Andrea Pierleoni

Open Targets: a platform for therapeutic target identification and validation

We have designed and developed a data integration and visualization platform that provides evidence about the association of known and potential drug targets with diseases. The platform is designed to support identification and prioritization of biological targets for follow-up. Each drug target is linked to a disease using integrated genome-wide data from a broad range of data sources. The platform provides either a target-centric workflow to identify diseases that may be associated with a specific target, or a disease-centric workflow to identify targets that may be associated with a specific disease. Users can easily transition between these target- and disease-centric workflows. The Open Targets Validation Platform is accessible at https://www.targetvalidation.org.

Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

Background Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform.

A novel polyclonal antibody library for expression profiling of poorly characterized, membrane and secreted human proteins ☆

The YOMICS™ antibody library (http://www.yomics.com/) presented in this article is a new collection of 1559 murine polyclonal antibodies specific for 1287 distinct human proteins. This antibody library is designed to target marginally characterized membrane-associated and secreted proteins. It was generated against human proteins annotated as transmembrane or secreted in GenBank, EnsEMBL, Vega and Uniprot databases, described in no or very few dedicated PubMed-linked publications. The selected proteins/protein regions were expressed in E. coli, purified and used to raise antibodies in the mouse. The capability of YOMICS™ antibodies to specifically recognize their target proteins either as recombinant form or as expressed in cells and tissues was confirmed through several experimental approaches, including Western blot, confocal microscopy and immunohistochemistry (IHC). Moreover, to show the applicability of the library for biomarker investigation by IHC, five antibodies against proteins either known to be expressed in some cancers or homologous to tumor-associated proteins were tested on tissue microarrays carrying tumor and normal tissues from breast, colon, lung, ovary and prostate. A consistent differential expression in cancer was observed. Our results indicate that the YOMICS™ antibody library is a tool for systematic protein expression profile analysis that nicely complements the already available commercial antibody collections.

TCTN2: a novel tumor marker with oncogenic properties

Tectonic family member 2 (TCTN2) encodes a transmembrane protein that belongs to the tectonic family, which is involved in ciliary functions. Previous studies have demonstrated the role of tectonics in regulating a variety of signaling pathways at the transition zone of cilia. However, the role of tectonics in cancer is still unclear. Here we identify that TCTN2 is overexpressed in colorectal, lung and ovary cancers. We show that different cancer cell lines express the protein that localizes at the plasma membrane, facing the intracellular milieu. TCTN2 over-expression in cancer cells resulted in an increased ability to form colonies in an anchorage independent way. On the other hand, downregulation of TCTN2 using targeted epigenetic editing in cancer cells significantly reduced colony formation, cell invasiveness, increased apoptosis and impaired assembly of primary cilia. Taken together, our results indicate that TCTN2 acts as an oncogene, making it an interesting cancer-associated protein and a potential candidate for therapeutic applications.

Abstract C96: A novel polyclonal antibody library for expression profiling of poorly characterized membrane and secreted human proteins.

The YOMICS™ antibody library (http://www.yomics.com/) presented in this article is a new collection of 1559 murine polyclonal antibodies specific for 1287 distinct human proteins. This antibody library is designed to target marginally characterized membrane-associated and secreted proteins. It was generated against human proteins annotated as transmembrane or secreted in GenBank, EnsEMBL, Vega and Uniprot genome databases, described in no or very few dedicated PubMed-linked publications. The selected proteins/protein regions were expressed in E. coli, purified and used to raise antibodies in the mouse. The capability of YOMICS™ antibodies to specifically recognize their target proteins either as recombinant form or as expressed in cells and tissues was confirmed through several experimental approaches, including Western blot, confocal microscopy and immunohistochemistry (IHC). Moreover, to show the applicability of the library for biomarker investigation by IHC, five antibodies against proteins either known to be expressed in some cancers or homologous to tumor-associated proteins were tested on tissue microarrays carrying tumor and normal tissues from breast, colon, lung, ovary and prostate. A consistent differential expression in cancer was observed. Our results indicate that the YOMICS™ antibody library is a tool for systematic protein expression profile analysis that nicely complements the already available commercial antibody collections. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C96.

Abstract B180: Novel candidate tumor markers identified by a high-throughput “immuno-reverse-proteomics” approach.

Cancer biomarker discovery is an extremely active research field in both academia and pharmaceutical companies. Here we present a group of candidate tumor markers identified by a high-throughput immunohistochemistry approach based on the use of a library of 1600 mouse antibodies raised against marginally-characterized human proteins. Tissue microarray (TMA) representing breast, lung colon ovary and prostate cancers were used to identify novel tumor markers. Eighty-nine proteins were found over-expressed in one or more of the five tumors under analysis. The validation process is still in progress and so far has been undertaken on twenty-six of the eighty-nine hits. They are confirmed over-expressed in TMAs carrying 50 tumor samples for each tumor type and, in particular, nineteen proteins were found expressed in one or more tumors with frequencies ranging from 20% to 96%. Interestingly, some of the proteins are simultaneously over-expressed in specific patients9 groups, thus opening the possibility of tissue diagnostic/prognostic/predictive applications based on combinatorial markers. Moreover, in vitro characterization studies indicated that some markers play a pivotal role in tumor-related cell processes like cell proliferation (EXN32; EXN36; EXN7), invasiveness (EXN32; EXN36; EXN7; EXN4), clonal growth (EXN1) and pro-angiogenesis (EXN11). A panel of monoclonal antibodies highly specific the biomarkers is already available and proved to specifically detect the markers in tumor cells derived from surgical resections. The newly identified candidate markers are new tools for the development of specific drug therapies and diagnostic products. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B180.

Abstract 5579: Novel proteins highly expressed in tumor identified by a high throughput immunoproteomic approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cancer biomarker discovery is an extremely active research track in both academia and pharma industries. Several high throughput technologies were applied through the years to identify proteins specifically related to cancerous phenotype. However, most putative biomarkers identified from conventional proteomic/transcriptomic strategies still need to be clinically validated. Here we present a cancer biomarker discovery approach based on the use of a large library of antibodies raised against recombinant human proteins to detect tumor-associated proteins by immune-histochemistry analysis of clinical tumor tissues. Starting from the whole human genome, genes encoding proteins predicted as membrane- or secreted were selected and high through-put cloned and expressed. Recombinant proteins were used to build a polyclonal antibody library (YOMICS®) currently comprising more than 1700 murine immune sera. The ability of sera to recognize specific targets predominantly present in tumors was assessed by Tissue MicroArray (TMA, a miniaturized immunohistochemistry analysis) of tumor tissue samples and healthy controls from pedigreed patients affected by the most common human tumor types, including colon, ovary, breast, lung, ovary and prostate cancers. While the screening is still in progress, six antibodies were identified showing high reactivity on a high percentage (ranging from 40 to 95%) of tumor tissues on one or more cancer type, with negative staining on normal tissues. The corresponding protein targets, being novel tumor-associated proteins, were validated and characterized at cellular and molecular level by analyzing their expression, cellular localization and biological role in a panel of tumor cell lines. Interestingly, two of the six proteins are localized at the plasma membrane, while the other proteins are intracellular. Data so far available indicate that one of the six proteins confers an invasive phenotype to tumor cell lines. The newly identified proteins are promising candidates as new biomarkers and could be exploited to develop target-specific drug therapies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5579.