Biological Biomarkers of Response and Resistance to Immune Checkpoint Inhibitors in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) represents around 2% of cancer-related deaths worldwide per year. RCC is an immunogenic malignancy, and treatment of metastatic RCC (mRCC) has greatly improved since the advent of the new immunotherapy agents, including immune checkpoint inhibitors (ICIs). However, it should be stressed that a large proportion of patients does not respond to these therapies. There is thus an urgent need to identify predictive biomarkers of efficacy or resistance associated with ICIs or ICI/Tyrosine kinase inhibitor (TKI) combinations; this is a major challenge to achieve precision medicine for mRCC in routine practice. To identify potential biomarkers, it is necessary to improve our knowledge on the biology of immune checkpoints. A lot of efforts have been made over the last decade in the field of immuno-oncology. We summarize here the main data obtained in this field when considering mRCC. As for clinical biomarkers, clinician and scientific experts of the domain are facing difficulties in identifying such molecular entities, probably due to the complexity of immuno-oncology and the constant adaptation of tumor cells to their changing environment.

Complete Response in Metastatic Clear Cell Renal Cell Carcinoma Patients Treated with Immune-Checkpoint Inhibitors: Remission or Healing? How to Improve Patients' Outcomes?

Renal-cell carcinoma (RCC) accounts for 2% of cancer diagnoses and deaths worldwide. Clear-cell RCCs represent the vast majority (85%) of kidney cancers and are considered morphologically and genetically as immunogenic tumors. Indeed, the RCC tumoral microenvironment comprises T cells and myeloid cells in an immunosuppressive state, providing an opportunity to restore their activity through immunotherapy. Standard first-line systemic treatment for metastatic patients includes immune-checkpoint inhibitors (ICIs) targeting PD1, in combination with either another ICI or with antiangiogenic targeted therapy. During the past few years, several combinations have been approved with an overall survival benefit and overall response rate that depend on the combination. Interestingly, some patients achieve prolonged complete responses, raising the question of whether these metastatic RCC patients can be cured. This review will focus on recent therapeutic advances in RCC and the clinical and biological aspects underpinning the potential for healing.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

New insights into the mechanism of photodegradation of chitosan.

Chitosan films were subjected to accelerated artificial weathering at λ>300 nm and 60 °C in the presence of O2. The resulting variations in the chemical structure were characterized by IR spectroscopy and UV-vis spectroscopy, and a photooxidation mechanism was proposed based on the identified oxidation photoproducts. The formation of gluconolactone derivatives leading to chain scissions was shown. In addition, low molecular weight photoproducts, which accounted for chitosan deacetylation, were detected. Furthermore, crosslinking reactions occurred, as revealed by gel fraction characterization. Variations in the mechanical and surface properties were characterized by AFM, and the reduction in macroscopic properties was correlated with the structural changes observed at the molecular scale by a multiscale approach.

Camelid-derived single-chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications.

Hemostasis is a complex process involving the concerted action of molecular and vascular components. Its basic understanding as well as diagnostic and therapeutic aspects have greatly benefited from the use of monoclonal antibodies. Interestingly, camelid-derived single-domain antibodies (sdAbs), also known as VHH or nanobodies, have become available during the previous 2 decades as alternative tools in this regard. Compared to classic antibodies, sdAbs are easier to produce and their small size facilitates their engineering and functionalization. It is not surprising, therefore, that sdAbs are increasingly used in hemostasis-related research. In addition, they have the capacity to recognize unique epitopes unavailable to full monoclonal antibodies. This property can be used to develop novel diagnostic tests identifying conformational variants of hemostatic proteins. Examples include sdAbs that bind active but not globular von Willebrand factor or free factor VIIa but not tissue factor-bound factor VIIa. Finally, sdAbs have a high therapeutic potential, exemplified by caplacizumab, a homodimeric sdAb targeting von Willebrand factor that is approved for the treatment of thrombotic thrombocytopenic purpura. In this review, the various applications of sdAbs in thrombosis and hemostasis-related research, diagnostics, and therapeutic strategies will be discussed.

Is virtual reality effective to teach prevention of surgical site infections in the operating room? study protocol for a randomised controlled multicentre trial entitled VIP Room study.

INTRODUCTION: Some surgical site infections (SSI) could be prevented by following adequate infection prevention and control (IPC) measures. Poor compliance with IPC measures often occurs due to knowledge gaps and insufficient education of healthcare professionals. The education and training of SSI preventive measures does not usually take place in the operating room (OR), due to safety, and organisational and logistic issues. The proposed study aims to compare virtual reality (VR) as a tool for medical students to learn the SSI prevention measures and adequate behaviours (eg, limit movements…) in the OR, to conventional teaching. METHODS AND ANALYSIS: This protocol describes a randomised controlled multicentre trial comparing an educational intervention based on VR simulation to routine education. This multicentre study will be performed in three universities: Grenoble Alpes University (France), Imperial College London (UK) and University of Heidelberg (Germany). Third-year medical students of each university will be randomised in two groups. The students randomised in the intervention group will follow VR teaching. The students randomised in the control group will follow a conventional education programme. Primary outcome will be the difference between scores obtained at the IPC exam at the end of the year between the two groups. The written exam will be the same in the three countries. Secondary outcomes will be satisfaction and students' progression for the VR group. The data will be analysed with intention-to-treat and per protocol. ETHICS AND DISSEMINATION: This study has been approved by the Medical Education Ethics Committee of the London Imperial College (MEEC1920-172), by the Ethical Committee for the Research of Grenoble Alpes University (CER Grenoble Alpes-Avis-2019-099-24-2) and by the Ethics Committee of the Medical Faculty of Heidelberg University (S-765/2019). Results will be published in peer-reviewed medical journals, communicated to participants, general public and all relevant stakeholders.

In Vitro Characterization and Stability Profiles of Antibody-Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation.

Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody-fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N-hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.

Cleavage of anti-PF4/heparin IgG by a bacterial protease and potential benefit in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor-coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.

[Therapeutic antibodies in hemostasis. From the past to the future]. / Les anticorps thérapeutiques en hémostase - D'hier, d'aujourd'hui et de demain .

TITLE: Les anticorps thérapeutiques en hémostase - D'hier, d'aujourd'hui et de demain…. ABSTRACT: L'hémostase est un processus complexe qui implique de nombreux acteurs cellulaires et moléculaires. En pathologie, les thromboses d'une part, et les pathologies hémorragiques constitutionnelles dominées par l'hémophilie d'autre part, ont bénéficié ces dernières années du développement d'anticorps thérapeutiques qui révolutionnent aujourd'hui la prise en charge des malades.

Bacteriological screening of breast milk samples destined to direct milk donation: Prospective evaluation between 2007 and 2016.

This study analyzes the bacteriological quality of breast milk samples destined to direct milk donation to preterm infants under 34 Gestational Weeks (GW) hospitalized in a neonatology and a neonatal intensive care unit of a French university hospital. All samples of breast milk destined to direct milk donation between April 2007 and December 2016 were included. A sample was defined as compliant if its total flora was less than 106 Colony Forming Units per milliliter (CFU/mL) and in the absence of Staphylococcus aureus and other pathogens. A total of 777 samples were taken from 629 mothers. The overall non-compliance rate for the initial sample was 21.3%; 63 samples (10.0%) had a total flora ≥ 106 CFU/mL, 63 (10.0%) were contaminated by a pathogenic bacteria and 8 (1.3%) were non-compliant because of both. An increase of the non-compliance rate was observed between 2008 and 2016 (10.2%-26.1%). The increase of the total flora non-compliance rate began in 2011, in link with the doubling of the number of samples taken, to reach a peak in 2013 then decreased in link with development of portable pump. No statistically significant difference of the presence of S. aureus in breast milk was observed. For the other pathogenic bacteria, the rate increased significantly in 2014. The increase of the non-compliance rate could be explained by a decrease of best practices in milk collection. Education of mothers should be strengthened.

Incidence of paradoxical reactions in patients treated with tocilizumab for rheumatoid arthritis: Data from the French registry REGATE.

OBJECTIVES: Assess the frequency of paradoxical reactions encountered in daily practice under tocilizumab, using the REGATE (Registry-RoActemra) registry. The secondary objectives were to determine the type of paradoxical reaction and the consequences of these reactions. METHODS: The REGATE registry is an independent prospective registry, promoted by the French Society of Rheumatology, consisting of patients treated with tocilizumab for rheumatoid arthritis. The paradoxical reaction was retained if it was a paradoxical precipitation of a condition for which tocilizumab was indicated, if tocilizumab was being used for an alternative indication, and if it appeared after at least one tocilizumab infusion. RESULTS: Among the 1491 patients included with at least one follow-up visit (3429 patient-years), a paradoxical reaction occurred in 9 patients (0.60% of patients; 2.62/1000 patient-years). These were 7 de novo pathologies (3 vasculitis, 3 uveitis, 1 lupus) and 2 exacerbations of pre-existing conditions (1 vasculitis, 1 lupus). Permanent discontinuation of tocilizumab was chosen for 5 patients. CONCLUSIONS: In the REGATE registry, the occurrence of paradoxical reactions in patients treated with tocilizumab was rare.

Antibody-drug conjugates: Design and development for therapy and imaging in and beyond cancer, LabEx MAbImprove industrial workshop, July 27-28, 2017, Tours, France.

The annual "Antibody Industrial Symposium", co organized by LabEx MAbImprove, MabDesign and Polepharma, was held in Tours, France on June 27-28, 2017. The focus was on antibody-drug-conjugates (ADCs), new entities which realize the hope of Paul Ehrlich's magic bullet. ADCs result from the bioconjugation of a highly cytotoxic drug to a selective monoclonal antibody, which acts as a vector. Building on knowledge gained during the development of three approved ADCs, brentuximab vedotin (Adcetris®), ado trastuzumab emtansine (Kadcyla®) and inotuzumab ozogamicin (Besponsa®), and the many ADCs in development, this meeting addressed strategies and the latest innovations in the field from fundamental research to manufacturing.

Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparin-induced thrombocytopenia antibodies.

The laboratory diagnosis of heparin-induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet-activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRApos ), with relatively high levels of PF4-specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 µg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4-SRApos ). Importantly, levels of PF4-specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre-test probability of HIT was intermediate/high in all 'SRApos ' or 'SRA-PF4pos ' patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA.

Monitoring Cerebral and Renal Oxygenation Status during Neonatal Digestive Surgeries Using Near Infrared Spectroscopy.

BACKGROUND: Depending on the initial pathology, hypovolemia, intra-abdominal hypertension, and sepsis are often encountered in neonatal digestive surgery. Accurate newborn monitoring during and after surgery is essential to adapt resuscitation protocols. Near infrared spectroscopy (NIRS) is non-invasive and can detect hypoperfusion which indicates a low circulatory blood flow, regardless of the cause. OBJECTIVE: Evaluating changes in cerebral and renal regional oxygen saturation during neonatal digestive surgeries, conducted according to normal practices, with commonly used monitoring parameters. Analyzing retrospectively the inter-relationships between NIRS values and mean arterial pressure (MAP) values as well as pre-ductal SpO2. METHODS: Prospective, descriptive, monocentric study. All neonates referred for surgery were included. NIRS allows the measurement of cerebral and renal oxygenation fluctuations, as well as calculating difference in intraoperative and postoperative values. RESULTS: Nineteen patients were included. Cerebral regional oxygen saturation (C rSO2) values were stable while renal regional oxygen saturation (R rSO2) values tended to decrease with time during surgery. Indeed, 72% of rSO2 decline episodes occurred after the first 30 min of surgery, without any significant statistical differences for the next 90 min of surgery. After surgery, the lowest average C and R rSO2 values were evidenced during the first 6 h, with 60% of C rSO2 and R rSO2 anomalies occurring in that time frame. There was no significant statistical difference observed in the following 18 h. There was a significant correlation between R rSO2 and SpO2 values (p < 0.01), but not with C rSO2 values. There was no correlation with the MAP either for the C rSO2 values or R rSO2 ones. CONCLUSION: NIRS is a promising non-invasive bedside tool to monitor cerebral and tissue perfusion, analyzing tissue microcirculation. NIRS has its interest to guide neonatal digestive surgeries (bowel manipulation, viscera reduction) and may represent an early warning for identifying patients requiring resuscitation during or after these surgeries.

Benefits of Rapid Molecular Diagnosis of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections in Women Attending Family Planning Clinics.

We evaluated the benefits of on-demand systematic screening for Chlamydia trachomatis and Neisseria gonorrhoeae using the Xpert CT/NG assay in 589 women attending family planning clinics. The sexually transmitted infection prevalence was 16.5% with 15.1% C. trachomatis and 3.1% N. gonorrhoeae infections. The on-demand test allowed for a quicker management of patients at high risk for sexually transmitted infections.