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Dario Neri

Publications and source records attributed to Dario Neri.

At least 55 records · Page 3Linked to original sources
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↗
PubMed2003

Activation of T cells via tumor antigen specific chimeric receptors: the role of the intracellular signaling domain.

T cells engineered to express hybrid receptors with antibody defined specificity can successfully be targeted to tumor cells. In order to select intracellular domains of chimeric receptors capable of efficiently activate T cells in vitro and in vivo, we compared the function of receptors, which share the same extracellular antigen-binding part, joined to different intra-cellular signal transduction units. The antigen binding domain of the receptors was a single-chain fragment of a monoclonal antibody, which recognize a High Molecular Weight Melanoma-Associated Antigen with high affinity. The intracellular tails were derived from the T-cell receptor zeta chain (TCR-zeta), from the B-cell receptor Ig-alpha molecule and from a mutated Ig-alpha molecule able of stronger signal transduction. We compared the activity of the different chimeric receptors at a single-cell level by using a T-cell line that expressed an activation-dependent EGFP-reporter gene. Upon cross-linking with immobilized antibodies, all receptors were able to induce EGFP expression in the majority of the T cells. In contrast, EGFP expression was induced by contact to melanoma cells in vitro only in T cells that expressed the chimeric receptor that contained the TCR-zeta intracellular tail. In these T cells, the co-expression of chimeric receptors that contain a mutated Ig-alpha tail lowers the threshold of T-cell activation and facilitates tumor recognition in vitro and in vivo. Given their specificity and efficiency, T cells grafted with these type of receptors may represent potential candidates for cancer passive immunotherapy.

Animals↗
PubMed2003

Modulation of gene expression by extracellular pH variations in human fibroblasts: a transcriptomic and proteomic study.

Homeostasis of the intracellular ionic concentration, in particular that of hydrogen ions, is pivotal to the maintenance of cell function and viability. Nonetheless, pH fluctuations in both the intracellular and the extracellular compartments can occurr during development, in physiological processes and in disease. The influence of pH variations on gene expression has been studied in different model systems, but only for a limited number of genes. We have performed a broad range analysis of the patterns of gene expression in normal human dermal fibroblasts at two different pH values (in the presence and in the absence of serum), with the aim of getting a deeper insight into the regulation of the transcriptional program as a response to a pH change. Using the Affymetrix gene chip system, we found that the expression of 2068 genes (out of 12 565) was modulated by more than two-fold at 24, 48 or 72 h after the shift of the culture medium pH to a more acidic value, stanniocalcin 1 being a remarkable example of a strongly up-regulated gene. Genes displaying a modulated pattern of expression included, among others, cell cycle regulators (consistent with the observation that acidic pH abolishes the growth of fibroblasts in culture) and relevant extracellular matrix (ECM) components. Extracellular matrix protein 2, a protein with a restricted pattern of expression in adult human tissues, was found to be remarkably overexpressed as a consequence of serum starvation. Since ECM components, whose expression is controlled by pH, have been used as targets for biomolecular intervention, we have complemented the Affymetrix analysis with a two-dimensional polyacrylamide gel electrophoresis analysis of proteins which are differentially secreted by fibroblasts at acidic or basic pH. Mass spectrometric analysis of more than 650 protein spots allowed the identification of 170 protein isoforms or fragments, belonging to 40 different proteins. Some proteins were only expressed at basic pH (including, for instance, tetranectin), while others (e.g., agrin) were only detectable at acidic pH. Some of the identified proteins may represent promising candidate targets for biomedical applications, e.g., for antibody-mediated vascular targeting strategies.

Cells, Cultured↗
PubMed2003

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.

Mass Spectrometry↗
PubMed2003

Expression of extradomain-B-containing fibronectin in subretinal choroidal neovascular membranes.

PURPOSE: To investigate the presence of the fibronectin isoform containing the extradomain B (B-FN), a marker-protein of angiogenesis, in surgically excised human choroidal neovascular membranes (CNVM) to evaluate whether B-FN could be used as a therapeutic target for specific antibody-photosensitizer immunoconjugates. DESIGN: Laboratory investigation. METHODS: The setting was an institutional practice. The study population consisted of 15 eyes (15 patients) with CNVM undergoing membrane excision (four eyes with age-related macular degeneration, seven with pathologic myopia and four with multifocal choroiditis). The control group consisted of eight eye bank eyes (four subjects) without choroidal neovascularization. Light microscopic immunohistochemistry on cryostat sections of tissues was obtained. B-FN was detected by a human recombinant antibody, CGS-1, and compared with immunostaining for endothelial cells with factor VIII-related antigen. The main outcome measure was the presence of CGS-1 positively stained cells or areas of the extracellular matrix. Staining of CGS-1 was scored on a scale from 0 to 3. RESULTS: Fourteen of 15 neovascular membranes stained strongly with CGS-1 (score 2 or 3). One membrane from a patient with pathologic myopia was negatively stained (score 0). CGS-1 positive staining was detected around endothelial cells and in the extracellular matrix of CNVMs. The retina of eyes without choroidal neovascularization was negative with CGS-1 in all eight donor eyes, while the choroid contained some weakly CGS-1 positive cells (score 0 and 1, respectively). CONCLUSIONS: The extradomain B is abundantly expressed in CNVMs, but its expression is more restricted in eyes harboring no apparent choroidal neovascularization. In the future, B-FN might serve as a target for the delivery of antibody-photosensitizer immunoconjugates to newly developed vessels to enhance the selectivity of photodynamic therapy.

Adult↗
PubMed2003

Expression of the extra domain B of fibronectin, a marker of angiogenesis, in head and neck tumors.

OBJECTIVES/HYPOTHESIS: The extra domain B (ED-B) of fibronectin, a naturally occurring marker of tissue remodeling and angiogenesis, is expressed in the majority of aggressive solid human tumors, whereas it is not detectable in normal vessels and tissues. STUDY DESIGN: In view of the diagnostic and therapeutic clinical applications of the L19 antibody, which is specific for the ED-B domain of fibronectin, a prospective immunohistochemical analysis of different head and neck tumors was performed. METHODS: In all, 82 head and neck tissue biopsy specimens were immunohistochemically analyzed using the L19 antibody. They consisted of 53 different malignant tumors, 8 benign tumors, 10 nontumoral lesions, and 11 normal control tissues. RESULTS: A strong positive staining with the L19 antibody could be observed in 87% of the investigated malignant tumors, in only 38% of the benign tumors, and in 20% of the nontumoral lesions (P <.0001). The extra domain B was completely absent in the normal control tissue samples. CONCLUSIONS: The results show that ED-B is abundantly expressed around the neovasculature and in the stroma of the majority of malignant tumors of the head and neck but is undetectable in normal tissues. The ED-B domain of fibronectin is a good-quality tumor-stroma-associated antigen that warrants clinical trials with antibody-based pharmaceuticals, including immunoscintigraphic investigations and radioimmunoguided surgery with the radiolabeled L19 antibody.

Antibodies, Monoclonal↗
PubMed2003

Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer.

PURPOSE: 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)]. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Adult↗
PubMed2002

An antibody-calmodulin fusion protein reveals a functional dependence between macromolecular isoelectric point and tumor targeting performance.

PURPOSE: Human monoclonal antibodies are promising agents for the development of improved anticancer therapeutics, because, unlike low-molecular-weight chemotherapeutic agents, they can selectively localize to solid tumors. In particular, the scFv(L19) antibody fragment, specific for the EDB domain of fibronectin, a marker of angiogenesis, has demonstrated an impressive tumor targeting performance in a variety of tumor-bearing animals and in patients with cancer. The purpose of this study was to develop a tumor pretargeting strategy, based on a novel anti-EDB fusion protein. METHODS AND MATERIALS: We have fused the scFv(L19) to calmodulin, a small acidic protein for which specific binding peptides with a dissociation constant in the picomolar range are available. The resulting fusion protein has been expressed in mammalian cells and purified to homogeneity, before being characterized by quantitative biodistribution analysis in mice bearing the F9 murine teratocarcinoma. RESULTS: Surprisingly, we have found that the fusion of scFv(L19) to calmodulin completely abrogated the tumor targeting ability of the antibody in vivo, although both scFv(L19) and calmodulin moieties within the fusion protein retained unaltered binding affinities toward their respective ligand. Furthermore, a systematic analysis of 13 derivatives of scFv(L19) recently produced in our laboratories showed that the 10 derivatives that retain the tumor targeting ability of the parental antibody have isoelectric points (pI) between 5.0 and 9.0, whereas scFv(L19)-calmodulin (pI = 4.49) and two other derivatives of scFv(L19) with pI >9.0 were unable to target tumors in vivo. CONCLUSIONS: Because the EDB domain of fibronectin is a component of the modified extracellular matrix, predominantly located at the abluminal side of tumor blood vessels, our data suggest that extreme pI values of antibody-based pharmaceuticals may inhibit protein extravasation, perhaps by virtue of electrostatic interactions with endothelial cells and/or components of the extracellular matrix.

Animals↗
PubMed2002

Tumor-targeting properties of antibody-vascular endothelial growth factor fusion proteins.

A major problem of antibody-based targeting of solid tumors is the poor penetration of antibodies into tumor tissue. Vasoactive immunoconjugates have been proposed as a means of increasing antibody uptake in tumors. In principle, VEGF (also known as vascular permeability factor) could selectively alter vascular permeability, leading to improved tumor targeting. A possible role for VEGF in the targeting of tumor neovasculature has been postulated, based on the overexpression of VEGF receptors in tumor endothelial cells. However, quantitative biodistribution studies on this topic are not available. In this report, we describe the cloning, expression, characterization and biodistribution in tumor-bearing mice of antibodies fused to either VEGF(120) or VEGF(164) The MAb fragments chosen for analysis were scFv(L19), specific for the ED-B domain of fibronectin, a marker of angiogenesis, and scFv(HyHEL-10), a negative control antibody of irrelevant specificity in mice. Neither unconjugated VEGF nor scFv(HyHEL-10)-VEGF fusion proteins showed accumulation in the tumor (tumor:blood ratios approx. 1 at 4 hr and 24 hr postinjection). By contrast, scFv(L19)-VEGF(120) but not scFv(L19)-VEGF(164) showed significant accumulation in tumors (tumor:blood ratio = 9.3 at 24 hr) but was not superior to unconjugated scFv(L19). Preinjection of unlabeled scFv(L19)-VEGF(120) prior to administration of radiolabeled fusion protein led to increased accumulation of radiolabeled scFv(L19)-VEGF(120) in the tumor but only at very high concentrations (20 microg/mouse).

Animals↗
PubMed2002

Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin.

We recently demonstrated that a human recombinant scFv, L19, reacting with the ED-B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human "small immunoprotein" (SIP) and a complete human IgG1 and performed biodistribution studies in tumor-bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv)(2), SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2-5 times higher than that of the (scFv)(2), reaching a maximum 4-6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor-blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv)(2) and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED-B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials.

Animals↗
PubMed2002

Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix.

Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesis-related, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P =.003,.003, and.002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion protein, a mixture of IL-2 and L19, or saline alone). Immunohistochemical analysis of tumor infiltrates demonstrated a significantly higher number of T lymphocytes, natural killer cells, and macrophages, as well as increased interferon-gamma (IFN-gamma) accumulation, in tumors from animals treated with L19-IL-2 compared to tumors from control groups. The fact that ED-B is 100% homologous in human and mouse, thus ensuring that L19 reacts equally well with human and murine antigen, should ultimately expedite transfer of this reagent to clinical trials.

Animals↗
PubMed2002

Differentiation between high- and low-grade astrocytoma using a human recombinant antibody to the extra domain-B of fibronectin.

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.

Antibodies↗
PubMed2002

Two general methods for the isolation of enzyme activities by colony filter screening.

We describe two general methodologies, based on filter-sandwich assays, for isolating enzymatic activities from a large repertoire of protein variants expressed in the cytoplasm of E. coli cells. The enzymes are released by the freezing and thawing of bacterial colonies grown on a porous master filter and diffuse to a second "reaction" filter that closely contacts the master filter. Reaction substrates can be immobilized either on the filter or on the enzyme itself (which is then, in turn, captured on the reaction filter). The resulting products are detected with suitable affinity reagents. We used biotin ligase as a model enzyme to assess the performance of the two methodologies. Active enzymes were released by the bacteria, locally biotinylated the immobilized target substrate peptide, and allowed the sensitive and specific detection of individual catalytically active colonies.

Bacterial Proteins↗
PubMed2002

Affinity-capture reagents for protein arrays.

The simultaneous identification and quantitative measurement of the production levels of thousands of different proteins in a biological specimen remains an unachieved goal of modern proteomic research. Advances in the development of microarray-based platforms for highly parallel detection of proteins have therefore received a considerable impulse during the last few years. Here, we review the existing reagents for affinity capture of protein targets, as well as the techniques used for their immobilization on solid supports and methods for the detection of binding events, underlining the problems and the opportunities in this continuously evolving research field.

Biotechnology↗