The Octet biosensors provide a high-throughput alternative to the well-established surface plasmon resonance (SPR) and SPR imaging (SPRi) biosensors to characterize antibody-antigen interactions. ...However, the utility of the Octet biosensors for accurate and reproducible measurement of binding rate constants of monoclonal antibodies (mAbs) is limited due to challenges such as analyte rebinding, and mass transport limitation (MTL). This study focuses on addressing these challenges and provides experimental conditions to reliably measure kinetics of mAb-antigen interactions. The mAb capture density of less than 0.6 nm was found to be optimal to measure a wide range of binding affinities on Octet HTX biosensor. The titration kinetic and single cycle kinetic assays performed on Octet HTX generated reproducible binding kinetic parameters and correlated with the values measured on Biacore 4000 and MASS-1. Kinetic assays performed on 0.1 nm density mAb surfaces significantly reduced MTL and enabled characterization of picomolar affinity mAbs. Finally, kinetic analysis performed on 150 antibodies to 10 antigens with molecular weights ranging from 21kD to 105kD showed concordance between Octet HTX, Biacore 4000 and MASS-1 (R2 > 0.90). The data presented in this study suggest that under optimal experimental conditions, Octet biosensor is capable of generating kinetic values comparable to SPR/SPRi biosensors.
•High density antibody capture surface exhibited analyte rebinding and mass transport limitation (MTL).•Kinetic assays performed on 0.1 nm density antibody surface significantly reduced MTL and enabled characterization of picomolar affinity interactions.•A total of 165 fully human antibodies binding to 14 different antigens with molecular weight ranging from 14kD to 105kD were characterized on Octet HTX, Biacore 4000 and MASS-1 and the results revealed that measured binding kinetic values were comparable across different biosensors.
Surface plasmon resonance (SPR) is a well-established method to characterize biomolecular interactions and is widely used in drug discovery and development. Here, we demonstrate that capture surfaces ...profoundly impact the binding kinetics parameters that are measured for antibody-antigen interactions. Six unique antibody-antigen interactions were characterized using eight different anti-human IgG capture surfaces. The antigen binding affinities for six different human monoclonal antibodies (hmAbs) captured using three different goat anti-human Fc (AHC) polyclonal antibody (pAb) surfaces were in reasonable agreement (3-7-fold weaker) with those measured by kinetic exclusion assay (KinExA). In contrast, up to 81, 32, 489, 2826, and 219-fold weaker antigen binding affinities were measured using mouse AHC mAb, Protein G, Protein A, Protein A/G, and Protein L surfaces, respectively. Protein A, Protein A/G and Protein G interacted with the Fab of hmAbs, possibly affecting antigen binding to hmAbs captured over these surfaces. Additional studies revealed that mouse AHC mAb binds hmAbs with a weak affinity (5.5–36.3 nM) and t½ values of 1.4–3.3min, compared to the sub-nanomolar affinities of the goat AHC pAbs. These results emphasize the value of measuring binding kinetics of the capture molecule before immobilizing them onto the sensor surface to perform capture kinetics assays on label-free biosensors.
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•The antibody-antigen binding kinetics of six picomolar affinity human monoclonal antibodies (hmAbs) were measured on a surface plasmon resonance (SPR) biosensor using eight different hmAb capture surfaces, four anti-human Fc (AHC) and four non-antibody anti-human IgG (AHG) and compared with binding kinetics determined by kinetic exclusion assays (KinExA).•Amongst the capture reagents tested, three different goat AHC polyclonal antibodies (pAbs) measured binding affinities best correlated with KinExA data, whereas the Protein L surface was not reliable for determining binding kinetics data.•Protein A, Protein A/G and Protein G surfaces interacted with the Fab region of hmAbs, which may influence Ag binding.•Three goat AHC pAbs bound to hmAbs with sub-nanomolar affinities and t½ values ranging from 11.7 to 337 min; whereas, the mouse AHC mAb showed weaker affinity for human IgGs with KD and t½ values ranging from 5.5 to 36.3 nM and 1.4–3.3 min, respectively.•Selection of a high affinity, Fc-specific capture molecule that exhibits a slow dissociation rate, even under low AHC surface (<8 RU) density, is essential to reliably characterize antibody-antigen interactions using capture kinetics analysis on a label-free optical biosensor.
I propose a treatment selection model that introduces unobserved heterogeneity in both choice sets and preferences to evaluate the average effects of a program offer. I show how to exploit the model ...structure to define parameters capturing these effects and then computationally characterize their identified sets under instrumental variable variation in choice sets. I illustrate these tools by analyzing the effects of providing an offer to the Head Start preschool program using data from the Head Start Impact Study. I find that such a policy affects a large number of children who take up the offer, and that they subsequently have positive effects on test scores. These effects arise from children who do not have any preschool as an outside option. A cost–benefit analysis reveals that the earning benefits associated with the test score gains can be large and outweigh the net costs associated with offer take up.
Severe inflammatory airway diseases are associated with inflammation that does not resolve, leading to structural changes and an overall environment primed for exacerbations.
We sought to identify ...and inhibit pathways that perpetuate this heightened inflammatory state because this could lead to therapies that allow for a more quiescent lung that is less predisposed to symptoms and exacerbations.
Using prolonged exposure to house dust mite in mice, we developed a mouse model of persistent and exacerbating airway disease characterized by a mixed inflammatory phenotype.
We show that lung IL-33 drives inflammation and remodeling beyond the type 2 response classically associated with IL-33 signaling. IL-33 blockade with an IL-33 neutralizing antibody normalized established inflammation and improved remodeling of both the lung epithelium and lung parenchyma. Specifically, IL-33 blockade normalized persisting and exacerbating inflammatory end points, including eosinophilic, neutrophilic, and ST2+CD4+ T-cell infiltration. Importantly, we identified a key role for IL-33 in driving lung remodeling because anti–IL-33 also re-established the presence of ciliated cells over mucus-producing cells and decreased myofibroblast numbers, even in the context of continuous allergen exposure, resulting in improved lung function.
Overall, this study shows that increased IL-33 levels drive a self-perpetuating amplification loop that maintains the lung in a state of lasting inflammation and remodeled tissue primed for exacerbations. Thus IL-33 blockade might ameliorate symptoms and prevent exacerbations by quelling persistent inflammation and airway remodeling.
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Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders ...the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and κ light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics.
Traditional approaches for development of antibodies are poorly suited to combating the emergence of novel pathogens, as they require multiple steps of laborious optimization and process adaptation for clinical development. Here, we describe the simultaneous use of two state-of-the-art technologies to rapidly generate and validate antibodies against Middle East Respiratory Syndrome coronavirus (MERS-CoV), following a highly optimized process that links immunization to production of clinical material grade antibodies and developed promising clinical candidates for prophylaxis and treatment of MERS-CoV, and a humanized mouse model of infection that was used to evaluate our therapeutics. This study forms the basis for a rapid response to address the public threat resulting from emerging coronaviruses or other pathogens that pose a serious threat to human health in the future.
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, ...tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOP-mutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP.
Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated ...therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This paper studies the validity of nonparametric tests used in the regression discontinuity design. The null hypothesis of interest is that the average treatment effect at the threshold in the ...so-called sharp design equals a pre-specified value. We first show that, under assumptions used in the majority of the literature, for any test the power against any alternative is bounded above by its size. This result implies that, under these assumptions, any test with nontrivial power will exhibit size distortions. We next provide a sufficient strengthening of the standard assumptions under which we show that a version of a test suggested in Calonico, Cattaneo, and Titiunik (2014) can control limiting size.
The surface plasmon resonance (SPR) biosensors are being routinely used in different stages of drug discovery and development. However, the lack of high throughput SPR biosensors continues to be a ...primary bottleneck for the rapid kinetic screening of large panels of monoclonal antibodies (mAbs). To further increase the throughput of the Biacore 4000 biosensor, we have developed three kinetic screening assays to characterize mAb-antigen interactions – (i) 16-mAb capture kinetic, (ii) single cycle kinetic (SCK), and (iii) parallel kinetic (PK). The performance of all three kinetic assays was evaluated by characterizing the binding of kinetically diverse human mAbs to four antigens with molecular weights of 14kD, 29kD, 38kD, and 48kD and binding affinities ranging from 130pM to 200 nM. The binding rate constants measured using all three kinetic assays were reproducible across multiple experiments and correlated with the values generated using the conventional 8-mAb capture kinetic assay on the Biacore 4000 (R2 > 0.94). Moreover, the 16-mAb capture assay decreased experiment time and analyte consumption by 35% and 50%, respectively. This work illustrates the significance of the 16-mAb capture kinetic, SCK, and PK assays to increase the throughput of Biacore 4000 and to support rapid kinetic screening of mAbs.
•Three new capture kinetic assays were developed to increase throughput - 16-mAb capture kinetic, single cycle kinetic (SCK) and parallel kinetic (PK).•All three capture kinetic assays generate reliable and reproducible binding kinetic parameters.•The 16-mAb capture kinetic assay reduces experiment time and analyte consumption by 35% and 50%, respectively.•Both SCK and PK reduce assay development time by avoiding surface regeneration, which further extends the throughput of Biacore 4000.
Rapid growth in the field of biotherapeutics has led to an increased demand for high-throughput, label-free biosensors exhibiting high sensitivity. To support the current needs, Sierra Sensors ...introduced a surface plasmon resonance imaging (SPRi) based biosensor, Molecular Affinity Screening System (MASS-1). We assessed the potential utility of MASS-1 to support Regeneron's therapeutic antibody discovery. A large panel of antibody-antigen interactions was characterized using MASS-1 and the kinetic data were compared with the Biacore 4000 biosensor. Less than 10% deviation in the binding rate constants measured across eight flow channels of MASS-1 was observed. The single injection cycle kinetic assay allowed rapid measurement of binding rate constants for antibody-antigen interactions. MASS-1 sensitivity was independent of protein immobilization level and kinetic analysis performed using ultra-low density mAb surfaces allowed characterization of picomolar affinity interactions without mass transport limitation. High-throughput characterization of a panel of 189 monoclonal antibodies to 13 different antigens with molecular weights ranging from 14kD to 105kD revealed that binding kinetic parameters measured on MASS-1 were comparable to those measured on Biacore 4000. Our data demonstrate that MASS-1 measures reliable binding kinetic parameters and has an appropriate combination of throughput and sensitivity to support discovery and development of therapeutic antibodies.
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•Less than 10% variation in rate constants measured across different flow channels.•SICK assay allows characterization of antibody-antigen interactions in 2 cycles.•MASS-1 measures reliable kinetic values and has an appropriate combination of throughput and sensitivity which allows the design of kinetic assays using ultra-low density antibody surface with Rmax < 5RU.
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