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Cytotoxic CD8+ T cells are considered as one of the main populations of effector immune cells in antitumor immunity. The absence of CD8+ T cells in the central tumor area has become a major obstacle for solid tumor immunotherapy. Thus, novel therapeutic strategies that could promote CD8+ T cells to accumulate in the central tumor area are urgently needed. To this aim, we want to propose a novel candidate INGM01 a highly glycosylated mucin-like protein localized on the cell surface and so far poorly characterized. High INGM01 expression is reported to be associated to a higher survival rate in some cancers (PreCOG database). We found that INGM01 is physiologically expressed on the surface of different cancer human cell lines and its over-expression by cDNA transfection significantly increased motility and migration phenotypes, such as improved scratch recovery in the wound healing assay, altered cytoskeleton organization with loss of actin branches, reduced E-cadherin expression and activated Fak pathway through phosphorylation of its Y925. We also found that INGM01 is specifically over-expressed in tumor-infiltrating CD8+ and CD4+ lymphocytes, as judged by IHC and IF analysis of cryopreserved tissues and by FACS analysis of T cells isolated from different cancers (e.g. colon, kidney), while it is marginally expressed in T-cells resident in adjacent normal tissues. Microscopy analysis showed that INGM01 localizes in anchoring sites of CD8+ T-cells attacking the cancer cells forming a cluster of lymphocytes on their surface. INGM01 expression in T lymphocyte is significantly induced by CD3/CD28 receptor activation and by the microenvironmental milieu conditioned by cancer cells, through the production of soluble factors. Indeed, INGM01 is localized in the uropod of both CD3/28 activated and ex vivo isolated tumor infiltrating CD4+ and CD8+ T cells confirming a role of INGM01 in motility of the T-cells. We found INGM01 co-expressed with other uropod-associated proteins, such as ICAM-I, LFA-1 and CXCR-3, involved in the acquisition of the polarity needed for T-cell chemotaxis and for migration. By Boyden chamber, with and without a HUVEC cell monolayer, we found that INGM01 positive CD8+ and Jurkat cells migrate towards wells containing conditioned medium of cancer cells and this process is significantly impaired by INGM01 silencing indicating its role in chemotaxis and trans-endothelial migration. Furthermore, INGM01 contributes to the cytotoxic function of CD8+ T-cells, since its silencing causes a reduction of expression of Th1 effector cytokines, such as IFN-γ, TNFα. Our hypothesis is that, after a deeper analysis of the interaction/s and ligand/s involving INGM01, it might be possible to generate affinity agents or bi-spefic antibodies able to enhance the intratumoral infiltration of cytotoxic CD8+ T-cells expressing INGM01 and their migration towards cancer cells, to promote their killing. Citation Format: Elisa Pesce, Chiara Cordiglieri, Cristina Manara, Stefania Oliveto, Susanna Campagnoli, Tiziano Donnarumma, Manuele Martinelli, Mariacristina Crosti, Elisa De Camilli, Stefano Biffo, Sergio Abrignani, Mauro Bombaci, Renata Maria Grifantini, Andrea Gobbini. A novel candidate for immunotherapy mediating the balance between the immune system and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2829.

The identification of markers targetable by specific mAbs represents a high medical need in cancer therapy. Our objective is to discover novel tumor-associated proteins showing promise as targets for monoclonal antibody (mAb) therapy, and to generate and validate highly specific mAbs for therapeutic applications. The approach we used to discover novel tumor markers is based on a high through-put immune-histochemical (IHC) screening of tumor and normal tissues using collections of murine polyclonal and mAbs raised against recombinant human proteins. In the course of such analysis, we discovered and validated different surface exposed proteins over-expressed in one or more cancers. Here, we report a surface exposed protein mainly over-expressed in ovary and breast cancers. Interestingly, the protein is highly expressed in high grade breast cancers. Approximately 40% of cancers in which the protein is over-expressed belong to the triple negative subtype. In line with IHC data, an expression profile analysis in different cells lines showed that this protein is expressed in ovarian and breast cancer cell lines. In breast cell lines, high expression was found in triple negative cells positive to the androgen receptor. Gene silencing experiments combined to phenotypic analysis, showed that loss of protein expression significantly reduces cell proliferation and invasiveness. Several mAbs able to recognize the target protein on the surface of breast and ovary cancer cell lines have been selected and validated in a number of immunoassays. The specificity of the mAb binding was confirmed by gene silencing and competition assays with peptides encompassing the mAb epitopes. Specific mAbs able to detect the protein in cancer cells in IHC are under validation. These antibodies show limited reactivity on normal human tissues and are negative on PBMC from normal donors. Moreover, these mAbs are efficiently internalized by cancer cells, suggesting that they are amenable to the development of Antibody-Drug-Conjugate. Finally, they efficiently recognize the macaca protein ortholog, thus facilitating future safety studies in non-human primates. Overall, results so far accumulated highlight the potential of this novel tumor-associated protein and available mAbs for the development of targeted therapy against ovarian cancer and triple negative breast cancer. Other promising targets and related monoclonal antibodies will be described during the meeting. Citation Format: Matteo Parri, Susanna Campagnoli, Alberto Grandi, Elisa De Camilli, Aurelien Lacombe, Boquan Jin, Serenella Eppenberger-Castori, Giuseppe Viale, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3800.

Suppression of the host's immune system plays a major role in cancer progression. The tumor microenvironment (TME) not only plays a pivotal role during cancer progression and metastasis but also has profound effects on therapeutic efficacy. Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances and provided a new weapon against cancer. This therapy has elicited durable clinical responses and, in a fraction of patients, long-term remissions. Several immune checkpoint inhibitors have been approved by the Food and Drug Administration (FDA), whose therapeutic effect is due to blocking the activity of proteins expressed in T lymphocytes (CTLA4, PD-1) and the interaction with surface protein expressed on tumor cells (PD-L1 and PD-L2). In our recent research activities we have identified 89 tumor-associated proteins by systematic immunohistochemistry analysis of tissue microarray (TMA) representing breast, colon, lung ovary and prostate cancer by using a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted proteins only marginally characterized. Among them, an interesting protein, which decorated the plasma membrane of breast cancer cells, was selected. This protein has been described to interact with the CD270/BTLA/CD160 pathway that regulates T-cell activation. It has also the capability to interfere with T-cell mediated allo-responses. Our characterization data showed that the protein is localized on the surface of several cancer cell lines. Moreover, it has the ability to stimulate cytokine production and secretion in PBMC from healthy donors. Several monoclonal antibodies have been generated in our lab against this protein and selected for the ability to bind the surface of cancer cells. Moreover, the ability of these monoclonal antibodies to interfere with the immune cells activity and to interfere with tumor growth in animal models is under evaluation. Results will be provided during the meeting. The currently approved therapeutic targets regimens for triple negative breast cancer and ovarian cancer have limited efficacy. Therefore, the identification of other tumor-associated proteins targetable by specific mAbs is essential to improve patient survival. Citation Format: Susanna Campagnoli, Matteo Parri, Alberto Grandi, Elisa De Camilli, Giuseppe Viale, Renata Maria Grifantini, Piero Pileri. A novel potential target for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4870.

The study focuses on two novel potential therapeutic targets identified by an immune-histochemistry (IHC) screening with a large collection of polyclonal antibodies (approximately 1600) raised against marginally characterized human proteins. Here we describe the molecular characterization of two surface-exposed proteins (EXN91 and EXN36) associated to different cancer types. EXN91 is an adhesion molecule and it acts as a signaling receptor, cell communication and motility. Prevalence studies showed that the protein is mainly detected in colon cancer (CRC)with high frequency (approximately 90%), both in early and advanced colon cancer stages, with a statistical association for early cancer stages. Moreover, the protein is clearly surface exposed in liver metastasis from colon. Experiments are ongoing to assess whether EXN91 is also expressed in precancerous conditions of colon and rectum that have the potential to develop into cancer. Interestingly, EXN91 is over-expressed in KRAS and BRAF mutant CRC with high frequency. Moreover, an expanded IHC analysis revealed that it is also over-expressed and surface exposed in a number of other cancers, including HCC, RCC, bladder and endometrium cancer. Preliminary data showed that EXN91 behaves as an auto-antigen being able to induce specific autoantibodies in CRC patients. EXN36 is mainly over-expressed in ovary and breast cancers (frequency of approximately 30-40%), including triple negative breast cancer. EXN36 prevalence studies in different cancer entities are ongoing. Moreover, EXN36 is involved in cell proliferation, migration and invasiveness. Murine monoclonal antibodies (mAb) able to recognize EXN91 and EXN36 on the surface of cancer cells show potential for specific therapeutic indications. In particular, a murine mAb recognizes EXN91 on the surface of colon cancer cells, including KRAS and BRAF mutant CRC. This antibody inhibits growth of colon cancer in xenograft mouse models. Concerning anti-EXN36 antibodies, they are able to bind the surface of different breast and ovary cancer cells. EXN36 and EXN91 mAbs show a high number of binding sites on the cell surface. The mAb specificity has been confirmed in different immunoassays by gene silencing and peptide competition assays. Moreover, they show limited IHC reactivity in normal tissues. The anti-EXN36 and antiEXN91 antibodies are efficiently internalized by cancer cells, suggesting that they can be exploited for the development of Antibody-Drug-Conjugate (ADC). Initial results from in vitro studies show that these antibodies, when tested in drug screening assays with Protein G conjugated to commercially available drugs show significant anti-tumor activity with a specific conjugation chemistry. Overall, the results indicate that EXN36, EXN91 and their mAbs could be developed for the targeted therapy of different cancer indications at high medical need. Citation Format: Matteo Parri, Susanna Campagnoli, Alberto Grandi, Elisa De Camilli, Paolo Sarmientos, Boquan Jin, Guido Grandi, Giuseppe Viale, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for antibody-drug conjugate therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2015-3575

We identified 89 novel candidate markers for prevalent cancers by a systematic Tissue microarray analysis (TMA) of a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Among them, here we report a novel cancer-associated protein, referred as EXN32, so far only marginally characterized. EXN32 is a metallo-protease highly conserved across species, known to be involved in ovarian folliculogenesis. An anti-EXN32 monoclonal antibody (mAb) detects the protein in a high percentage of in breast, lung, ovary, and colo-rectal (CRC) cancers (high-to-medium intensity ranging from 30 to 70% in different cancer types. An analysis of high density tissue microarrays (300-700 highly characterized clinical specimens per each cancer) shows that EXN32 is over-expressed in all cancer stages and grades. In breast cancer, a EXN32 it is over-expressed in Her-2+, PR+ and ER+ patients and also in triple negative cases. In colon cancer, EXN32 is over-expressed both in B-RAF and K-RAS mutant and wild type cancers. We also found that EXN32 over-expression is statistically associated to a low activation of cadherin and beta-catenin pathways. Moreover, its expression is also associated with other prognostic and predictive markers. This evidence opens the way to further investigations on the EXN32 role in the marker-associated pathways. A characterization of the protein biological role showed that alteration of EXN32 expression strongly hampers cell proliferation, migration and invasiveness. The molecular pathways in which the protein is involved show that it plays a role in the Unfolded Protein Response and defence against oxidative stress. Our findings reveal that EXN32 is strongly affected by reticular stress induced by thapsigargin. In addition, EXN32 silencing during reticular stress protect cells from expressing the stress marker Bip/Grp78. In addition, EXN32 is involved also in handling of oxidative stress. Indeed, EXN32 silencing in cells exposed to 1% O2 hypoxia, causing a mitochondrial delivery of oxidants and hence sustaining oxidative stress, leads to inhibition of the Nrf2-mediated anti-oxidant response and to reduction of accumulation of HIF-1, the master transcription factor instructing cells to respond to hypoxic stress. We are investigating the possibility that EXN32 may participate as a molecular starter to the survival response induced by extracellular stresses. Results indicate EXN32 as a potential target for the design of novel drugs, such as aptamers, small molecules and protease inhibitors. For instance, molecules such as 2-PMPA and analogues are expected to inhibit EXN32 activity. Moreover, EXN32 and its specific mAb represent interesting tools for the molecular characterization of specific cancer subtypes. Investigations are ongoing to understand the prognostic and predictive value of EXN32. Citation Format: Matteo Parri, Alberto Grandi, Susanna Campagnoli, Elisa De Camilli, Alice Santi, Boquan Jin, Paolo Sarmientos, Guido Grandi, Giuseppe Viale, Paola Chiarugi, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. A novel potential therapeutic target for breast, lung, ovary and colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4384A. doi:10.1158/1538-7445.AM2015-4384A

In our recent research activities, we identified 89 novel candidate markers for prevalent cancers by a systematic Tissue microarray analysis (TMA) of a large collection of polyclonal antibodies (pAbs, approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Monoclonal antibodies were generated towards 20 distinct antigens that are being characterized and validated for diagnostic and therapeutic applications. Among them, here we describe the molecular characterization of a novel cancer -associated protein (referred as EXN32) belonging to the metallo-protease family and so far only marginally characterized. Immuno-histochemical analysis of Tissue Microarrays carrying clinical specimens from prevalent cancers and matched normal samples with the anti-EXN32 pAb showed that the protein is over-expressed in breast, lung, ovary,and colon cancers, with frequencies ranging from 30 to 70%. A characterization of the protein biological role showed that alteration of EXN32 expression significantly hampers cell proliferation, migration and invasiveness. Initial studies aimed at identifying the molecular pathways in which the protein is involved show that it plays a role in the response to the reticular stress and hypoxia. A highly specific murine monoclonal antibody (mAb) specifically recognizes the protein in the four cancer types. This mAb is currently used to discover statistical associations between EXN32 with specific clinic-pathological parameters using high density tissue microarrays carrying highly characterized clinical specimens (300-700 per each cancer). Available data on breast and colon show that EXN32 is over-expressed in all cancer stages and grades. In breast cancer, a EXN32 bio-distribution analysis versus clinically relevant surface receptors shows that the protein is over-expressed in Her-2+, PR+ and ER+ patients and also in triple negative cases. In colon cancer, EXN32 over-expression shows statistical correlation with a panel of key molecular markers such as beta-catenin, e-cadherin, TGFbeta and others, and open the way to further investigations on the EXN32 role in their associated pathways. Overall, results indicate EXN32 as a potential target for the design of novel drugs, such as aptamers, si-RNA and small molecules. The EXN32 specific mAb represents an interesting tool for the molecular characterization of specific cancer subtypes. Investigations are ongoing to understand the predictive value of EXN32 for state-of art and innovative therapeutic strategies. Citation Format: Matteo Parri, Susanna Campagnoli, Alberto Grandi, Elisa De Camilli, Valentina Farini, Serenella Eppenberger, Paola Chiarugi, Paolo Sarmientos, Boquan Jin, Guido Grandi, Giuseppe Viale, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. A novel potential cancer marker and therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1769. doi:10.1158/1538-7445.AM2014-1769

In our recent research activities, we identified a panel of novel candidate markers for prevalent cancers by a systematic immune-histochemical screening with a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Of utmost interest were those proteins over-expressed detected in one or more tumors showing surface exposure on cancer cells. Among them, here we describe the molecular characterization of a novel surface-associated protein (EXN36) belonging to the lectin-binding protein family for which very little is presently known. We found that the protein is mainly over-expressed in breast and ovary cancers (frequency of approximately 30-40% of tested cases, based on analysis of 50 patients) and it is involved in cell proliferation, migration and invasiveness, as demonstrated by gene silencing. With the intent of exploiting EXN36 as therapeutic target for monoclonal antibody (mAb) therapy, highly specific murine mAbs were generated and proved to recognize the protein on the surface of selected breast and ovary cell lines. Of particular interest are two mAbs, which, upon binding, are efficiently internalized by breast and ovary cancer cells. These two mAbs influence the viability of breast and ovary cells in vitro. One of them shows antibody-dependent cell-mediated cytotoxicity in vitro in breast cancer cells. An analysis of their biodistribution in normal human tissues showed that these mAbs have limited cytoplasmic reactivity in some tissues. Overall, the functional properties of the anti-EXN36 mAbs make them amenable for the generation of antibody-drug conjugates (ADCs). Moreover, they could be exploited for immune-nanoparticles targeted delivery systems. In vivo efficacy in breast and ovary xenograft cancer models are ongoing to evaluate their therapeutic activity either as naked antibodies or ADCs. Overall, data indicate that EXN36 and the specific mAbs represent innovative and promising tools for a targeted mAb therapy of breast and colon cancer, either alone or in combinatorial therapeutic strategies. Citation Format: Alberto Grandi, Susanna Campagnoli, Matteo Parri, Elisa De Camilli, Boquan Jin, Paolo Sarmientos, Guido Grandi, Luigi Terracciano, Giuseppe Viale, Piero Pileri, Renata Maria Grifantini. Discovery of a novel target for monoclonal antibody therapy of breast and ovary cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2014-4622

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

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

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.