L19-IL2 Immunocytokine in Combination with the Anti-Syndecan-1 46F2SIP Antibody Format: A New Targeted Treatment Approach in an Ovarian Carcinoma Model.

Epithelial ovarian cancer (EOC) is the fifth most common cancer affecting the female population. At present, different targeted treatment approaches may improve currently employed therapies leading either to the delay of tumor recurrence or to disease stabilization. In this study we show that syndecan-1 (SDC1) and tumor angiogenic-associated B-fibronectin isoform (B-FN) are involved in EOC progression and we describe the prominent role of SDC1 in the vasculogenic mimicry (VM) process.

Anti-cancer Therapies Employing IL-2 Cytokine Tumor Targeting: Contribution of Innate, Adaptive and Immunosuppressive Cells in the Anti-tumor Efficacy.

Antibody-cytokine fusion proteins (immunocytokine) exert a potent anti-cancer effect; indeed, they target the immunosuppressive tumor microenvironment (TME) due to a specific anti-tumor antibody linked to immune activating cytokines. Once bound to the target tumor, the interleukin-2 (IL-2) immunocytokines composed of either full antibody or single chain Fv conjugated to IL-2 can promote the recruitment and activation of natural killer (NK) cells and cytotoxic CD8 T lymphocytes (CTL). This recruitment induces a TME switch toward a classical T helper 1 (Th1) anti-tumor immune response, supported by the cross-talk between NK and dendritic cells (DC).

Tumor Vasculature Targeted TNFα Therapy: Reversion of Microenvironment Anergy and Enhancement of the Anti-tumor Efficiency.

Tumor cells and tumor-associated stromal cells such as immune, endothelial and mesenchimal cells create a Tumor Microenvironment (TME) which allows tumor cell promotion, growth and dissemination while dampening the anti-tumor immune response. Efficient anti-tumor interventions have to keep into consideration the complexity of the TME and take advantage of immunotherapy and chemotherapy combined approaches. Thus, the aim of tumor therapy is to directly hit tumor cells and reverse endothelial and immune cell anergy.

Targeting Syndecan-1, a molecule implicated in the process of vasculogenic mimicry, enhances the therapeutic efficacy of the L19-IL2 immunocytokine in human melanoma xenografts.

Anti-angiogenic therapy of solid tumors has until now failed to produce the long lasting clinical benefits desired, possibly due to the complexity of the neoangiogenic process. Indeed, a prominent role is played by "vasculogenic" or "vascular" mimicry (VM), a phenomenon in which aggressive cancer cells form an alternative microvascular circulation, independently of endothelial cell angiogenesis. In this study we observed, in melanoma patient cell lines having vasculogenic/stem-cell like phenotype and in melanoma tumors, the syndecan-1 co-expression with VM markers, such as CD144 and VEGFR-2.

Periostin is a novel therapeutic target that predicts and regulates glioma malignancy.

Background: Periostin is a secreted matricellular protein critical for epithelial-mesenchymal transition and carcinoma metastasis. In glioblastoma, it is highly upregulated compared with normal brain, and existing reports indicate potential prognostic and functional importance in glioma. However, the clinical implications of periostin expression and function related to its therapeutic potential have not been fully explored.

Schedule-dependent therapeutic efficacy of L19mTNF-α and melphalan combined with gemcitabine.

L19-tumor necrosis factor alpha (L19mTNF-α; L), a fusion protein consisting of mouse TNFα and the human antibody fragment L19 directed to the extra domain-B (ED-B) of fibronectin, is able to selectively target tumor vasculature and to exert a long-lasting therapeutic activity in combination with melphalan (M) in syngeneic mouse tumor models. We have studied the antitumor activity of single L19mTNF-α treatment in combination with melphalan and gemcitabine (G) using different administration protocols in two histologically different murine tumor models: WEHI-164 fibrosarcoma and K7M2 osteosarcoma. All responding mice showed significant reduction in myeloid-derived suppressor cells (MDSCs) and an increase in CD4(+) and CD8(+) T cells in the tumor infiltrates, as well as significant reduction in regulatory T cells (Treg) at the level of draining lymph nodes.

A novel human anti-syndecan-1 antibody inhibits vascular maturation and tumour growth in melanoma.

Introduction: Syndecan-1 is a cell membrane protein that, after its shedding by heparanase enzymes, is accumulated in the extracellular matrix of some tumours, e.g. myeloma and lung carcinoma, where it modulates several key processes of tumourigenesis such as cancer cell proliferation and apoptosis, angiogenesis and metastasis.

Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin.

Selective targeted delivery of the TNF-alpha receptor p75 and uteroglobin to the vasculature of inflamed tissues: a preliminary report.

Background: Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. We hypothesized that the specific targeting of TNF-alpha antagonists to inflamed tissues could increase drug efficacy and reduce side effects.

Results: Using uteroglobin (UG), a potent anti-inflammatory protein, as a scaffold, we prepared a bispecific tetravalent molecule consisting of the extracellular ligand-binding portion of the human TNF-alpha receptor P75 (TNFRII) and the scFv L19.

Identification of a novel cell binding site of periostin involved in tumour growth.

Introduction: Periostin (PN), a member of the fasciclin family of proteins, is a TGF-β-induced extracellular matrix protein involved in cell survival, angiogenesis, invasion and metastasis. It is considered a potent angiogenic factor and a marker of tumour progression in many types of human cancer. Many different kinds of cells bind to PN by means of the integrins αvβ3 and αvβ5, but the periostin epitope recognised by these integrins is not formally demonstrated.

Alternative splicing of the angiogenesis associated extra-domain B of fibronectin regulates the accessibility of the B-C loop of the type III repeat 8.

Background: Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA.

Therapy-induced antitumor vaccination in neuroblastomas by the combined targeting of IL-2 and TNFalpha.

L19-IL2 and L19TNFalpha are fusion proteins composed of L19(scFv), specific for the angiogenesis-associated ED-B containing fibronectin isoform and IL-2 or TNFalpha. Because of the tumor targeting properties of L19, IL-2 and TNFalpha concentrate at therapeutic doses at the tumor vascular level. To evaluate the therapeutic effects of L19-IL2 and L19mTNFalpha in neuroblastoma (NB)-bearing mice, A/J mice bearing Neuro2A or NIE115 NB were systemically treated with L19-IL2 and L19mTNFalpha, alone or in combination protocols.

Use of uteroglobin for the engineering of polyvalent, polyspecific fusion proteins.

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-alpha, we expressed various biologically active uteroglobin fusion proteins.

A novel human fibronectin cryptic sequence unmasked by the insertion of the angiogenesis-associated extra type III domain B.

The angiogenesis-associated extra-domain B (EDB) of fibronectin (FN) is a complete type III repeat of 91 amino acids. Its expression is modulated by the alternative splicing pattern of the FN pre-mRNA. FN containing the EDB (B-FN) is undetectable in tissues of healthy adults, with rare exceptions such as the female reproductive system where tissue remodeling and angiogenesis are recurrent physiological processes.

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: identification of differentially expressed and MAR-binding proteins.

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH).

Differential proteomic analysis of nuclear matrix in muscle-invasive bladder cancer: potential to improve diagnosis and prognosis.

Introduction: Although several molecular markers for bladder cancer have been identified, at present little information on prognostic biomarkers is available in the literature. Prognostication of this tumor is largely based on clinicopathological characteristics. Our aim was to identify nuclear matrix (NM) proteins that might serve to better characterize the phenotype of the invasive bladder cancer and to investigate their diagnostic and prognostic roles.

Therapy-induced antitumor vaccination by targeting tumor necrosis factor alpha to tumor vessels in combination with melphalan.

Treatment of tumor-bearing mice with mouse (m)TNF-alpha, targeted to tumor vasculature by the anti-ED-B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6 days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice.

Both CD133+ and CD133- medulloblastoma cell lines express ligands for triggering NK receptors and are susceptible to NK-mediated cytotoxicity.

Adoptive cellular immunotherapy has been proposed as an additional treatment of medulloblastoma, an intracranial tumor characterized by a particularly poor prognosis. However, little is known on the ability of the immune system to effectively attack this tumor. In this study, we show that activated human NK cells efficiently kill medulloblastoma cell lines in vitro.

Targeted delivery of tumor necrosis factor-alpha to tumor vessels induces a therapeutic T cell-mediated immune response that protects the host against syngeneic tumors of different histologic origin.

Purpose: We sought to demonstrate that a single systemic administration of L19mTNFalpha (a fusion protein constituted by the scFv L19 specific for the oncofetal ED-B domain of fibronectin and tumor necrosis factor alpha, TNFalpha) in combination with melphalan induced complete and long-lasting tumor eradication in tumor-bearing mice and triggered the generation of a specific T cell-based immune response that protects the animals from a second tumor challenge, as well as from challenges with syngeneic tumor cells of different histologic origin.

Experimental Design And Results: Treatment with L19mTNFalpha, in combination with melphalan, induced complete tumor regression in 83% of BALB/c mice with WEHI-164 fibrosarcoma and 33% of animals with C51 colon carcinoma. All cured mice rejected challenges with the same tumor cells and, in a very high percentage of animals, also rejected challenges with syngeneic tumor cells of different histologic origin.

Nuclear matrix protein expression in prostate cancer: possible prognostic and diagnostic applications.

Different lines of evidence suggest that the nuclear matrix (NM), the protein scaffold of the nucleus, represents a functional unit playing a pivotal role in the spatial and temporal coordination of the events of gene activation. Any change in the gene expression pattern, which occurs during carcinogenesis, may partially depend on an impairment of the regulatory functions of the NM. Therefore, increasing interest has been addressed to the study of NM modifications associated with malignant transformations and to potential clinical applications.

PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis.

The capability of NK lymphocytes to kill tumor cells depends on different receptors/ligands interactions. In order to identify the cellular ligands recognized by "orphan" triggering receptors, mice were immunized with NK susceptible target cells. mAbs were selected that inhibited NK cytotoxicity and recognized two different molecules of 70 and 60-65 kDa.

Identification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts a protective role from an NK cell-mediated lysis.

In this study, in an attempt to identify neuroblastoma-associated surface antigens, we generated mAbs against the ACN neuroblastoma cell line. A mAb was selected (5B14) that reacted with all neuroblastoma cell lines analyzed and allowed detection of tumor cell infiltrates in bone marrow aspirates from neuroblastoma patients. In cytofluorimetric analysis, unlike anti-disialoganglioside mAb, 5B14 mAb did not display reactivity with normal bone marrow hematopoietic cell precursors, thus representing a highly specific marker for identifying neuroblastoma cells.

Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.

A modified Neuhoff's colloidal Coomassie Blue G-250 stain is reported, dubbed "blue silver" on account of its considerably higher sensitivity, approaching the one of conventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.

Selective targeted delivery of TNFalpha to tumor blood vessels.

We sought to enhance the selective toxicity of tumor necrosis factor alpha (TNFalpha) to permit its systemic use in cancer therapy. Because ligand-targeted therapeutics have proven successful in improving the selective toxicity of drugs, we prepared a fusion protein (L19mTNFalpha) composed of mouse TNFalpha and a high-affinity antibody fragment (L19 scFv) to the extradomain B (ED-B) domain of fibronectin, a marker of angiogenesis. L19mTNFalpha was expressed in mammalian cells, purified, and characterized.

Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells.