Targeted delivery of neutralizing anti-C5 antibody to renal endothelium prevents complement- dependent tissue damage

Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney

Targeting CD34(+) cells of the inflamed synovial endothelium by guided nanoparticles for the treatment of rheumatoid arthritis

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34 + endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34 + endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation

Phage Display Technology for Human Monoclonal Antibodies

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection

Prevention of arthritis by locally synthesized recombinant antibody neutralizing complement component C5.

Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production

Spleen Stiffness Probability Index (SSPI): A simple and accurate method to detect esophageal varices in patients with compensated liver cirrhosis.

Abstract Introduction and objectives Recent findings pointed out that even low-risk esophageal varices (EVs) are markers of severe prognosis. Accordingly, we analyzed spleen stiffness (SS) as a non-invasive method to predict EVs of any grade in a cohort of patients with compensated liver cirrhosis. Method We measured SS and liver stiffness (LS) using point-Shear-Wave Elastography (pSWE) with Philips Affiniti 70 system in 210 cirrhotic patients who had undergone endoscopic screening for EVs. We compared SS and LS predictive capability for EVs of any grade. Results SS was higher in cirrhotic patients with EVs if compared to patients without EVs (p Conclusion SS showed significantly higher performance than other parameters, proving to be the best non-invasive test in the screening of EVs: by directly applying SS cut-off of 31 kPa, our department could have safely avoided endoscopy in 36% of patients. Despite cut-off analyses, it was possible to create a probability model that could further stratify low-risk from high-risk patients (for any grade of EVs)

Adverse reactions of direct-acting antiviral agents in HCV patients: Our experience

Background and Aims: Direct-acting Antiviral Agents (DAAs) have shown a high rate of Sustained Virologic Response (SVR) in the treatment of hepatitis C, with few and mild adverse reactions. The aim of this study was to evaluate: (1) The efficacy of DAAs; (2) The incidence of adverse reactions; (3) The interactions with other drugs in our centre. Method: We performed an observational study of all HCV patients treated with DAAs in our centre from 01/01/2015 to 01/08/2017. We recorded: patient demographic information, grade of fibrosis by point-ShearWave Elastography on Philips IU22 before and 12 months after treatment, HCV genotype, DAAs adverse reactions and therapeutic scheme, and concomitant drugs. Results: We treated 142 HCV consecutive patients with second generation DAAs. We obtained a Sustained Virologic Response at 12 weeks (SVR12) in 93.5% of patients, 9 failures and 3 treatment interruptions due to adverse effects. Only 3 patients did not showany adverse reaction. In almost half of the cases fatigue and a reduction of at least 2 g/dL of haemoglobin levels were observed. Another frequent adverse effect was hyperglycemia (31.7%), observed in around 70% of the patients with type 2 diabetes treated with insulin or oral hypoglicemic agents and only in 25% of the non-diabetic patients. Hyperglycemia was more frequent in patients taking Daclatasvir + Sofosbuvir (45.7% vs 31.7% \u2013 p < 0.001) without a correlation with a specific antidiabetic drugs. Moreover, higher degrees of fibrosis were associated with a higher number of adverse reactions (p < 0.001). Patients who take Ribavirin display a high rate of anaemia also present with only DAA, even if less severe (67% vs 28% \u2013 p < 0.001). Conclusion: In this real life study, DAAs confirmed the excellent therapeutic success already known in literature. However,we found a higher rate of adverse effects especially in more advanced liver disease. In particular we have noticed a relevant rate of hyperglycemia, an adverse effect that has not been reported in literature and that appears to be related to the drug\u2019s class or the hepatic effect of HCV clearance rather than drug-drug interactions