of Diagnostic Vectors to Support Targeted Therapies and Theranostics: Essential Tools in Personalized Cancer Therapy

Novel technologies are being developed to improve patient therapy through the identification of targets and surrogate molecular signatures that can help direct appropriate treatment regimens for efficacy and drug safety. This is particularly the case in oncology whereby patient tumor and biofluids are routinely isolated and analyzed for genetic, immunohistochemical, and/or soluble markers to determine if a predictive biomarker signature (i.e., mutated gene product, differentially expressed protein, altered cell surface antigen, etc.) exists as a means for selecting optimal treatment. These biomarkers may be drug-specific targets and/or differentially expressed nucleic acids, proteins, or cell lineage profiles that can directly affect the patient’s disease tissue or immune response to a therapeutic regimen. Improvements in diagnostics that can prescreen predictive response biomarker profiles will continue to optimize the ability to enhance patient therapy via molecularly defined disease-specific treatment. Conversely, patients lacking predictive response biomarkers will no longer needlessly be exposed to drugs that are unlikely to provide clinical benefit, thereby enabling patients to pursue other therapeutic options and lowering overall healthcare costs by avoiding futile treatment. While patient molecular profiling offers a powerful tool to direct treatment options, the difficulty in identifying disease-specific targets or predictive biomarker signatures that stratify a significant fraction within a disease indication remains challenging. A goal for drug developers is to identify and implement new strategies that can rapidly enable the development of beneficial disease-specific therapies for broad patient-specific targeting without the need of tedious predictive biomarker discovery and validation efforts, currently a bottleneck for development timelines. Successful strategies may gain an advantage by employing repurposed, less-expensive existing agents while potentially improving the therapeutic activity of novel, target-specific therapies that may otherwise have off-target toxicities or less efficacy in cells exhibiting certain pathways. Here, we discuss the use of co-developing diagnostic-targeting vectors to identify patients whose malignant tissue can specifically uptake a targeted anti-cancer drug vector prior to treatment. Using this system, a patient can be predetermined in real-time as to whether or not their tumor(s) can specifically uptake a drug-linked diagnostic vector, thus inferring the uptake of a similar vector linked to an anti-cancer agent. If tumor-specific uptake is observed, then the patient may be suitable for drug-linked vector therapy and have a higher likelihood of clinical benefit while patients with no tumor uptake should consider other therapeutic options. This approach offers complementary opportunities to rapidly develop broad tumor-specific agents for use in personalized medicine.

Molecular Analysis of Human Interleukin-9 Receptor Transcripts in Peripheral Blood Mononuclear Cells IDENTIFICATION OF A SPLICE VARIANT ENCODING FOR A NONFUNCTIONAL CELL SURFACE RECEPTOR

Genetic studies on mouse models of asthma have identified interleukin-9 (IL9) as a determining factor in controlling bronchial hyperresponsiveness, a hallmark of the disease. Recently, the human IL9 receptor (hIL9R) gene locus has also been implicated in determining susceptibility to bronchial hyperresponsiveness and asthma. In order to evaluate the structure and function of the encoded product, we analyzed receptor transcripts derived from peripheral blood mononuclear cells of 50 unrelated donors. Sequence analysis of the entire coding region identified a splice variant that contains an in frame deletion of a single residue at codon 173 (ΔQ). This variant could be permanently expressed in a cytokine-dependent murine T-cell line but lacked the ability to induce proliferation in response to human IL9. In situ analyses of cells expressing the wild-type and ΔQ receptors found both forms to be expressed at the cell surface, but the ΔQ receptor was unable to bind hIL9 and could not be recognized by N-terminal specific antibodies. These findings demonstrate that hIL9RΔQ presents an altered structure and function and suggests its potential role in down-regulating IL9 signaling in effector cells and associated biological processes.

A First-in-human Phase I Study of MORAb-004, a Monoclonal Antibody to Endosialin in Patients with Advanced Solid Tumors

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. Clin Cancer Res; 21(6); 1281–8. ©2014 AACR.

Morphogenics as a tool for target discovery and drug development.

Abstract: Mutations in DNA mismatch repair (MMR) genes lead to genetically hypermutable cells. Germline mutations in MMR genes in man have been linked to the genetic predisposition to hereditary nonpolyposis colon cancer and a number of other inherited and sporadic malignancies. The ability to modulate the MMR process (referred to as morphogenics) in model systems offers a powerful tool for generating functional diversity in cells and multicellular organisms via the perpetual genomewide accumulation of randomized point and slippage mutation(s). Morphogenics is a platform process that employs a dominant negative MMR gene to create genetic diversity within defined cellular systems and results in a wide range of phenotypes, thus enabling the development and improvement of pharmaceutical products and the discovery of new pharmaceutical targets. Libraries of morphogenics‐derived siblings are generated through random mutagenesis from naturally occurring DNA polymerase‐induced mutations that occur during DNA replication. Morphogenic cells are screened in high‐throughput assays to identify subclones with desired phenotypes for pathway discovery and/or product development. Morphogenics has been successfully applied to a wide range of hosts, including mammalian cells, transgenic mice, plants, yeast, and bacteria. Manipulation of these systems via morphogenics has led to the discovery of novel disease‐associated phenotypes in targeted model systems. Moreover, morphogenics has been successfully applied to antibody‐producing cell lines to yield subclones producing antibodies with enhanced binding affinities for therapeutic use, as well as to derive subclones with enhanced titers that are suitable for scaleable manufacturing. The selective manipulation of the MMR process via morphogenics is a platform technology that offers many advantages for the discovery of druggable targets, as well as for the development of novel pharmaceutical products.

Generation and characterization of high affinity human monoclonal antibodies that neutralize staphylococcal enterotoxin B

BackgroundStaphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies.MethodsTo develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo.ResultsThe high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD50 (0.2 μg) , protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge.ConclusionWe have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.

Antibodies to GM-CSF

Hybridoma lines that secrete human monoclonal antibodies with high binding specificity and biological activity, particularly neutralizing activity against granulocyte-macrophage colony stimulating factor, and methods of generating the hybridoma lines are provided. Target antigens and epitopes are also provided. The antibodies may be used in therapeutic methods, for example in the treatment of cancer, infectious disease, or autoimmune disease.