Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines

Sviluppo di in anticorpo monoclonale anti-Asaia (anti-Asaia monoclonal antibody)

La presente invenzione riguarda lo sviluppo di un anticorpo monoclonale prodotto contro batteri del genere Asaia. Il succitato anticorpo è caratterizzato da elevata specificità ed è stato ottenuto mediante la fusione di cellule mielomatose murine con splenociti ottenuti da topi Balb/c immunizzati con il batterio di nostro interesse, ovvero Asaia, secondo la tecnica messa a punto da Kohler e Milstein nel 1974 (Nature 256: 495-497)

Tailoring DNA vaccines: designing strategies against HER2-positive cancers

The crucial role of HER2 in epithelial transformation and its selective overexpression on cancer tissues makes it an ideal target for cancer immunotherapies such as passive immunotherapy withTrastuzumab.There are, however, a number of concerns regarding the use of monoclonal antibodies which include resistance, repeated treatments, considerable costs, and side effects that make active immunotherapies against HER2 desirable alternative approaches.The efficacy of anti-HER2 DNA vaccination has been widely demonstrated in transgenic cancer-prone mice, which recapitulate several features of human breast cancers. Nonetheless, the rational design of a cancer vaccine able to trigger a long-lasting immunity, and thus prevent tumor recurrence in patients, would require the understanding of how tolerance and immunosuppression regulate antitumor immune responses and, at the same time, the identification of the most immunogenic portions of the target protein. We herein retrace the findings that led to our most promising DNA vaccines that, by encoding human/rat chimeric forms of HER2, are able to circumvent peripheral tolerance. Preclinical data obtained with these chimeric DNA vaccines have provided the rationale for their use in an ongoing Phase I clinical trial (EudraCT 2011-001104-34)

Expression of transient receptor potential vanilloid-1 (TRPV1) in urothelial cancers of human bladder: relation to clinico-pathological and molecular parameters.

AIMS: To evaluate the expression of transient receptor potential vanilloid type-1 channel protein (TRPV1) in normal and neoplastic urothelial tissues and to correlate TRPV1 expression with clinicopathological parameters and disease-specific survival. METHODS AND RESULTS: TRPV1 expression was analysed in normal and neoplastic urothelial samples at both mRNA and protein levels by quantitative real time polymerase chain reaction (qPCR) and immunohistochemistry, respectively. TRPV1 downregulation was found in urothelial cancer (UC) specimens, which correlated with tumour progression. Moreover, TRPV1 mRNA levels were associated with clinicopathological parameters to assess the role of TRPV1 downregulation as a negative prognostic factor for survival. Kaplan-Meier survival analysis demonstrated a significantly shorter survival in patients showing TRPV1 mRNA downregulation. Multivariate Cox regression analysis indicated further that TRPV1 mRNA expression retained its significance as an independent risk factor. CONCLUSIONS: The progression of UC of human bladder is associated with a marked decrease in TRPV1 expression, with a progressive loss in high-grade muscle invasive UC. Downregulation of TRPV1 mRNA expression may represent an independent negative prognostic factor for bladder cancer patients

Sanguinarine suppresses basal-like breast cancer growth through dihydrofolate reductase inhibition

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC

Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

<div><p>We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines.</p> </div