Project: Characterization of new tumour markers and development of a new diagnostic tool for cancer based on circulating microvesicles
According to WHO, one third of the cancer burden could be cured if detected early and treated adequately. Diagnostic ambiguity of early lesions has significant adverse consequences for patients. Misclassifying a tumour as benign may be fatal, and diagnosing a benign lesion as malignant may result in significant morbidity. Currently there is no method available to definitively resolve these ambiguities._x000D_Thus the general objectives of the current EUROSTARS project are to set up and standardise new tools and methods, based on known and novel tumour markers (such as new oncoproteins and oncogenes), for diagnosing and monitoring cancer patients. The core activity to be performed in the project will be the development and characterization of new antibodies against tumour associated antigens, starting from a novel oncoprotein (TUCAP-1/TM9SF4) discovered by our research team and patented by Hansabiomed. TUCAP-1 belongs to transmembrane 9 superfamily of proteins, that are almost completely uncharacterised. A further activity to be performed in the project will be to test, develop and standardise an ELISA method for the screening, diagnosis and prognosis of human tumours. This method is based on another Hansabiomed patent consisting in a novel double sandwich ELISA test capable to provide a quantitative and qualitative analysis of tumour derived exosomes/microvescicles (MV), expressing specific tumoral markers (e.g. TUCAP-1, Cav-1, CD63, CD133, etc.), collected from plasma of patients with malignant tumours such as melanoma, leukaemia, osteosarcomas, breast, lung, and gastro-intestinal tract cancers. _x000D_In synthesis, the objective of the project is to achieve the following specific measurable goals:_x000D_- To produce, test and develop a panel of polyclonal and monoclonal antibodies against TUCAP-1;_x000D_- To screen, evaluate and develop the knowledge on TM9 superfamily proteins as potential new markers in oncology;_x000D_- To develop and standardize a double sandwich ELISA assay based on detection and characterization of tumors derived MV carrying tumor specific markers, for diagnosis, staging and prognosis of human tumors. _x000D__x000D_In detail, TUCAP-1, product of tm9sf4 gene, is a new tumour associated protein, highly expressed in metastatic lesions. This protein belongs to the TransMembrane 9 SuperFamily (TM9SF1, TM9SF2, TM9SF3 and TUCAP-1/TM9SF4) and is characterized by the presence of a large variable extracellular or lumenal N-terminal doCO followed by nine putative transmembrane doCOs in its conserved C terminal. Our preliminary experimental data show that TUCAP-1 is a key factor involved in tumour cell cannibalism. This phenomenon, characterized by the ability of tumour cannibal cells to phagocytose apoptotic cells, plastic beads, stained yeasts as well as live lymphocytes, has been observed in tumours of different histology, and always related to a poor prognosis. On the basis of these data we called this protein Tumour cannibalism associated protein (TUCAP-1). Moreover this protein seems to be involved in pH regulation of phago/endosomal vesicles, since TUCAP-1 is or could behave as an ion channel. These findings have a variety of implications, including that cannibalism is one of the CO mechanisms of tumour escape. Currently no commercial antibodies are available for detection of TUCAP-1 and TM9SF proteins._x000D_Exosomes are membrane microvesicles (MV) ranging from 50 to 100 nm in diameter. These MV of endosomal origin are secreted by cells under both physiological and pathological conditions, even though they are different in quantity and composition. Hansabiomed is currently developing a cheap test for detection and characterization of plasma derived exosomes. The current proposal will aim to standardize and ELISA based assay specifically designed for exosome analysis in cancer (for preclinical and clinical use). _x000D_The market applications for the planned developments are the following:_x000D_• Commercialization of a wide panel of antibodies (poly and monoclonal) against tumour specific antigens (currently not available);_x000D_• Development and commercialization of cheap and specific tools for preclinical and clinical research in human cancers;_x000D_• Pave the road for the development of a cheap and specific ELISA test, based on the analysis of plasma derived exosomes, for cancer screening, diagnosis, staging and prognosis of cancer patients._x000D_Based on the development achieved through the CIRTUMAN project, TUCAP-1 patent (further possible TM9SF related patents) together with the patent on the ELISA test for exosome detection in cancer applications will be proposed in out-license to big pharma and biotech Companies, who will be able to develop and market diagnostic solutions for clinical use._x000D_The consortium is composed of two Ps: an innovative biomedical research company Hansabiomed OÜ (HBM) in Estonia and a leading Italian company in the area of biotechnology services PRIMM srl.
| Acronym | CIRTUMAN (Reference Number: 4758) |
| Duration | 01/08/2009 - 31/01/2014 |
| Project Topic | Development of a new panel of antibodies against new specific tumour markers belonging to TM9SF proteins for pre-clinical and clinical use in oncology and development of a novel diagnostic and prognostic double sandwich ELISA test for human cancers based on circulating microvesicles (exosomes) |
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Project Results (after finalisation) |
The large majority of CIRTUMAN’s expected results have been successfully achieved, with some additional unexpected achievement. CO objectives were: _x000D_i) screening, development, applicability of a panel of mAbs and pAbs against TUCAP-1/TM9SF4 and other TM9SF proteins; _x000D_ii) development of an ELISA-like assay for detection, characterization and quantification of tumor derived microvesicles from biological samples (cell culture supernatants and patients’ fluids);_x000D_iii) preliminary evaluation of developed tools (Abs) and assays in cancer diagnostics and setting up prototype products for research uses. _x000D__x000D_WP1/WP2 - We obtained both good quality poly and monoclonal Abs, despite difficulties encountered in producing mAbs targeting conformational epitopes, which are known to be difficult to develop. In particular we obtained: _x000D_• A pAb against TM9SF4 for WB and suitable for ExoTEST and 3 different clones of mouse anti-TM9SF4 mAbs specifically targeting the protein in cell and exosomal lysates (patented);_x000D_• IHC evidence for a role of TUCAP1/TM9SF4 in tumor cannibalism and intercellular communication between malignant melanoma and keratinocytes, most likely via exosomes;_x000D_• IHC evidence that TUCAP1/TM9SF4 is a highly specific and sensitive marker of malignancy in gastric and colorectal cancer tissues._x000D_• pAbs against TM9SF3 for WB and suitable for ExoTEST applications and a good mAb against TM9SF2, detecting the protein on exosomes and exploitable on ELISA-like assays;_x000D_• IHC analysis of TM9SF3 expression in gastric and colon cancer suggesting TM9SF3 as a marker of malignancy._x000D_We also obtained information on molecular pathways involving TUCAP-1 in cancer. Results led to a TUCAP-1 mAbs patent and to a paper on the function and role of TUCAP-1 in tumors, now submitted for publication._x000D_WP2 did not suggest, apart from TM9SF3, an appealing role of other TM9SF proteins as potential cancer biomarkers. _x000D__x000D_WP3/WP4 - We produced all elements required in an ELISA assay for tumor exosome immune-capture, quantification and characterization and several prototypes of commercial assays. In particular:_x000D_• Exosome standard preparations (from HDs and tumor primary cell lines) as calibrators for ELISA assays;_x000D_• A pAb against RAB5 suitable for ExoTEST applications; 3 clones of anti CD81 mAbs; 3 clones of anti Caveolin-1 mAbs;_x000D_• Identification of a significant number of exosome associated tumor/tissue biomarkers;_x000D_• Identification of several tumor/tissue related proteins that when co-expressed on exosomes provide a potent diagnostic potentiality to the assays (full details on the large number of biomarkers tested are reported in the periodic progress reports to EAS)._x000D_• A clone bank for most interesting mAbs stored in liquid nitrogen at PRIMM;_x000D_• Initial small batches of poly and mono clonal Abs;_x000D_• Several prototypes of commercial ExoTEST kits for research uses;_x000D_• Packaging prototype, data sheets and handbooks for each prototype kit. _x000D_We also developed exosome based assays for prostate cancer and for colorectal cancer having improved sensitivity and specificity over currently available ones, applicable for screening/diagnosis/monitoring of oncologic patients. An assay for exosome based diagnostics of prostate cancer has been patented (colorectal assay patent on the way) and a paper on the same topic submitted to a peer reviewed journal. Products developed have been included since Jan 2014 in the HansaBioMed catalogue, which is the largest and more advanced catalogue of reagents and tools for exosome research today on the market (full list of commercialized products on company’s website)._x000D__x000D_WP5 - We effectively managed the project, timely provided detailed reports, set up adequate IP protection strategies (resulting in several new patent applications filed), effectively communicated project results at major international scientific events and biotech forums, resulting in high company global visibility and initiating strategic collaborations, one with Roche Diagnostics. |
| Network | Eurostars |
| Call | Eurostars Cut-Off 2 |
Project partner
| Number | Name | Role | Country |
|---|---|---|---|
| 2 | Hansabiomed OÜ | Coordinator | Estonia |
| 2 | PRIMM srl | Partner | Italy |