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 specificitie

Tumor-targeting properties of novel immunocytokines based on murine IL1β and IL6

There is an increasing biotechnological interest in ‘arming' therapeutic antibodies with bioactive payloads. Many antibody-cytokine fusion proteins (immunocytokines) have been described and some of these biopharmaceuticals have progressed to clinical studies. Here, we describe for the first time the expression and in vivo characterization of immunocytokines based on murine IL1β and IL6. These potent pro-inflammatory cytokines were fused at the N-terminus or at the C-terminus of the monoclonal antibodies F8 (specific to the alternatively-spliced extra-domain A domain of fibronectin, a marker of tumor angiogenesis). All immunocytokines retained the binding properties of the parental antibody and were homogenous, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, except for the N-terminal fusion of IL1β which revealed the presence of glycosylated species. When analyzed by quantitative biodistribution analysis using radioiodinated protein preparations, F8 fusions with IL6 revealed a preferential accumulation at the tumor site for both cytokine orientations, whereas IL1β fusions exhibited lower tumor to organ ratios and a slower blood clearance profile. The fusion proteins with the cytokine payload at the C-terminus were studied in therapy experiments in immunocompetent mice bearing F9 tumors. Immunocytokines based on IL1β resulted in 10% body weight loss at a 5-µg dose, whereas IL6-based products caused a 5% body weight loss at a 225-µg dose. Both F8-IL1β and F8-IL6 exhibited a <50% inhibition of tumor growth rate, which was substantially lower than the one previously reported for F8-TNF, a closely related pro-inflammatory immunocytokine. This study indicates that IL6 can be efficiently delivered to the tumor neo-vasculature by fusion with the F8 antibody. While F8-IL6 was not as potent as other F8-based immunocytokines that exhibit similar biodistribution profiles, the fusion protein sheds light on the different roles of pro-inflammatory cytokines in boosting immunity against the tumo

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 futur

A critical evaluation of the tumor-targeting properties of bispecific antibodies based on quantitative biodistribution data

Bispecific and bifunctional antibodies are attracting considerable interest as innovative anti-cancer therapeutics, but their ability to selectively localize at the tumor site has rarely been studied by quantitative biodistribution studies in immunocompetent animal models or in patients. Here, we describe the production of a novel bifunctional antibody, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin) fused to the extracellular portion of CD86 (co-stimulatory molecule B7.2). However, the fusion molecule was unable to target tumors in vivo. These data suggest that bispecific antibodies do not always localize on tumors and should therefore be characterized by imaging or biodistribution studie

The targeted delivery of IL17 to the mouse tumor neo-vasculature enhances angiogenesis but does not reduce tumor growth rate

There has been a long controversy as to whether interleukin-17 (IL17) has an impact on tumor growth. In order to assess whether IL17 may affect tumor growth, it would be convenient to achieve high levels of this pro-inflammatory cytokine at the tumor neo-vasculature, since IL17 is known to promote angiogenesis. Here, we have generated and tested in vivo a fusion protein, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis) and of murine IL17 (mIL17). The resulting immunocytokine (termed F8-mIL17) was shown to selectively localize at the tumor neo-vasculature and to vigorously promote tumor angiogenesis, without however reducing or enhancing tumor growth rate both in immunocompetent and in immunodeficient mic

CLIP and the City: Addressing the Artificial Encoding of Cities in Multimodal Foundation Deep Learning Models

In this project, we propose and explore a computational pipeline to examine urban cultural landscapes through the lens of artificial intelligence, and for questioning modes of embedding culture in machine learning models. By employing machine learning models that extract features and textual properties from images, we aim to uncover the connections between a city's history, architecture, and urban development. The city of Rome serves as a significant case study for this research. To achieve this objective, we feed 360° panoramic images into large vision-language models (e.g. OpenCLIP), to question how mainstream culture is expressed in these models. In this machine-triggered urban experiment, we investigate overlaps between history and machinic interpretation and whether relevant temporal correlations can be captured through generic street images only. Finally, by spatially analysing the captured data, we identify clusters and discontinuities in the urban layout aiming at visually depicting the interplay of forces behind its development. As in a forensic exercise, the paper seeks to uncover the complex social and historical dynamics of urban environments, exploiting only contemporary images of their settings and a generic embedding of culture. It explores potential cultural biases embedded in machine learning models by comparing Rome - culturally relevant for the western world - with other cities around the world; leveraging innovative computational pipelines and globally covering datasets to provide a novel research line for urban studies

Analisi dell'eterogeneità dinamica a tempi brevi in un sistema polimerico sottoraffreddato

Oggetto della tesi è lo studio delle proprietà di un liquido in equilibrio metastabile: tali sono i fluidi che, portati al di sotto della loro temperatura di cristallizzazione, non passano in fase cristallina (energeticamente favorita), ma permangono nella fase fluida in un minimo relativo dell'energia libera. Se ulteriormente raffreddato, un liquido nella regione metastabile risponde sempre più lentamente alle sollecitazioni esterne finché la viscosità raggiunge valori tali da rendere il sistema apparentemente solido: si parla di arresto strutturale e di transizione vetrosa. L'analisi microscopica di liquido sottoraffreddato evidenzia la presenza di strutture, dette gabbie, costituite dai primi vicini dell'unità cinetica (atomo o molecola) entro cui quest'ultima resta intrappolata. A causa di tali strutture, la dinamica dei costituenti del fluido è estremamente eterogenea: atomi (o molecole) circondati da una gabbia compatta tendono a muoversi poco, altri possono trovare delle vie di fuga da queste strutture e si muovono molto più rapidamente; questa proprietà va sotto il nome di eterogeneità dinamica. Si individua pertanto una famiglia di unità cinetiche che compiono spostamenti ben superiori alla media in un arco di tempo fissato: i monomeri mobili. Nella tesi saranno studiate le proprietà di una parte di questa famiglia, ossia dei monomeri che procedono nel moto per successione di salti, intesi come spostamenti confrontabili con le dimensioni della particella in un arco di tempo fissato tipico del regime di gabbia. Particolare attenzione sarà dedicata alla funzione densità di probabilità dei tempi di latenza (i.e. tempi che intercorrono tra due salti successivi) ed alla durata dei salti stessi. Per avere una visione più ampia, verrà analizzata la probabilità relativa al numero di salti compiuti per monomero saltante e cercheremo di comprendere se e quanto eventuali salti consecutivi (conteggiati come singoli eventi ripetuti) possano andare ad influire sulle quantità sopra citate. Inoltre verranno studiate le stringhe, ovvero strutture monodimensionamli di molecole soggette ad uno o più salti consecutivi, per capire se ed in qual misura esse possano contribuire al rilassamento strutturale del sistema

Isolation and characterization of an inhibitory human monoclonal antibody specific to the urokinase-type plasminogen activator, uPA

The serine protease urokinase (uPA, urokinase-type plasminogen activator) is over-expressed in certain tumors and is considered to be the strongest single indicator of poor prognosis in patients with metastatic breast cancer. In this article, we describe the isolation and affinity maturation of a fully human recombinant antibody (termed DS2), specific to the human uPA and capable of inhibiting its enzymatic activity with an IC50 value in the low nanomolar range. The novel antibody cross-reacts with murine uPA. It was expressed both as scFv fragment and in IgG format, allowing a systematic comparative immunofluorescence (IF) analysis of the uPA expression patterns in a large panel of human und murine tumors and of normal human tissues. Although uPA was strongly expressed in virtually all tumor specimens tested, it exhibited only a weak expression in certain normal tissues (mainly in the colon, lung, spleen and bone marrow). IgG(DS2) was not able to inhibit cancer growth in immunocompromised mice bearing subcutaneous human MDA-MB-231 or DoHH-2 tumors. However, an ex vivo IF analysis confirmed the ability of the DS2 antibody to preferentially localize at the tumor site compared with normal organs. Collectively, these data suggest that uPA blocking antibodies may not be indicated for cancer growth inhibition strategies, but may serve as valuable tools for the implementation of pharmacodelivery strategies against a variety of different tumor

Tumour-targeting properties of antibodies specific to MMP-1A, MMP-2 and MMP-3

Purpose: Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are upregulated in many diseases, but several attempts to use radiolabelled MMP inhibitors for imaging tumours have proved unsuccessful in mouse models, possibly due to the limited specificity of these agents or their unfavourable pharmacokinetic profiles. In principle, radiolabelled monoclonal antibodies could be considered for the selective targeting and imaging of individual MMPs. Methods: We cloned, produced and characterized high-affinity monoclonal antibodies specific to murine MMP-1A, MMP-2 and MMP-3 in SIP (small immunoprotein) miniantibody format using biochemical and immunochemical methods. We also performed comparative biodistribution analysis of their tumour-targeting properties at three time points (3h, 24h, 48h) in mice bearing subcutaneous F9 tumours using radioiodinated protein preparations. The clinical stage L19 antibody, specific to the alternatively spliced EDB domain of fibronectin, was used as reference tumour-targeting agent for in vivo studies. Results: All anti-MMP antibodies and SIP(L19) strongly stained sections of F9 tumours when assessed by immunofluorescence methods. In biodistribution experiments, SIP(SP3), specific to MMP-3, selectively accumulated at the tumour site 24 and 48h after intravenous injection, but was rapidly cleared from other organs. By contrast, SIP(SP1) and SIP(SP2), specific to MMP-1A and MMP-2, showed no preferential accumulation at the tumour site. Conclusion: Antibodies specific to MMP-3 may serve as vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to sites of diseas

The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model

BACKGROUND Endometriosis is still a highly underdiagnosed disease, and the current medical and surgical treatment of endometriosis is associated with a high recurrence rate. This study investigates the use of derivatives of the human antibody F8, specific to the alternatively spliced extra-domain A of fibronectin (Fn), for the imaging and treatment of endometriosis. METHODS Immunohistochemistry and immunofluorescence was used to evaluate antigen expression in endometriotic tissue of human endometriosis and of a syngeneic mouse model of the disease. The in vivo targeting performance of a fluorescent derivative of the F8 antibody was assessed by imaging mice with endometriosis using a near-infrared fluorescence imager, 24 h following i.v. injection of the antibody conjugate. Furthermore, the mouse model was used for therapy experiments using two recombinant F8-based immunocytokines [F8-interleukin-10 (IL10) and F8-IL2] or saline for the treatment groups. RESULTS A very strong vascular expression of splice isoforms of Fn and of tenascin-C was observed in human endometriotic lesions by immunohistochemistry and immunofluorescence techniques. After i.v. administration, a selective accumulation of the F8 antibody in endometriotic lesions could be observed in a syngeneic mouse model. These targeting data were used as a basis for therapy experiments with a pro-inflammatory (F8-IL2) and an anti-inflammatory (F8-IL10) cytokine fusion protein of the F8 antibody. The average lesion size in the F8-IL10 treatment group was clearly reduced compared with the saline control group and with the F8-IL2 group, for which no therapeutic effects were observed. CONCLUSIONS The F8 antibody targets endometriotic lesions in vivo in a mouse model of endometriosis and may be used for the non-invasive imaging of the disease and for the pharmacodelivery of anti-inflammatory cytokines, such as IL1