Biomedical subjects

Dario Neri

Publications and source records attributed to Dario Neri.

At least 19 recordsLinked to original sources

PubMed2005

Engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy.

A number of cytokines are either approved drugs or are in advanced clinical trials, yet these biopharmaceuticals do not typically localize efficiently in solid tumors and manifest their therapeutic potential at the expense of severe side effects. The targeted delivery of cytokines to solid tumors is a promising avenue for increasing the therapeutic index of these biopharmaceuticals. We engineered a fusion protein between scFv(L19), a human antibody fragment specific to the EDB domain of fibronectin, and a cysteine-free mutant of murine interferon-gamma. The resulting fusion protein was capable of targeting new blood vessels in solid tumors, and the targeting efficiency was strikingly increased in tumor-bearing knockout mice lacking the interferon-gamma receptor. ScFv(L19)-interferon-gamma displayed a strong antitumor effect in both subcutaneous and metastatic murine F9 teratocarcinomas, but was not efficacious as single agent when used to treat C51 and CT26 tumors. The potency of this fusion protein could be substantially enhanced by combination with doxorubicin and other immunocytokines. These findings are of clinical relevance, as the EDB domain is a marker of angiogenesis, with identical sequence in mouse and man, which is abundantly expressed in a variety of aggressive solid tumors but is undetectable in most normal tissues.

Adenocarcinoma↗

PubMed2005

In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature.

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography-tandem mass spectrometry for identification. In the present study, in vivo biotinylation procedure led to the identification of hundreds of proteins in different mouse organs, including some showing a restricted pattern of expression in certain body tissues. Furthermore, biotinylation of mice with F9 subcutaneous tumors or orthotopic kidney tumors revealed both quantitative and qualitative differences in the recovery of biotinylated proteins, as compared to normal tissues. This technology is applicable to proteomic investigations of the differential expression of accessible proteins in physiological and pathological processes in animal models, and to human surgical specimens using ex vivo perfusion procedures.

Animals↗

PubMed2005

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping.

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.

Biotinylation↗

PubMed2005

Design, construction, and characterization of a large synthetic human antibody phage display library.

Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.

Antibodies, Monoclonal↗

PubMed2005

A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins.

2-D peptide mapping is a novel technique for the relative quantification of membrane proteins (Scheurer S. et al., Proteomics 2005, in press). Using closely related metastatic and nonmetastatic teratocarcinoma cell lines as a model system, we have performed a comparative analysis of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques, with the aim to increase the number of proteins identified by 2-D peptide mapping. Our experience indicates that the LC-MALDI TOF/TOF technique is superior to LC-ESI MS/MS in terms of the number of proteins identified and confidence in protein identification. Furthermore, the best results were obtained by tryptic digestion of proteins eluted from a streptavidin column using a cleavable biotin derivative.

Animals↗

PubMed2005

Tumour vascular targeting.

It is now accepted that the growth of solid tumours is dependent on their capacity to acquire a blood supply, and much effort has been directed towards the development of agents (known as anti-angiogenics) that disrupt this process. More recently, it has become apparent that targeted destruction of the established tumour vasculature is another avenue for exciting therapeutic opportunities. In this article, we present evidence that vascular targeting is an effective antitumour strategy in animal models, describe strategies for identifying putative tumour vascular targets and discuss future prospects for vascular targeting in the clinic.

Angiogenesis Inhibitors↗

PubMed2004

Molecular imaging of atherosclerotic plaques using a human antibody against the extra-domain B of fibronectin.

Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E-null (ApoE-/-) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE-/- mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.

Adult↗

PubMed2004

Covalent DNA display as a novel tool for directed evolution of proteins in vitro.

We present a novel method for the directed evolution of polypeptides, which combines in vitro compartmentalization and covalent DNA display. A library of linear DNA fragments is co-packaged with an in vitro transcription/translation mixture in the compartments of a water-in-oil emulsion. Experimental conditions are adjusted so that, in most cases, one compartment contains one DNA molecule. The DNA fragments encode fusion proteins containing a DNA-methyltransferase (M.Hae III), which can form a covalent bond with a 5-fluorodeoxycytidine base at the extremity of the DNA fragment. The resulting library of DNA-protein fusions is extracted from the emulsion and DNA molecules displaying a protein with desired binding properties are selected from the pool of DNA-protein fusions by affinity panning on target antigens. We applied this methodology in model selection experiments, using specific ligands for the capture of peptides and globular proteins bound to DNA. We observed enrichment factors >1000-fold for selections performed in separate emulsions and up to 150-fold for selections performed using mixtures of DNA molecules. M.Hae III could be fused to small globular proteins (such as calmodulin and fibronectin domains), which are ideally suited for the generation of combinatorial libraries and for the isolation of novel binding specificities.

DNA Adducts↗

PubMed2004

Molecular targeting of angiogenesis for imaging and therapy.

Angiogenesis, i.e. the proliferation of new blood vessels from pre-existing ones, is an underlying process in many human diseases, including cancer, blinding ocular disorders and rheumatoid arthritis. The ability to selectively target and interfere with neovascularisation would potentially be useful in the diagnosis and treatment of angiogenesis-related diseases. This review presents the authors' views on some of the most relevant markers of angiogenesis described to date, as well as on specific ligands which have been characterised in pre-clinical animal models and/or clinical studies. Furthermore, we present an overview on technologies which are likely to have an impact on the way molecular targeting of angiogenesis is performed in the future.

Animals↗

PubMed2004

Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy.

We present a new antibody-directed enzyme prodrug therapy strategy (ADEPT) based on a post-proline cleaving endopeptidase and prodrugs, in which cytotoxic moieties are linked to a proline-containing peptide. Human prolyl endopeptidase was expressed in Escherichia coli and purified to homogeneity. The enzyme was active in buffer and in human serum but was rapidly thermally inactivated by incubation at 37 degrees C, thus preventing applications in vivo. While prolyl endopeptidase display on filamentous phage abolished viral infectivity and prevented directed evolution strategies based on phage display, we robotically screened 10752 individual colonies of mutant enzymes using a fluorogenic assay to improve enzyme stability. A single amino acid mutation (Glu289 --> Gly) improved protein stability, resulting in a half-life of 16 h at 37 degrees C in phosphate buffer. Two prodrugs were synthesized, in which an N-protected glycine-proline dipeptide was covalently coupled to doxorubicin and melphalan. (Benzyloxycarbonyl)glycylprolylmelphalan, but not the more sterically hindered doxorubicin prodrug, could be efficiently activated by prolyl endopeptidase [specific activity = 813.3 nmol min(-1) (mg of enzyme)(-1) at 25 degrees C]. The melphalan prodrug was essentially nontoxic to CHO, F9 teratocarcinoma, MCF7 breast adenocarcinoma, and p3U1 mouse myeloma cells up to millimolar concentrations, while prodrug incubation with the engineered prolyl endopeptidase mutant led to a cell killing profile superimposable to the one of melphalan. The prolyl endopeptidase mutant was then chemically coupled to the human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis. The resulting immunoconjugate retains antigen binding and enzymatic activity, thus opening the way to anticancer ADEPT applications.

Animals↗

PubMed2004

Encoded self-assembling chemical libraries.

The isolation of molecules capable of high-affinity and specific binding to biological targets is a central problem in chemistry, biology and pharmaceutical sciences. Here we describe the use of encoded self-assembling chemical (ESAC) libraries for the facile identification of molecules that bind macromolecular targets. ESAC technology uses libraries of organic molecules linked to individual oligonucleotides that mediate the self-assembly of the library and provide a code associated with each organic molecule. After panning ESAC libraries on the biomolecular target of interest, the 'binding code' of the selected compounds can be 'decoded' by a number of experimental techniques (e.g., hybridization on oligonucleotide microarrays). The potential of this technology was demonstrated by the affinity maturation (>40-fold) of binding molecules to human serum albumin and bovine carbonic anhydrase, leading to binders with dissociation constants in the nanomolar range.

Albumins↗

PubMed2004

Molecular targeting of angiogenesis.

The majority of pharmacological approaches for the treatment of solid tumors suffer from poor selectivity, thus limiting dose escalation (i.e., the doses of drug which are required to kill tumor cells cause unacceptable toxicities to normal tissues). The situation is made more dramatic by the fact that the majority of anticancer drugs accumulate preferentially in normal tissues rather than in neoplastic sites, due to the irregular vasculature and to the high interstitial pressure of solid tumors. One avenue towards the development of more efficacious and better tolerated anti-cancer drugs relies on the targeted delivery of therapeutic agents to the tumor environment, thus sparing normal tissues. Molecular markers which are selectively expressed in the stroma and in neo-vascular sites of aggressive solid tumors appear to be particularly suited for ligand-based tumor targeting strategies. Tumor blood vessels are accessible to agents coming from the bloodstream, and their occlusion may result in an avalanche of tumor cell death. Furthermore, endothelial cells and stromal cells are genetically more stable than tumor cells and can produce abundant markers, which are ideally suited for tumor targeting strategies. This review focuses on recent advances in the development of ligands for the selective targeting of tumor blood vessels and new blood vessels in other angiogenesis-related diseases.

Angiogenesis Inhibitors↗

PubMed2004

Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells: A transcriptomic and proteomic study.

Hypoxia is a characteristic feature of many human pathologies, including cancer. The sustained proliferation rate of tumor cells leads to alterations of the tumor microenvironment, that progressively becomes more acidic, nutrient-deprived, and hypoxic. The reduced partial pressure of oxygen triggers the onset of an adaptive response, aimed at increasing the local oxygen concentration by several complementary actions. Although directly exposed to the blood stream, endothelial cells lining the vascular lumen in tumors also can be exposed to hypoxia and therefore can contribute to the onset of the adaptive response that leads to tumor angiogenesis. Aiming at getting a detailed insight into the oxygen-dependent regulation of the transcriptional program of vascular endothelial cells and at identifying new relevant markers that may be used as targets for therapeutic intervention in tumor angiogenesis, we have performed a broad-range transcriptomic analysis, using the Affymetrix HG-U133A Gene Chips, of mRNA expression levels in human umbilical cord vein endothelial cells (HUVEC), exposed in vitro to hypoxia for different time periods. The transcriptomic analysis was complemented by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA levels and alternative splicing for some selected extracellular matrix protein genes, and by a proteomic analysis, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and tandem mass spectrometry for protein separation and identification, of hypoxic and normoxic HUVEC whole-cell lysates and subcellular fractions. Our analysis confirmed previous findings on genes whose expression is regulated by oxygen concentration but also identified new genes (e.g., CXCR4, claudin 3, CD24, tetranectin, Del-1, procollagen lysyl hydroxylase 1 and 2) which are transcriptionally upregulated in hypoxic conditions.

Alternative Splicing↗

PubMed2004

Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution.

The interaction between streptavidin and biotin is one of the most widely used tools in chemistry and biology. However, the release of biotinylated proteins from streptavidin resins remains a major problem, due to the extraordinary stability of this complex. We present a new protocol for the quantitative elution of biotinylated proteins from streptavidin Sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method was demonstrated by the quantitative recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin.

Animals↗

PubMed2004

Diagnostic and therapeutic applications of recombinant antibodies: targeting the extra-domain B of fibronectin, a marker of tumor angiogenesis.

Angiogenesis, the sprouting of new blood vessels from preexisting ones, is a phenomenon associated to several human pathologies, including different potentially blinding ocular disorders, rheumatoid arthritis, and cancer. Indeed, ongoing angiogenesis is a characteristic feature of the majority of aggressive solid tumors, and is also a pre-requisite for the progression towards the metastatic phenotype. One established marker of angiogenesis is represented by an isoform of the oncofoetal fibronectin (FN), containing an additional domain inserted by alternative splicing of the FN pre-mRNA and called extra-domain B (ED-B). This isoform has been found to be present almost exclusively in the modified extra-cellular matrix surrounding newly-formed blood vessels in tumors (and other animal models of ocular pathologies), being completely absent from the normal vasculature in adult organs. This article reviews the recombinant antibodies raised against ED-B and the different methodologies used for the generation of these antibodies. Moreover, new diagnostic and therapeutic applications based on the delivery of bioactive molecules to tumor blood vessels by means of ED-B targeting will be discussed.

Animals↗

PubMed2003

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. L19mTNFalpha was an immunoreactive and biologically active homotrimer. Radiolabeled L19mTNFalpha selectively targeted tumor neovasculature in tumor-bearing mice, where it accumulated selectively and persistently (tumor-to-blood ratio of the percentage of injected dose per gram [%ID/g] of 700, 48 hours from injection). L19mTNFalpha showed a greater anticancer therapeutic activity than both mTNFalpha and TN11mTNFalpha, a control fusion protein in which an antibody fragment, irrelevant in the tumor model used, substituted for L19. This activity was further dramatically enhanced by its combination with melphalan or the recently reported fusion protein L19-IL2. In conclusion, L19mTNFalpha allows concentrating therapeutically active doses of TNFalpha at the tumor level, thus opening new possibilities for the systemic use of TNFalpha in cancer therapy.

Adenocarcinoma↗

PubMed2003

Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha.

The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of cytokines in cancer therapy consists of fusing them to monoclonal antibodies capable of a selective localization at the tumor site. We have constructed fusion proteins of interleukin-12 (IL-12) and tumor necrosis factor (TNF-alpha) with L19, an antibody fragment specific to the extradomain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. In this article, we have evaluated the tumor-targeting properties and the anticancer activities of combinations of the two antibody-cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-alpha. Although all fusion proteins were active in vitro, the triple fusion protein failed to localize to tumors in vivo and to show significant therapeutic effects. By contrast, the combination of IL-12-L19 and L19-TNF-alpha displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF-alpha (2 micro g + 2 micro g/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF-alpha appears to be synergistic in vivo.

Animals↗

PubMed2003

Unexpected observation of concentration-dependent dissociation rates for antibody-antigen complexes and other macromolecular complexes in competition experiments.

The dissociation rate constant of bimolecular complexes between macromolecules (k(off)) is often measured in solution by competition experiments and is generally expected to follow first-order kinetics.When measuring k(off) constants by competition for three complexes of high-affinity recombinant antibody fragments with the cognate antigen and for one calmodulin/peptide complex, a surprising dependence between apparent dissociation rate and concentration of competitor (antigen or calmodulin-binding peptide) was observed. Our results may be characteristic for macromolecules consisting of two domains (such as single-chain Fv fragments) and may reflect a transient opening of the two domains which are involved in the binding reaction, and which are connected by a polypeptide linker.

Antibodies↗