Gene splicing profiles are frequently altered in cancer, and the splice variants of fibronectin (FN) that contain the extra-domains A (EDA) or B (EDB), referred to as EDA+FN or EDB+FN, are highly ...upregulated in tumor vasculature. Transforming growth factor β (TGF-β) signaling has been attributed a pivotal role in glioblastoma, with TGF-β promoting angiogenesis and vessel remodeling. By using immunohistochemistry staining, we observed that the oncofetal FN isoforms EDA+FN and EDB+FN are expressed in glioblastoma vasculature.
single-cell gene expression profiling of tumors by using CD31 and α-smooth muscle actin (αSMA) as markers for endothelial cells, and pericytes and vascular smooth muscle cells (VSMCs), respectively, confirmed the predominant expression of FN, EDA+FN and EDB+FN in the vascular compartment of glioblastoma. Specifically, within the CD31-positive cell population, we identified a positive correlation between the expression of EDA+FN and EDB+FN, and of molecules associated with TGF-β signaling. Further, TGF-β induced EDA+FN and EDB+FN in human cerebral microvascular endothelial cells and glioblastoma-derived endothelial cells in a SMAD3- and SMAD4-dependent manner. In turn, we found that FN modulated TGF-β superfamily signaling in endothelial cells via the EDA and EDB, pointing towards a bidirectional influence of oncofetal FN and TGF-β superfamily signaling.
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 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The “blue silver” exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the “blue silver” exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of bovine serum albumin (BSA) gave a detection limit (signal‐to‐noise ratio > 3) of 1 ng in a single zone. The somewhat lower sensitivity of “blue silver” as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two‐dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.
Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN ...isoform that contains the "extra-domain-B" (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues in vivo. We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B.
We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6.
The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity.
ED-B fibronectin is expressed only during angiogenic processes and in tissues undergoing growth and/or extensive remodeling. We demonstrated previously the possibility to target and selectively ...deliver therapeutic substances to tumor vasculature in experimental animal models using a human recombinant antibody fragment, L19, specific for the ED-B domain of fibronectin. Here we evaluate the possibility of targeting primary tumors and metastatic lesions in cancer patients through immunoscintigraphy using (123)I-labeled dimeric L19 L19(scFv)(2).
Twenty patients (34-79 years of age) with lung, colorectal, or brain cancer, whose tumors had been confirmed by imaging techniques and/or histologically, were admitted to the immunoscintigraphic investigation.
The dimeric L19 antibody selectively localized in tumor lesions in aggressive types of lung cancer and colorectal cancer. Because ED-B fibronectin is expressed only during angiogenic processes and in tissues undergoing growth and/or extensive remodeling, L19(scFv)(2) is able to distinguish between quiescent and actively growing lesions. No side effects were observed.
The ability of L19(scFv)(2) to target tumors in patients provides the foundations for new therapeutic applications, in which the L19 antibody is engineered to selectively deliver bioactive molecules to primary tumors as well as to metastases.
Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the ...synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A-containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.
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.
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. In adoptive immunity transfer experiments, the splenocytes from tumor-cured mice protected naive mice both from C51 colon carcinoma and from WEHI-164 fibrosarcoma. Similar results were also obtained in adoptive immunity transfer experiments using severely immunodepressed mice. Experiments using depleted splenocytes showed that T cells play a major role in tumor rejection.
The results show that the selective targeting of mTNFalpha to the tumor enhances its immunostimulatory properties to the point of generating a therapeutic immune response against different histologically unrelated syngeneic tumors. These findings predicate treatment approaches for cancer patients based on the targeted delivery of TNFalpha to the tumor vasculature.
Different fibronectin (FN) isoforms are generated by the alternative splicing of the primary FN transcript. We previously demonstrated that the isoform containing the extra domain B sequence of ...fibronectin (B-FN), a complete type-III-homology repeat, is a marker of angiogenesis that accumulates around neovasculature only during angiogenic processes. We produced a single-chain human recombinant antibody (scFv), L19, which reacts specifically with B-FN and selectively targets tumor vasculature
in vivo
. We used this scFv and an antibody against a pan-endothelial marker (Factor VIII) in a double-staining procedure on specimens of low- and high-grade astrocytomas to determine the percentage of B-FN-positive vessels, (denominating the resulting value angiogenic index AI). Compared to vascular density and proliferative activity (evaluated using antibodies to Factor VIII and Ki67, respectively), AI correlated better with tumor grade (1.6 ± 2.6% and 92.0 ± 8.7% of B-FN-positive vessels in low- and high-grade astrocytomas, respectively) and was a more precise diagnostic tool than either of the two conventional methods. In fact, discriminating analysis using these three parameters showed that only AI accurately classified 100% of the cases studied, compared to 64% and 89% correctly diagnosed by vascular density and of proliferating cells, respectively.
Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as ...antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.
We have shown previously that the MHC class II-negative murine TS/A adenocarcinoma is rejected in vivo if induced to express MHC class II molecules by transfection of the MHC class II transactivator ...CIITA. In this study, we explored the immunologic basis of tumor rejection and the correlation between histopathology of tumor tissue and immune rejection.
Stable TS/A-CIITA transfectants were generated and injected into mice. In vivo cell depletion, immunohistochemistry of tumor tissues, and immune functional assays were done to assess the cellular and immunologic basis of rejection.
Ninety-two percent of mice injected with TS/A-CIITA rejected the tumor and were completely resistant to challenge with parental TS/A. Only CD4+ and CD8+ cells were required for rejection. The tumor microenvironment in TS/A-CIITA-injected mice changed dramatically when compared with the TS/A parental-injected mice. Rapid infiltration with CD4+ T cells followed by dendritic cells, CD8+ T cells, and granulocytes was observed. Importantly, TS/A-CIITA cells could act as antigen-presenting cells because they process and present nominal antigens to CD4+ T cells. Tumor-specific CD4+ T cells of TS/A-CIITA-injected mice had the functional characteristics of Th1 cells and produced IFN-gamma and this was relevant for generation and maintenance of protective antitumor response, because IFN-gamma knockout mice were no longer rejecting TS/A-CIITA tumor cells.
CIITA-dependent MHC class II expression confers to TS/A tumor cells the capacity to act as a protective vaccine against the tumor by triggering tumor antigen presentation to T helper cells, antitumor polarization of cellular and soluble components of the tumor microenvironment, and establishment of antitumor immune memory.
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