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2022 ◽  
Vol 3 (1) ◽  
pp. 01-05
Author(s):  
Nightingale Syabbalo

Asthma is a heterogeneous chronic airway disease comprising of distinct phenotypes characterized by different immunopathophysiologic pathways, clinical features, disease severity, and response to treatment. The phenotypes of asthma include eosinophilic, neutrophilic, mixed cellularity, and paucigranulocytic asthma. Eosinophilic asthma is principally a T helper type 2 (Th2)-mediated airway disease. However, several other immune and structural cells secrete the cytokines implicated in the pathogenesis of eosinophilic asthma. Innate type 2 lymphoid cells, mast cells, basophils, and eosinophils secrete Th2 cytokines, such as interleukin-4 (IL-4), IL-13, and IL-5. Additionally, airway epithelial cells produce alarmin cytokines, including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). Alarmins are the key initiators of allergic inflammation at the sentinel mucosal surfaces. Innovative biotherapeutic research has led to the discovery of monoclonal antibodies which target and inhibit the immunopathological effects of the cytokines involved in the pathogenesis of eosinophilic asthma. Parenteral biologics targeting the inciting interleukins, include mepolizumab and reslizumab (anti-IL-5), benralizumab (anti-IL-5Rα), dupilumab (anti-4Rα), and tezelizumab (anti-TSLP). They have been shown to significantly reduce annualized exacerbation rates, improve asthma control, lung function, and quality of life. Currently, there are no pulmonary delivered aerosol biologics for topical treatment of asthma. CSJ117 is a potent neutralizing antibody Fab fragment against TSLP, formulated as a PulmoSol TM engineered powder, and is delivered to the lungs by a dry powder inhaler. Phase 2 placebo-controlled clinical trial evaluated the efficacy and safety of CSJ117. CSJ117 delivered as an inhaler attenuated the late asthmatic response (LAR), and the early asthmatic response (EAR) after allergen inhalation challenge (AIC) at day 84 of treatment. The maximum decrease in FVE1 from pre-AIC were significantly lower in the CSJ117 group compared to placebo (P = 029), during LAR. CSJ117 also significantly reduced fractional exhaled nitric oxide before AIC at day 83; and significantly reduced the allergen-induced increase in % sputum eosinophil count. Pulmonary delivery of biologics directly to the airway mucosal surface has several advantages over parenteral routes, particularly in treating airway diseases such as asthma. Inhaler delivered biologics, such as CSJ117 are innovative and attractive methods of future precision treatment of asthma, and other respiratory diseases.


2022 ◽  
Author(s):  
Bruce Campbell ◽  
Sandra Engle ◽  
Patricia Bourassa ◽  
Robert Aiello

Pathological retention of LDL in the intima is involved in atherosclerosis, although the retention mechanisms are not well-understood. Previously, we reported Sterile Alpha Motif Domain Containing 1 (SAMD1), a protein secreted by intimal smooth muscle cells in atherosclerotic lesions, appears to bind LDL in extracellular matrix around intimal cells. Fab-fragment inhibitors of apparently irreversible SAMD1/LDL binding reduced LDL retention in carotid injury models, but did not have a significant effect on early spontaneous lesion initiation. The normal function of SAMD1 is unknown, but it may have multiple epigenetic roles; our histology of mouse atherosclerosis models revealed extensive SAMD1 expression, possibly related to cell phenotype modulation and antigen presentation. For this report, we generated SAMD1-/-, SAMD1-/+, and SAMD1-/+ apoE-/- mice to further explore SAMD1's role in atherosclerosis. SAMD1 was found in tissues throughout the SAMD1+/+ and SAMD1-/+embryos. Homozygous loss of SAMD1 was embryonic lethal: at embryonic day 14, organs were partially developed and/or degraded; heads and brains were malformed; no blood vessels were observed; red blood cells were scattered and pooled, primarily near the embryo surface; and cell death was occurring. Development appeared normal in heterozygous SAMD1 embryos, but postnatal genotyping showed a reduced ability to thrive. Growth of atherosclerotic lesions in SAMD1-/+ apoE-/- after 35 weeks was not significantly less than in mice SAMD1+/+ apoE-/- mice.


2022 ◽  
Author(s):  
Tobias V Lanz ◽  
R Camille Brewer ◽  
Peggy P Ho ◽  
Kevin M Jude ◽  
Daniel Fernandez ◽  
...  

Abstract Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal fluid (CSF) of MS patients contribute to inflammation and secrete oligoclonal immunoglobulins. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies.


2021 ◽  
Vol 9 (4) ◽  
pp. 59-62
Author(s):  
Ui Beom Park ◽  
Hyun Tae Lee ◽  
Yujin Kim ◽  
Tae Jun Jeong ◽  
Nahyun Gu ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wantanee Treewattanawong ◽  
Thassanai Sitthiyotha ◽  
Surasak Chunsrivirot

AbstractSARS-CoV-2 is responsible for COVID-19 pandemic, causing large numbers of cases and deaths. It initiates entry into human cells by binding to the peptidase domain of angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain of S1 subunit of spike protein (SARS-CoV-2-RBD). Employing neutralizing antibodies to prevent binding between SARS-CoV-2-RBD and ACE2 is an effective COVID-19 therapeutic solution. Previous studies found that CC12.3 is a highly potent neutralizing antibody that was isolated from a SARS-CoV-2 infected patient, and its Fab fragment (Fab CC12.3) bound to SARS-CoV-2-RBD with comparable binding affinity to ACE2. To enhance its binding affinity, we employed computational protein design to redesign all CDRs of Fab CC12.3 and molecular dynamics (MD) to validate their predicted binding affinities by the MM-GBSA method. MD results show that the predicted binding affinities of the three best designed Fabs CC12.3 (CC12.3-D02, CC12.3-D05, and CC12.3-D08) are better than those of Fab CC12.3 and ACE2. Additionally, our results suggest that enhanced binding affinities of CC12.3-D02, CC12.3-D05, and CC12.3-D08 are caused by increased SARS-CoV-2-RBD binding interactions of CDRs L1 and L3. This study redesigned neutralizing antibodies with better predicted binding affinities to SARS-CoV-2-RBD than Fab CC12.3 and ACE2. They are promising candidates as neutralizing antibodies against SARS-CoV-2.


2021 ◽  
Author(s):  
Federica Liccardo ◽  
Matteo Lo Monte ◽  
Brunella Corrado ◽  
Martina Veneruso ◽  
Simona Celentano ◽  
...  

Currently, a major technical limitation of microscopy based image analysis is the linkage error – which describes the distance between e.g. the target epitope of cellular protein to the fluorescence emitter, which position is finally detected in a microscope. With continuously improving resolution of today′s (super–resolution) microscopes, the linkage errors can severely hamper the correct interpretation of images and is usually introduced in experiments by the use of standard intracellular staining reagents such as fluorescently labelled antibodies. The linkage error of standard labelled antibodies is caused by the size of the antibody and the random distribution of fluorescent emitters on the antibody surface. Together, these two factors account for a fluorescence displacement of ~40nm when staining proteins by indirect immunofluorescence; and ~20nm when staining with fluorescently coupled primary antibodies. In this study, we describe a class of staining reagents that effectively reduce the linkage error by more than five–fold when compared to conventional staining techniques. These reagents, called Fluo–N–Fabs, consist of an antigen binding fragment of a full-length antibody (Fab / fragment antigen binding) that is selectively conjugated at the N-terminal amino group with fluorescent organic molecules, thereby reducing the distance between the fluorescent emitter and the protein target of the analysis. Fluo–N–Fabs also exhibit the capability to penetrate tissues and highly crowded cell compartments, thus allowing for the efficient detection of cellular epitopes of interest in a wide range of fixed samples. We believe this class of reagents realize an unmet need in cell biological super resolution imaging studies where the precise localization of the target of interest is crucial for the understanding of complex biological phenomena.


2021 ◽  
Vol 8 ◽  
Author(s):  
Gian Pietro Pietri ◽  
Marta Tontini ◽  
Barbara Brogioni ◽  
Davide Oldrini ◽  
Stefania Robakiewicz ◽  
...  

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.


2021 ◽  
Vol 118 (41) ◽  
pp. e2115001118
Author(s):  
Xudong Wu ◽  
Tom A. Rapoport

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.


2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Habibe Yılmaz ◽  
Güliz Ak ◽  
Gülçe Özmen ◽  
Ercüment Karasulu ◽  
Şenay Hamarat Şanlıer

Abstract. SPR methods are increasingly accepted by the biopharmaceutical industry in quality control (QC), R&D and clinical research. However, there are few reports on method validation and system suitability testing (SST), in particular on concentration determination methods related to the potency of the drug. System compliance must be demonstrated with certain tests in order, for the method validation to be ensured, as agreed by the scientific community and the industry, and specified in the guidelines. To contribute to the alleviation of the deficiency in this matter, we developed a system suitability testing of an SPR-based active concentration measurement method using Fab-fragment as a model. High amounts of anti-FAB1 were captured onto sensor chip surfaces via biotin/neutravidin interaction. The unknown concentration of FAB1 measured via calibration curve dependent concentration measurement. Controls were run between unknown samples to check the reliability of the method. The standard four-parameter logistic equation was used to determine the unknown concentration. CV % of samples and Difference % of controls form standards were calculated from obtained data. The CV % and Diff. % values were less than or equal to 10 % at each parameter. The 100 % level of unknown sample measured as 105 %. Obtained CV % values and repeated analyses showed that the system and method are suitable for the concentration analysis of the FAB1 fragment. The system suitability parameters evaluated in the study can be applied for all SPR analyzes.   Resumen. Los métodos SPR (Surface Plamon Resonance) son cada vez más aceptados por la industria biofarmacéutica en el control de calidad (QC), investigación y desarrollo (R & D) e investigación clínica. Sin embargo, existen pocos informes sobre la validación de los métodos y las pruebas de idoneidad del sistema (SST), en particular sobre los métodos de determinación de la concentración relacionados con la potencia del fármaco. La idoneidad del sistema debe demostrarse para garantizar la validación del método, según la normatividad acordada por la comunidad científica y la industria. Con el fin de contribuir al alivio de la deficiencia en este asunto, se desarrolló una prueba de idoneidad del sistema con un método de medición de concentración activa basado en SPR utilizando el fragmento Fab como modelo. Se capturaron grandes cantidades de anti-FAB1 en las superficies del chip sensor mediante la interacción biotina/neutravidina. Se estableció la concentración desconocida de FAB1 a través de la determinación de la concentración dependiente de la curva de calibración. Se ejecutaron controles entre muestras desconocidas para comprobar la fiabilidad del método. Se utilizó una ecuación estándar de cuatro parámetros para determinar la concentración desconocida. A partir de los datos obtenidos se determinó el porcentaje de CV de las muestras y la diferencia respecto a los controles. Los valores de % CV y % Diff. fueron menores o iguales al 10 % en cada parámetro. Los valores de % CV obtenidos y los análisis repetidos mostraron que el sistema y el método son adecuados para el análisis de concentración del fragmento FAB1. Los parámetros de idoneidad del sistema evaluados en el estudio se pueden aplicar a todos los análisis SPR.


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