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Introduction and objectives: Carbohydrate antigen 125 (CA125) has been shown to be useful for risk stratification in patients admitted with acute heart failure (AHF). We sought to determine a CA125 cutpoint for identifying patients at low risk of 1-month death or the composite of death/HF readmission following admission for AHF.Methods: The derivation cohort included 3231 consecutive patients with AHF. CA125 cutoff values with 90% negative predictive value (NPV) and sensitivity up to 85% were identified. The adequacy of these cutpoints and the risk of 1-month death/HF readmission was then tested using the Royston-Parmar method. The best cutpoint was selected and externally validated in a cohort of patients hospitalized from BIOSTAT-CHF (n = 1583).Result: In the derivation cohort, the median [IQR] CA125 was 57 [25.3-157] U/mL. The optimal cutoff value was < 23 U/mL (21.5% of patients), with NPVs of 99.3% and 94.1% for death and the composite endpoint, respectively. On multivariate survival analyses, CA125 < 23 U/mL was independently associated with a lower risk of death (HR, 0.20; 95%Cl, 0.08-0.50; P < .001), and the combined endpoint (HR, 0.63; 95%Cl, 950.45-0.90; P = .009). The ability of this cutpoint to discriminate patients at a low 1-month risk was confirmed in the validation cohort (NPVs of 98.6% and 96.6% for death and the composite endpoint). The predicted ability of this cutoff remained significant at 6 months of follow-up.Conclusion: In patients admitted with AHF, CA125 < 23 U/mL identified a subgroup at low risk of short-term adverse events, a population that may not require intense postdischarge monitoring. (C) 2021 Sociedad Espanola de Cardiologia. Published by Elsevier Espafia, S.L.U. All rights reserved.

Biologic Therapies for Severe Asthma Patients with severe asthma are at increased risk for a decreased quality of life, fixed airway obstruction, hospitalization, and death. Biologics may be required to reduce the disease burden. This review discusses the mechanisms, efficacy, and safety of biologics for severe asthma.

Purpose The aim of the present paper is to review the role of HER2 antibodies, affibodies and nanobodies as vehicles for imaging and therapy approaches in breast cancer, including a detailed look at recent clinical data from antibody drug conjugates and nanobodies as well as affibodies that are currently under development. Results Clinical and preclinical studies have shown that the use of monoclonal antibodies in molecular imaging is impaired by slow blood clearance, associated with slow and low tumor uptake and with limited tumor penetration potential. Antibody fragments, such as nanobodies, on the other hand, can be radiolabelled with short-lived radioisotopes and provide high-contrast images within a few hours after injection, allowing early diagnosis and reduced radiation exposure of patients. Even in therapy, the small radioactively labeled nanobodies prove to be superior to radioactively labeled monoclonal antibodies due to their higher specificity and their ability to penetrate the tumor. Conclusion While monoclonal antibodies are well established drug delivery vehicles, the current literature on molecular imaging supports the notion that antibody fragments, such as affibodies or nanobodies, might be superior in this approach.

Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry into classification of leukemia and lymphoma. In our opinion, this now enables introduction of markers for more dynamic parameters, such as proliferative and (anti)apoptotic markers, which have proven their value in the field of histopathology in the diagnostic process of solid tumors and lymphoma. Although use of proliferative and (anti)apoptotic markers as objective parameters in the diagnostic process of myeloid malignancies was studied in the past decades, this did not result in the incorporation of these biomarkers into clinical diagnosis. This review addresses the potential of these markers for implementation in the current, state-of-the-art multiparameter analysis of myeloid malignancies. The reviewed studies clearly recognize the importance of proliferation and apoptotic mechanisms in the pathogenesis of bone marrow (BM) malignancies. The literature is, however, contradictory on the role of these processes in myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms, and acute myeloid leukemia. Furthermore, several studies underline the need for the analysis of the proliferative and apoptotic rates in subsets of hematopoietic BM cell lineages and argue that these results can have diagnostic and prognostic value in patients with myeloid malignancies. Recent developments in multiparameter flow cytometry now allow quantification of proliferative and (anti)apoptotic indicators in myeloid cells during their different maturation stages of separate hematopoietic cell lineages. This will lead to a better understanding of the biology and pathogenesis of these malignancies.

Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry into classification of leukemia and lymphoma. In our opinion, this now enables introduction of markers for more dynamic parameters, such as proliferative and (anti)apoptotic markers, which have proven their value in the field of histopathology in the diagnostic process of solid tumors and lymphoma. Although use of proliferative and (anti)apoptotic markers as objective parameters in the diagnostic process of myeloid malignancies was studied in the past decades, this did not result in the incorporation of these biomarkers into clinical diagnosis. This review addresses the potential of these markers for implementation in the current, state-of-the-art multiparameter analysis of myeloid malignancies. The reviewed studies clearly recognize the importance of proliferation and apoptotic mechanisms in the pathogenesis of bone marrow (BM) malignancies. The literature is, however, contradictory on the role of these processes in myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms, and acute myeloid leukemia. Furthermore, several studies underline the need for the analysis of the proliferative and apoptotic rates in subsets of hematopoietic BM cell lineages and argue that these results can have diagnostic and prognostic value in patients with myeloid malignancies. Recent developments in multiparameter flow cytometry now allow quantification of proliferative and (anti)apoptotic indicators in myeloid cells during their different maturation stages of separate hematopoietic cell lineages. This will lead to a better understanding of the biology and pathogenesis of these malignancies.

Advanced Breast Cancer; Efficacy; Margetuximab Cáncer de mama avanzado; Eficacia; Margetuximab Càncer de mama avançat; Eficàcia; Margetuximab Importance ERRB2 (formerly HER2)–positive advanced breast cancer (ABC) remains typically incurable with optimal treatment undefined in later lines of therapy. The chimeric antibody margetuximab shares ERBB2 specificity with trastuzumab but incorporates an engineered Fc region to increase immune activation. Objective To compare the clinical efficacy of margetuximab vs trastuzumab, each with chemotherapy, in patients with pretreated ERBB2-positive ABC. Design, Setting, and Participants The SOPHIA phase 3 randomized open-label trial of margetuximab plus chemotherapy vs trastuzumab plus chemotherapy enrolled 536 patients from August 26, 2015, to October 10, 2018, at 166 sites in 17 countries. Eligible patients had disease progression on 2 or more prior anti-ERBB2 therapies and 1 to 3 lines of therapy for metastatic disease. Data were analyzed from February 2019 to October 2019. Interventions Investigators selected chemotherapy before 1:1 randomization to margetuximab, 15 mg/kg, or trastuzumab, 6 mg/kg (loading dose, 8 mg/kg), each in 3-week cycles. Stratification factors were metastatic sites (≤2, >2), lines of therapy (≤2, >2), and chemotherapy choice. Main Outcomes and Measures Sequential primary end points were progression-free survival (PFS) by central blinded analysis and overall survival (OS). All α was allocated to PFS, followed by OS. Secondary end points were investigator-assessed PFS and objective response rate by central blinded analysis. Results A total of 536 patients were randomized to receive margetuximab (n = 266) or trastuzumab (n = 270). The median age was 56 (27-86) years; 266 (100%) women were in the margetuximab group, while 267 (98.9%) women were in the trastuzumab group. Groups were balanced. All but 1 patient had received prior pertuzumab, and 489 (91.2%) had received prior ado-trastuzumab emtansine. Margetuximab improved primary PFS over trastuzumab with 24% relative risk reduction (hazard ratio [HR], 0.76; 95% CI, 0.59-0.98; P = .03; median, 5.8 [95% CI, 5.5-7.0] months vs 4.9 [95% CI, 4.2-5.6] months; October 10, 2018). After the second planned interim analysis of 270 deaths, median OS was 21.6 months with margetuximab vs 19.8 months with trastuzumab (HR, 0.89; 95% CI, 0.69-1.13; P = .33; September 10, 2019), and investigator-assessed PFS showed 29% relative risk reduction favoring margetuximab (HR, 0.71; 95% CI, 0.58-0.86; P

Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates., Probing the energy landscape and thermodynamics of biomolecules for drug design.

European journal of nuclear medicine and molecular imaging 48(5), 1371-1389 (2021). doi:10.1007/s00259-020-05094-1, Published by Springer-Verl., Heidelberg [u.a.]

Background Risankizumab is a humanized IgG monoclonal antibody that selectively inhibits interleukin-23 through binding the p19 subunit. In Phase 3 trials, risankizumab demonstrated superior efficacy compared with adalimumab and ustekinumab in patients with moderate-to-severe plaque psoriasis. Here, we evaluated the impact of baseline characteristics on efficacy of risankizumab compared with ustekinumab in patients with moderate-to-severe plaque psoriasis. Methods This analysis included all patients initially randomized to risankizumab or ustekinumab from the replicate, double-blinded, randomized, placebo-controlled phase 3 trials, UltIMMa-1 (NCT02684370) and UltIMMa-2 (NCT02684357). Patients received either risankizumab (150 mg) or ustekinumab (weight-based; 45 or 90 mg per label) at weeks 0, 4, 16, 28 and 40. Efficacy was assessed as the proportion of patients achieving >= 90% improvement in Psoriasis Area and Severity Index (PASI 90) at weeks 16 and 52 by baseline patient demographics, disease characteristics and prior biologic exposure. Mean per cent improvement in PASI was calculated by body weight and body mass index at week 52. Missing efficacy data were imputed as non-responders for categorical variables and last observation carried forward for continuous variables. Logistic regression analyses assessed for interactions between treatment and five independent variables (age, sex, weight, baseline PASI score and presence of psoriatic arthritis) at both weeks 16 and 52. Results Baseline patient demographics, disease characteristics and prior biologic exposure were similar between patients randomized to risankizumab (n = 598) and ustekinumab (n = 199). At weeks 16 and 52, risankizumab demonstrated superior efficacy compared with ustekinumab across these patient characteristics (P < 0.01). Logistic regression analyses demonstrated that risankizumab was superior to ustekinumab at weeks 16 and 52 in all models tested (P < 0.0001 for all). Conclusions Risankizumab demonstrated consistent and superior efficacy compared with ustekinumab regardless of patient demographics, disease characteristics or prior biologic exposure.

Mature lymphoproliferative diseases are a heterogeneous group of neoplasms arising from different stages of B-cell and T-cell development. With improved understanding of the molecular processes in lymphoma and novel treatment options, arises a growing need for the molecular characterisation of tumours. Molecular imaging with single-photon-emission CT and PET using specific radionuclide tracers can provide whole-body information to investigate cancer biology, to evaluate phenotypic heterogeneity, to identify resistance to targeted therapy, and to assess the biodistribution of drugs in patients. In this Review, we evaluate the existing literature on molecular imaging in lymphoma, other than 18F-fluordeoxyglucose molecular imaging. The aim is to examine the contribution of molecular imaging to the understanding of the biology of lymphoma and to discuss potential implications for the diagnostics and therapy of this disease. Finally, we discuss possible applications for molecular imaging of patients with lymphoma in the clinical context.

Background: CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods: The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results: High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion: Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC.

Copyright © 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND)., Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A Neisseria meningitidis (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be O-acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the O- and N-acetyl moieties through either H-bonding or CH–π interactions. In silico docking showed that 3-O-acetylation of the upstream residue is essential for antibody binding, while O-acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies., This work was sponsored by GlaxoSmithKline Biologicals and has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement 675671.

BackgroundC reactive protein (CRP) levels are suggested as serum biomarkers in the diagnosis and prognosis of psoriatic arthritis (PsA). However, increased CRP levels are found in less than 50% of PsA patients even in the presence of active disease.ObjectivesTo evaluate the role of CRP levels in interventional clinical trials in PsA patients to better understand the trial generalisability, relationship with disease activity and predictive value for treatment response and decision making.MethodsA systematic review was conducted via PubMed, Cochrane and Embase. We focused on phase III trials in PsA.ResultsEight of 22 studies applied minimum baseline CRP levels for inclusion. Baseline CRP levels were wide-ranging (0.1–238 mg/L) and lower in studies without CRP in the enrolment criteria. All 22 studies used the American College of Rheumatology (ACR20) response and other endpoints that integrated CRP levels. One of seven studies that evaluated individual ACR-score components revealed a decrease in CRP levels along with improvement of other endpoints. Subanalyses show conflicting evidence on CRP levels as predictor of disease course.ConclusionCRP levels were inconsistently used as inclusion criterion in clinical trials, often limiting generalisability of the data. The use of composite scores such as ACR20 or Disease Activity Score-28-CRP is also limited since baseline levels of CRP affects their sensitivity to change. High CRP levels may be an individual predictor for disease progression and response to treatment, but the current conflicting findings and selective patient trial inclusions, do not allow CRP to play a very prominent role in treatment decision making.

Objective To evaluate the effect of ixekizumab on self-reported functioning and health in patients with active nonradiographic axial spondyloarthritis (SpA). Methods COAST-X was a randomized, controlled trial conducted in patients with nonradiographic axial SpA over 52 weeks. Participants were randomized at a ratio of 1:1:1 to receive 80 mg of ixekizumab subcutaneously every 4 weeks or 2 weeks or placebo for 52 weeks. Self-reported functioning and health end points included the Medical Outcomes Study Short Form 36 (SF-36) health survey, Assessment of Spondyloarthritis International Society (ASAS) health index, and European Quality of Life-5 Dimensions-5 Level (EQ-5D-5L) health-utility descriptive system. Results Compared to placebo, ixekizumab treatment resulted in improvement of SF-36 physical component summary scores from baseline, with a score of 4.7 improving to 8.9 with ixekizumab therapy every 4 weeks (P < 0.05) and a score of 9.3 with ixekizumab therapy every 2 weeks (P < 0.01); the greatest improvements were observed in the domains of physical functioning, role-physical, and bodily pain at weeks 16 and 52. A higher proportion of patients receiving ixekizumab therapy every 2 weeks reported >= 3 improvements based on the ASAS health index from baseline to weeks 16 and 52 (P < 0.05). Significantly more patients receiving ixekizumab every 4 weeks reported improvements in "good health status" on the ASAS health index (ASAS score of

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of

Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated beta-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.

Introduction: The acquisition of humoral immunity in utero is essential for the fetus. The crucial protein, which is responsible for this part of immunity, is immunoglobulin-G (IgG). Immune functions of IgGs are mediated via the interaction of the crystallizable fragment (Fc) region of IgG with specific Fc gamma receptors (Fc gamma Rs). However, an atypical Fc gamma R, the neonatal Fc receptor (FcRn), is a key regulator of IgG transfer across the human placenta. During the last four decades ex vivo placental perfusion studies have contributed significantly to the study of mechanisms of IgG transfer across the multicellular placental barrier. Method: A PubMed search was conducted by using specific keywords: placenta, perfusion and IgG to review manuscripts using human placental perfusion to study the transplacental transfer of IgG. Relevant studies found in reference lists of these manuscripts were also added to the review, and references were included that supported or gave nuance to the discussion of the mechanisms of IgG kinetics in the placenta. Results and Discussion: We found twenty publications on the study of transplacental transfer of IgG using human ex vivo placental perfusion, by research groups with partly different settings. This review summarizes knowledge about placental IgG transfer, with a strong focus on the contributions from ex vivo placental perfusion studies.Superscript/Subscript Available

RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3−/− mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3−/− mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3−/− vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3−/− mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3−/− mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3−/− mice. Likewise following IAV infection of Ripk3−/− mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3−/− mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3−/− mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors.

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Tumour-associated macrophages (TAMs) are essential players in the tumour microenvironment (TME) and modulate various pro-tumorigenic functions such as immunosuppression, angiogenesis, cancer cell proliferation, invasion and metastasis, along with resistance to anti-cancer therapies. TAMs also mediate important anti-tumour functions and can clear dying cancer cells via efferocytosis. Thus, not surprisingly, TAMs exhibit heterogeneous activities and functional plasticity depending on the type and context of cancer cell death that they are faced with. This ultimately governs both the pro-tumorigenic and anti-tumorigenic activity of TAMs, making the interface between TAMs and dying cancer cells very important for modulating cancer growth and the efficacy of chemo-radiotherapy or immunotherapy. In this review, we discuss the interface of TAMs with cancer cell death from the perspectives of cell death pathways, TME-driven variations, TAM heterogeneity and cell-death-inducing anti-cancer therapies. We believe that a better understanding of how dying cancer cells influence TAMs can lead to improved combinatorial anti-cancer therapies, especially in combination with TAM-targeting immunotherapies. ispartof: CELLS vol:11 issue:23 ispartof: location:Switzerland status: published

Cancer cells exploit CD47 overexpression to inhibit phagocytic elimination and neoantigen processing via the myeloid CD47-SIRP alpha axis and thereby indirectly evade adaptive T cell immunity. Here, we report on a hitherto unrecognized direct immunoinhibitory feature of cancer cell-expressed CD47. We uncovered that in response to IFN gamma released during cognate T cell immune attack, cancer cells dynamically enhance CD47 cell surface expression, which coincides with acquiring adaptive immune resistance toward pro-apoptotic effector T cell mechanisms. Indeed, CRISPR/Cas9-mediated CD47-knockout rendered cancer cells more sensitive to cognate T cell immune attack. Subsequently, we developed a cancer-directed strategy to selectively overcome CD47-mediated adaptive immune resistance using bispecific antibody (bsAb) CD47xEGFR-IgG2s that was engineered to induce rapid and prolonged cancer cell surface displacement of CD47 by internalization. Treatment of CD47(pos) cancer cells with bsAb CD47xEGFR-IgG2s potently enhanced susceptibility to cognate CD8(pos) T cells. Targeting CD47-mediated adaptive immune resistance may open up new avenues in cancer immunotherapy.

Objectives The co-stimulatory CD40L–CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. Methods and Results Atherosclerotic plaques of apolipoprotein E-deficient (Apoe −/− ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe −/− mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. Conclusion Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

Nivolumab (Opdivo®) is a fully human immunoglobulin G4 isotype approved for the treatment of many cancers. It acts as an immune checkpoint inhibitor by blocking the interaction between PD-1 (Programmed Cell Death Protein 1) – an inhibitory receptor expressed on activated T cells- and its ligands, PD-L1 and PD-L2. The quantification of therapeutic proteins in their medicines and pharmaceutical preparations remains challenging because the protein content, a critical quality attribute, must be rigorously calculated using a validated stabilityindicating method, such as that indicated by the International Conference on Harmonization (ICH) quality guidelines, and this requires the analysis of the drug in the presence of its degraded products. In this work, we present an strategy based on the combined use of the UV and MS data to full file the requirement of the ICH-Q2 (R1) to develop and validated as stability indicated a (RP)UHPLC/UV-(HESI/Orbitrap™)MS method for the quantification of nivolumab in medicinal products. A comparative study of all figures of merit of the method using UV or MS data are shown and discussed. The results show that linearity was similar for the two detectors and was established over a range of 4–45 μg/mL and 1–45 μg/mL for the UV and (HESI/Orbitrap™)MS signals, respectively. The sensitivity of the method was higher when using the (HESI/Orbitrap™)MS signal (0.2 μg/mL) than with the UV(2.0 μg/mL). However, the UV signal provided better accuracy and precision than the (HESI/ Orbitrap™)MS signal, which did not meet the criteria for method robustness and system suitability. In spite of this, the MS signal plays a crucial role in this methodology by obtaining the molecular weight profile of the nivolumab isoforms, so enabling us to propose the glycans profile and detect structural modification due to degradation. The specificity of the method was evaluated by conducting forced degradation tests on samples of nivolumab in medicine form. The aim was to find out whether nivolumab suffers structural modifications when subject to stress. Structural modifications were detected by analysing the MS isoform profile, as changes of this kind promote new isoforms that are not chromatographically separated or detected by the UV signal. In this way, we demonstrated that the (RP)UHPLC/UV-(HESI/Orbitrap™)MS method was capable of detecting nivolumab degradation, and was suitable for use in nivolumab stability studies. Thus, the protein content in the daily surplus of the Opdivo® medicine, stored either at room temperature (20 ◦C) or refrigerated at 4 ◦C, could be tracked for 15 days., FPU18/03131 Ministry of Universities, Spain, (P20_01029) Junta de Andalucía (Spain) and European Regional Development Funds, postdoctoral position from the Junta de Andalucía, Spain, Project P20-01029 (I + D + i - Junta de Andalucía, Spain) Project B-FQM-308-UGR20 (Universidad de Granada, Proyectos I + D + i del Programa Operativo FEDER Andalucía 2020), CBUA/ Universidad de Granada

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations. ispartof: MBIO vol:12 issue:4 ispartof: location:United States status: accepted

Background: Glioblastoma is a grade IV astrocytoma with an average survival span for patients of 18 months after initial diagnosis and no standard treatment protocol is available. Therefore, there is a need to search for novel approaches to target glioblastoma. Objectives: This review intends to capture the role of immunoglobulin-M in cancer, more specifically in glioblastoma multiforme (GBM), and to compile the latest developments and immunological pathways relevant to glioblastoma. Methods: Information on glioblastoma, cancer microenvironment, cancer therapeutics, and how to improve the scenario were obtained from scientific literature databases such as Pubmed, Medline, Google Scholar, Science Direct, Springer, Wiley online library, and some data was harvested from regulatory and compliance databases such as clinicaltrials.gov, FDA database, and WHO Globocan. Results and Conclusions: Currently, only a limited number of therapies are approved for GBM, and no standard care is in place in case of disease relapse, necessitating a possible broader perspective in looking at the disease and its underlying mechanisms.

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies. ispartof: FRONTIERS IN IMMUNOLOGY vol:12 ispartof: location:Switzerland status: published

Chemotherapy; Mirvetuximab soravtansine; Ovarian cancer Quimioterapia; Mirvetuximab soravtansina; Cáncer de ovarios Quimioteràpia; Mirvetuximab soravtansina; Càncer d'ovaris Background Mirvetuximab soravtansine (MIRV) is an antibody-drug conjugate comprising a folate receptor alpha (FRα)-binding antibody, cleavable linker, and the maytansinoid DM4, a potent tubulin-targeting agent. The randomized, open-label, phase III study FORWARD I compared MIRV and investigator's choice chemotherapy in patients with platinum-resistant epithelial ovarian cancer (EOC). Patients and methods Eligible patients with 1-3 prior lines of therapy and whose tumors were positive for FRα expression were randomly assigned, in a 2 : 1 ratio, to receive MIRV (6 mg/kg, adjusted ideal body weight) or chemotherapy (paclitaxel, pegylated liposomal doxorubicin, or topotecan). The primary endpoint was progression-free survival [PFS, Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, blinded independent central review] in the intention-to-treat (ITT) population and in the prespecified FRα high population. Results A total of 366 patients were randomized; 243 received MIRV and 109 received chemotherapy. The primary endpoint, PFS, did not reach statistical significance in either the ITT [hazard ratio (HR), 0.98, P = 0.897] or the FRα high population (HR, 0.69, P = 0.049). Superior outcomes for MIRV over chemotherapy were observed in all secondary endpoints in the FRα high population including improved objective response rate (24% versus 10%), CA-125 responses (53% versus 25%), and patient-reported outcomes (27% versus 13%). Fewer treatment-related grade 3 or higher adverse events (25.1% versus 44.0%), and fewer events leading to dose reduction (19.8% versus 30.3%) and treatment discontinuation (4.5% versus 8.3%) were seen with MIRV compared with chemotherapy. Conclusions In patients with platinum-resistant EOC, MIRV did not result in a significant improvement in PFS compared with chemotherapy. Secondary endpoints consistently favored MIRV, particularly in patients with high FRα expression. MIRV showed a differentiated and more manageable safety profile than chemotherapy. This work was supported by ImmunoGen, Inc (no grant number).

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Addition of rituximab (R) to “CHOP” (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy improved outcome for diffuse large B-cell lymphoma (DLBCL) patients. Approximately 40% of patients who receive R-CHOP still succumb to disease due to intrinsic resistance or relapse. A potential negative regulator of DLBCL treatment outcome is the CD47 “don't eat me” immune checkpoint. To delineate the impact of CD47, we used a clinically and molecularly well-annotated cohort of 939 DLBCL patients, comprising both germinal center B-cell (GCB) and non-GCB DLBCL subtypes, treated with either CHOP or R-CHOP. High (above median) CD47 mRNA expression correlated with a detrimental effect on overall survival (OS) when DLBCL patients received R-CHOP therapy (P = 0.001), but not CHOP therapy (P = 0.645). Accordingly, patients with low CD47 expression benefited most from the addition of rituximab to CHOP [HR, 0.32; confidence interval (CI), 0.21–0.50; P < 0.001]. This negative impact of high CD47 expression on OS after R-CHOP treatment was only evident in cancers of non-GCB origin (HR, 2.09; CI, 1.26–3.47; P = 0.004) and not in the GCB subtype (HR, 1.16; CI, 0.68–1.99; P = 0.58). This differential impact of CD47 in non-GCB and GCB was confirmed in vitro, as macrophage-mediated phagocytosis stimulated by rituximab was augmented by CD47-blocking antibody only in non-GCB cell lines. Thus, high expression of CD47 mRNA limited the benefit of addition of rituximab to CHOP in non-GCB patients, and CD47-blockade only augmented rituximab-mediated phagocytosis in non-GCB cell lines. Patients with non-GCB DLBCL may benefit from CD47-targeted therapy in addition to rituximab.

Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks are blood-sucking parasites that secrete numerous immune-modulatory agents in their saliva to evade host immune responses. Evasin-3 is a small salivary protein that belongs to a class of chemokine-binding proteins isolated from the brown dog tick, Rhipicephalus sanguineus. Evasin-3 has been shown to have a high affinity for chemokines CXCL1 and CXCL8 and to diminish inflammation in mice. In the present study, solution NMR spectroscopy was used to investigate the structure of Evasin-3 and its CXCL8-Evasin-3 complex. Evasin-3 is found to disrupt the glycosaminoglycan-binding site of CXCL8 and inhibit the interaction of CXCL8 with CXCR2. Structural data were used to design two novel CXCL8-binding peptides. The linear tEv3 17-56 and cyclic tcEv3 16-56 dPG Evasin-3 variants were chemically synthesized by solid-phase peptide synthesis. The affinity of these newly synthesized variants to CXCL8 was measured by surface plasmon resonance biosensor analysis. The K-d values of tEv3 17-56 and tcEv3 16-56 dPG were 27 and 13 nm, respectively. Both compounds effectively inhibited CXCL8-induced migration of polymorphonuclear neutrophils. The present results suggest utility of synthetic Evasin-3 variants as scaffolds for designing and fine-tuning new chemokine-binding agents that suppress immune responses and inflammation.

Purpose of review Children with poor asthma control despite maximal maintenance therapy have problematic severe asthma (PSA). A step-wise approach including objective adherence monitoring and a detailed multidisciplinary team assessment to identify modifiable factors contributing to poor control is needed prior to considering therapy escalation. Pathophysiological phenotyping in those with true severe therapy-resistant asthma (STRA) and the current array of add-on therapies will be discussed. Recent findings Adherence monitoring using electronic devices has shown that only 20-30% of children with PSA have STRA and need additional therapies. Omalizumab and mepolizumab are licensed for children with STRA aged 6 years and older. Although robust safety and efficacy data, with reduced exacerbations, are available for omalizumab, biomarkers predicting response to treatment are lacking. Paediatric safety data are available for mepolizumab, but efficacy data are unknown for those aged 6-11 years and minimal for those 12 years and older. A sub-group of children with STRA have neutrophilia, but the clinical significance and contribution to disease severity remains uncertain. Summary Most children with PSA have steroid sensitive disease which improves with adherence to maintenance inhaled corticosteroids. Add-on therapies are only needed for the minority with STRA. Paediatric efficacy data of novel biologics and biomarkers that identify the optimal add-on for each child are lacking. If we are to progress toward individualized therapy for STRA, pragmatic clinical trials of biologics in accurately phenotyped children are needed.

This review discusses the challenges of quantitative protein bioanalysis by LC-MS at the protein level. We will notably address the possibilities and current limitations of protein sample preparation, separation by LC, the challenge of interpreting protein ESI-MS spectra and the options for protein quantification based on extracted ion chromatograms or deconvoluted spectra. The possibilities of high-resolution mass spectrometry (HRMS) with respect to improving the signal-to-noise (S/N) ratio and the challenges of analyzing complex mass spectra will be highlighted based on examples.