Renzo Nogarotto

Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

Characterization of the surfactin synthetase multi-enzyme complex

Three subunits (srfAORF1, srfAORF2 and srfAORF3) of the Bacillus subtilis surfactin synthetase multi-enzyme complex have been identified by SDS-PAGE and Western blot analyses. In accordance with the sequence analysis of the surfactin (srfA) operon, the protein subunits have a molecular mass of 402,000 Da, 401,000 Da and 144,000 Da, respectively. Confirmation of the identity of the proteins was obtained by analysing the total protein content of a number of mutant strains which harbour deletions or insertions either in the srfA promoter or in different positions within the srfA operon. The three subunits were partially purified by means of a series of chromatographic steps including ion-exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The partially purified proteins were used in activity assays to establish their amino-acid recognition specificity. In agreement with previously published results, this analysis showed that srfAORF1 recognizes glutamic acid and Leu, srfAORF2 recognizes Val, aspartic acid and Leu and srfAORF3 recognizes Leu. In addition, the subunits can activate and bind other amino acids, although with lower specificity. In particular, srfAORF1 binds Val, Ile and aspartic acid, srfAORF2 glutamic acid and Ile and srfAORF3 Ile and Val. Competition experiments as well as sequence comparison strongly suggest that the Leu binding sites of the three subunits can accept, beside Leu, Ile and Val. The kinetic parameters of srfAORF3 for Leu, Ile and Val have been determined.

Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis.

The most popular approach for proteomics analysis is based on the combination of two‐dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two‐dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture.

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.

Abstract 2857: Monoclonal antibodies against novel tumor markers for cancer diagnosis and treatment .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In our recent research activities, we identified a group of novel candidate tumor markers for prevalent cancers. Tissue microarray (TMA) representing breast, lung colon ovary and prostate cancers were analyzed by a high-throughput immunohistochemistry screening using of a library of 1600 mouse antibodies raised against membrane-associated and secreted human proteins for which little is known in the scientific literature. 89 proteins were found over-expressed in one or more of the five tumors under analysis. IHC analysis on TMA representing tissues from 50 patients per each of the five cancers allowed to select a group of 20 potential markers over-expressed in cancer with frequencies ranging from 20 to 96%, with concomitant marginal expression in normal tissues. With the aim at validating these proteins as novel markers for patient stratification, prognosis and response to therapy, highly specific monoclonal antibodies (mAbs) have been generated against them. Currently, mAbs against 4 markers able to selectively detect their target proteins in cancers by IHC have been already identified (positivity ranging from 15 up to 94%, and showing membranous or intracellular staining). The target proteins of these mAbs include:1) a scavenger receptor protein; 2) a putative metallo-protease, 3) a lectin-binding protein involved in the innate immune response, 4) a cadherin- homologous protein. The mAb clinical value is being examines using high-density TMA representing patients with known history and follow-up for breast (1553 patients), colon (1420 patients), lung (1527 patients), ovary (112 patients) and prostate (553 patients) cancers have been generated. The patient cohorts have been collected at the biobank of the institute for Pathology of Basel to select pools of prognostic/predictive monoclonal antibodies capable of discriminating i) patients with favorable or adverse prognosis, and ii) patients that respond/do not respond to specific therapeutic interventions. Available data indicated that these antibodies, alone and in combinations, show significant association with specific clinical features (formation of metastasis, progression-free survival, survival time). Interesting, some of available antibodies show therapeutic activity in preclinical cancer models. These antibodies are able to bind the surface of cancer cells in vitro and in vivo. Avalable data show that one mAb targeting the cadherin-like protein inhibits tumor growth in athymic nude mice bearing HCT15 and HT29 colon cancer xenograft models. Overall, these novel markers and their specific monoclonal antibodies could offer new opportunities for cancer diagnosis and treatment. Citation Format: Renata Maria Grifantini, Piero Pileri, Matteo Parri, Alberto Grandi, Susanna Campagnoli, Renzo Nogarotto, Elena De Camilli, Serenella Eppenberger, Luigi Terracciano, Giuseppe Viale, Guido Grandi, Paolo Sarmientos. Monoclonal antibodies against novel tumor markers for cancer diagnosis and treatment . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2857. doi:10.1158/1538-7445.AM2013-2857

Abstract 5533: Novel targets and monoclonal antibodies for cancer therapy.

Cancer therapeutic targets is an extremely active research field in both academia and pharmaceutical companies. In our recent research activities we identified by a high-throughput immunohistochemistry screening of Tissue microarray (TMA), a panel of 89 novel candidate tumor markers for prevalent cancers representing breast, lung colon ovary and prostate carcinomas. The novel marker candidates were found over-expressed in one or more of the five tumors under analysis, with significant frequency. Three of them seems to be promising therapeutic targets for monoclonal antibody therapy, being exposed on the surface of cancer cells. They include: 1) a lectin binding protein, over-expressed in breast, lung and ovary cancer; 2) a protein involved in iron homoeostasis and over-expressed in breast, colon, lung and ovary cancers, 3) a cadherin homologous protein detected in colon, lung and ovary cancers. Gene silencing using siRNA technology and/or over-expression experiments using marker-encoding plasmids significantly alter cell proliferation, migration, invasiveness and clonal growth in vitro, indicating that expression of the three proteins confers cell phenotypes relevant for tumor progression. Highly specific murine monoclonal antibodies (mAbs) were generated against the three proteins and proved to bind the surface of cancer cells lines. Of particular interest is a murine mAb targeting the cadherin-like protein that, upon binding to the cell surface, is efficiently internalized by cancer cells, suggesting that it is amenable to the development of antibody-drug conjugates. This mAb did not show any relevant IHC cross-reactivity in the 35 human tissues requested by FDA to demonstrate antibody specificity. Furthermore this mAb significantly inhibited tumor growth in athymic nude mice bearing HCT15 and HT29 colon cancer xenograft models. Finally, IHC analysis of approximately 300 colon cancer clinical samples indicates that the antibody stains a large fraction of colon cancer samples, and gives intense membranous staining in specific patients’ group. Overall, data indicate that this antibody could be developed as a novel tool for a targeted therapy of colo-rectal cancer, alone or in combination with other treatments Citation Format: Alberto Grandi, Matteo Parri, Susanna Campagnoli, Renzo Nogarotto, Elisa De Camilli, Ilaria Naldi, Caterina Cinti, Paolo Sarmientos, Guido Grandi, Luigi Terracciano, Giuseppe Viale, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2013-5533

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.

Protein Identification from 2-D Gels Using In Vitro Transcription Translation Products

The genomics revolution offers the opportunity to describe biological processes on the basis of global and quantitative gene expression patterns from cells or organisms representing different states. Although DNA microarrays are extremely powerful for measuring gene expression at the mRNA level (1), the nonpredictive correlation between mRNA and protein levels (2,3) and the discovery of post-translational mechanisms that either modify the structure and function of proteins or alter their half-life and rate of synthesis (4) indicate that direct measurement of protein expression is essential for a proper analysis of the biological processes.