Ilaria Naldi

Newly Engineered Magnetic Erythrocytes for Sustained and Targeted Delivery of Anti-Cancer Therapeutic Compounds

Cytotoxic chemotherapy of cancer is limited by serious, sometimes life-threatening, side effects that arise from toxicities to sensitive normal cells because the therapies are not selective for malignant cells. So how can they be selectively improved? Alternative pharmaceutical formulations of anti-cancer agents have been investigated in order to improve conventional chemotherapy treatment. These formulations are associated with problems like severe toxic side effects on healthy organs, drug resistance and limited access of the drug to the tumor sites suggested the need to focus on site-specific controlled drug delivery systems. In response to these concerns, we have developed a new drug delivery system based on magnetic erythrocytes engineered with a viral spike fusion protein. This new erythrocyte-based drug delivery system has the potential for magnetic-controlled site-specific localization and highly efficient fusion capability with the targeted cells. Here we show that the erythro-magneto-HA virosomes drug delivery system is able to attach and fuse with the target cells and to efficiently release therapeutic compounds inside the cells. The efficacy of the anti-cancer drug employed is increased and the dose required is 10 time less than that needed with conventional therapy.

Novel epigenetic target therapy for prostate cancer: a preclinical study.

Epigenetic events are critical contributors to the pathogenesis of cancer, and targeting epigenetic mechanisms represents a novel strategy in anticancer therapy. Classic demethylating agents, such as 5-Aza-2′-deoxycytidine (Decitabine), hold the potential for reprograming somatic cancer cells demonstrating high therapeutic efficacy in haematological malignancies. On the other hand, epigenetic treatment of solid tumours often gives rise to undesired cytotoxic side effects. Appropriate delivery systems able to enrich Decitabine at the site of action and improve its bioavailability would reduce the incidence of toxicity on healthy tissues. In this work we provide preclinical evidences of a safe, versatile and efficient targeted epigenetic therapy to treat hormone sensitive (LNCap) and hormone refractory (DU145) prostate cancers. A novel Decitabine formulation, based on the use of engineered erythrocyte (Erythro-Magneto-Hemagglutinin Virosomes, EMHVs) drug delivery system (DDS) carrying this drug, has been refined. Inside the EMHVs, the drug was shielded from the environment and phosphorylated in its active form. The novel magnetic EMHV DDS, endowed with fusogenic protein, improved the stability of the carried drug and exhibited a high efficiency in confining its delivery at the site of action in vivo by applying an external static magnetic field. Here we show that Decitabine loaded into EMHVs induces a significant tumour mass reduction in prostate cancer xenograft models at a concentration, which is seven hundred times lower than the therapeutic dose, suggesting an improved pharmacokinetics/pharmacodynamics of drug. These results are relevant for and discussed in light of developing personalised autologous therapies and innovative clinical approach for the treatment of solid tumours.

Microwave electromagnetic field regulates gene expression in T-lymphoblastoid leukemia CCRF-CEM cell line exposed to 900 MHz

Electric, magnetic, and electromagnetic fields are ubiquitous in our society, and concerns have been expressed regarding possible adverse effects of these exposures. Research on Extremely Low-Frequency (ELF) magnetic fields has been performed for more than two decades, and the methodology and quality of studies have improved over time. Studies have consistently shown increased risk for childhood leukemia associated with ELF magnetic fields. There are still inadequate data for other outcomes. More recently, focus has shifted toward Radio Frequencies (RF) exposures from mobile telephony. There are no persuasive data suggesting a health risk, but this research field is still immature with regard to the quantity and quality of available data. This technology is constantly changing and there is a need for continued research on this issue. To investigate whether exposure to high-frequency electromagnetic fields (EMF) could induce adverse health effects, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of 900 MHz MW-EMF generated by a transverse electromagnetic (TEM) cell at short and long exposure times. We evaluated the effect of high-frequency EMF on gene expression and we identified functional pathways influenced by 900 MHz MW-EMF exposure.

FAT1: A potential target for monoclonal antibody therapy in colon cancer

Background:Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents have limited efficacy.Methods:The atypical cadherin FAT1 was discovered as a novel CRC-associated protein by using a monoclonal antibody (mAb198.3). FAT1 expression was assessed in CRC cells by immunohistochemistry (IHC), immunoblots, flow cytometry and confocal microscopy. In addition, in vitro and in vivo tumour models were done to assess FAT1 potential value for therapeutic applications.Results:The study shows that FAT1 is broadly expressed in primary and metastatic CRC stages and detected by mAb198.3, regardless of KRAS and BRAF mutations. FAT1 mainly accumulates at the plasma membrane of cancer cells, whereas it is only marginally detected in normal human samples. Moreover, the study shows that FAT1 has an important role in cell invasiveness while it does not significantly influence apoptosis. mAb198.3 specifically recognises FAT1 on the surface of colon cancer cells and is efficiently internalised. Furthermore, it reduces cancer growth in a colon cancer xenograft model.Conclusions:This study provides evidence that FAT1 and mAb198.3 may offer new therapeutic opportunities for CRC including the tumours resistant to current EGFR-targeted therapies.

Innovative Erythrocyte-based Carriers for Gene Delivery in Porcine Vascular Smooth Muscle Cells: Basis for Local Therapy to Prevent Restenosis

Vascular restenosis is affecting 30-40% of patients treated by percutaneous coronary angioplasty (PTCA). The advent of stenting reduced but not abolished restenosis. The introduction of drug eluting stent (DES) further reduced restenosis, but impaired endothelization exposed to intracoronary thrombosis as late adverse event. It is widely accepted that the endothelial denudation and coronary wall damages expose Vascular Smooth Muscle Cells (VSMC) to multiple growth factors and plasma circulating agents thus activating migration and proliferative pathways leading to restenosis. Among the major players of this processes, phosphorylated Elk-1, forming the Elk-1/SRF transcription complex, controls the expression of a different set of genes responsible for cell proliferation. Therefore, it is feasible that gene-specific oligonucleotide therapy targeting VSMC migration and proliferation genes can be a promising therapeutic approach. While a plethora of vehicles is suitably working in static in vitro cultures, methods for in vivo delivery of oligonucleotides are still under investigation. Recently, we have patented a novel erythrocyte-based drug delivery system with high capability to fuse with targeted cells thus improving drug bioavailability at the site of action. Here, the potential of these engineered porcine erythrocytes to deliver a synthetic DNA Elk-1 decoy inside syngenic porcine VSMC was tested. The results of this study indicate that Elk-1 decoy is actually able to inhibit cell proliferation and migration of VSMC. Our data also suggest that erythrocyte-based carriers are more efficient in delivering these oligonucleotides in comparison to conventional vehicles. As a consequence, a lower dose of Elk-1 decoy, delivered by engineered erythrocytes, was sufficient to inhibit cell growth and migration. This approach represents the translational step to reach in vivo experiments in pigs after PTCA and/or stent implantation where oligonucleotide drugs will be site-specific administered by using erythrocyte-based carriers to prevent restenosis.

Magnetically driven drug delivery systems improving targeted immunotherapy for colon-rectal cancer

Abstract Colorectal cancer (CRC) is one of the major causes of cancer‐associated mortality worldwide. The currently approved therapeutic agents show a rather limited efficacy. We have recently demonstrated that the atypical cadherin FAT1 is a specific marker of CRC and that the FAT1‐specific monoclonal antibody mAb198.3 may offer new therapeutic opportunities for CRC, being efficiently internalized by cancer cells and reducing cancer growth in colon cancer xenograft models. In this study we explored the therapeutic efficacy of mAb198.3 using two drug delivery systems (DDS) for improving the targeted treatment of CRC. The mAb198.3 was either directly bound to super‐paramagnetic nanoparticles (spmNPs) or embedded into human erythrocyte‐based magnetized carriers, named Erythro‐Magneto‐Hemagglutinin Virosomes (EMHVs) to produce two different novel mAb198.3 formulations. Both DDS were endowed with magnetic properties and were anchored in the target tumor site by means of an external permanent magnet. The antibody loading efficiency of these two magnetically driven drug delivery systems and the overall therapeutic efficacy of these two formulations were assessed both in vitro and in a proof‐of‐concept in vivo study. We demonstrated that mAb198.3 bound to spmNPs or embedded into EMHVs was very effective in targeting FAT1‐positive colon cancer cells in vitro and accumulating in the tumor mass in vivo. Although both in vivo administered mAb198.3 formulations have approximately 200 lower antibody doses needed, these showed to achieve a relevant therapeutic effect, thus reducing cancer growth more efficiently respect to the naked antibody. These results indicate that the two proposed magnetically driven drug delivery systems have a considerable potential as platforms to improve bioavailability and pharmacodynamics of anti‐FAT mAb198.3 and raise new opportunities for a targeted therapy of CRC. Graphical abstract Figure. No caption available.

Cutting Edge Therapies for Cancer in the 21st Century

Cancer is a broad group of diseases involving unregulated cell growth with elevated death rates since more people live in old age with mass lifestyle changes occurring in the developing world. The causes of cancer are diverse, complex and still only partially understood. The chances of surviving the disease vary remarkably by the type and location of the malignancy and the extent of disease at the start of treatment. Early cancer detection is proviing to be a valid approach. Cancer can be detected in a number of ways, including the presence of certain signs and symptoms, screening tests or medical imaging. Cancer therapy is dynamically changing and revision and change in patient management is constant. Cancer is routinely treated with chemotherapy, radiation therapy and surgery. Tailored cancer targeted therapy is becoming an emerging objective of today.

Abstract C190: A novel monoclonal antibody for colon cancer therapy.

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 (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. Here we describe a monoclonal antibody (mAb) targeting a protein of the protocadherin- family in colo-rectal cancer (CRC). The antibody specifically recognizes its target protein in cancer tissues, with concomitant negligible or marginal reactivity in normal colon. The protein was not previously described as CRC target. IHC data from CRC samples of approximately 200 patients, (collected at the bio-bank of the institute for Pathology of Basel, Switzerland and at the European Institute of Pathology of Milan, Italy) showed that the antibody binds up to 80% CRC cases (stages from 1 to 4) with high or moderate intensity. In most cases, the antibody gives plasma membrane by Immune Histochemistry (IHC). An expanded analysis on approximately 1000 CRC cases is ongoing to assess whether the membranous IHC staining associates with specific clinic-pathological parameters. IHC analysis of 35 normal human tissues revealed that it gives intracellular staining in skeletal muscle, pituitary glands and prostate, whereas it gives background irrelevant staining in the other 32 tissues. The antibody is able to bind the surface of different colon cancer cells in vitro and in vivo. Upon binding it is efficiently internalized by colon cancer cells, suggesting that it can be exploited for the development of Antibody-Drug-Conjugate (ADC). Moreover, it inhibits tumor growth in athymic nude mice bearing HCT15 and HT29 colon cancer xenografts. Overall, data indicate that this monoclonal antibody could be developed for a targeted therapy of colo-rectal cancers, alone or in combinatorial strategies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C190. Citation Format: Alberto Grandi, Susanna Campagnoli, Matteo Parri, Elisa De Camilli, Ilaria Naldi, Lisa Gherardini, Caterina Cinti, Luigi Terracciano, Boquan Jin, Paolo Sarmientos, Giuseppe Viale, Guido Grandi, Piero Pileri, Renata Maria Grifantini. A novel monoclonal antibody for colon cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C190.

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