constant domain exchanged fab enables specific light chain pairing in heterodimeric bispecific seed antibodies

Abstract Background Bispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing. Methods We have designed a solution in which the CH1 and CL domain pair in one of the Fab fragments is replaced with a CH3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII). Results Bispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains. Conclusion Incorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties. General significance Our results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with CH3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

AbstractWe have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and Tm-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time

Determining appropriate interventions to mainstream nutritious orphan crops into African food systems

Nutritious ‘orphan’ crops could (re)diversify African food systems, but appropriate means to bring this about are required. A review of the literature on crop intervention options suggested success and failure factors in promotion, but indicated little about the relative importance of production-versus consumption-based measures and how these interact. An analysis of secondary crop production data indicated that addressing food policies could be valuable for orphan crop mainstreaming, but, as with literature review, did not provide clear guidance on the importance of different interventions. A survey of experts suggested that cross-disciplinary teams are important for developing mainstreaming strategies, but revealed no clear consensus on the importance of particular measures for specific orphan crops. We discuss the implications of these findings

On the missing single collision peak in low energy heavy ion scattering

We present experimental and simulation data on the oblique angle scattering of heavy Sn ions at 14 keV energy from a Mo surface. The simulations are performed with the binary collision approximation codes TRIM, TRIDYN, TRI3DYN, SDTrimSP, and IMSIL. Additional simulations were performed in the molecular dynamics framework with LAMMPS. Our key finding is the absence of an expected peak in the experimental energy spectrum of backscattered Sn ions associated with the pure single collision regime. In sharp contrast to this, however, all simulation codes we applied do show a prominent single collision signature both in the energy spectrum and in the angular scatter pattern. We discuss the possible origin of this important discrepancy and show in the process, that widely used binary collision approximation codes may contain hidden parameters important to know and to understand.</p

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity

Plasma-wall interaction studies within the EUROfusion consortium: Progress on plasma-facing components development and qualification

This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the Euratom research and training programme 2014-2018 under grant agreement No 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission.The provision of a particle and power exhaust solution which is compatible with first-wall components and edge-plasma conditions is a key area of present-day fusion research and mandatory for a successful operation of ITER and DEMO. The work package plasma-facing components (WP PFC) within the European fusion programme complements with laboratory experiments, i.e. in linear plasma devices, electron and ion beam loading facilities, the studies performed in toroidally confined magnetic devices, such as JET, ASDEX Upgrade, WEST etc. The connection of both groups is done via common physics and engineering studies, including the qualification and specification of plasma-facing components, and by modelling codes that simulate edge-plasma conditions and the plasma-material interaction as well as the study of fundamental processes. WP PFC addresses these critical points in order to ensure reliable and efficient use of conventional, solid PFCs in ITER (Be and W) and DEMO (W and steel) with respect to heat-load capabilities (transient and steady-state heat and particle loads), lifetime estimates (erosion, material mixing and surface morphology), and safety aspects (fuel retention, fuel removal, material migration and dust formation) particularly for quasi-steady-state conditions. Alternative scenarios and concepts (liquid Sn or Li as PFCs) for DEMO are developed and tested in the event that the conventional solution turns out to not be functional. Here, we present an overview of the activities with an emphasis on a few key results: (i) the observed synergistic effects in particle and heat loading of ITER-grade W with the available set of exposition devices on material properties such as roughness, ductility and microstructure; (ii) the progress in understanding of fuel retention, diffusion and outgassing in different W-based materials, including the impact of damage and impurities like N; and (iii), the preferential sputtering of Fe in EUROFER steel providing an in situ W surface and a potential first-wall solution for DEMO.European Commission; Consortium for Ocean Leadership 633053; Institute of Solid State Physics, University of Latvia as the Center of Excellence has received funding from the European Union’s Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamingPhase2 under grant agreement No. 739508, project CAMART