FHR4-based immunoconjugates direct complement-dependent cytotoxicity and phagocytosis towards HER2-positive cancer cells

Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b-binding protein C-terminal-alpha-/beta-chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent-positive regulator of the AP, the human factor H-related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VHH targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab-recognising epitopes [VHH(T) or VHH(P)], respectively, were used as HER2 anchoring moieties. Optimised high-FHR4 valence heteromultimeric immunoconjugates [FHR4/VHH(T) or FHR4/VHH(P)] were selected by sequential cell cloning and a selective multistep His-Trap purification. Optimised FHR4-heteromultimeric immunoconjugates successfully overcame FH-mediated complement inhibition threshold, causing increased C3b deposition on SK-OV-3, BT474 and SK-BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement-dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane-anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement-dependent cell-mediated cytotoxicity. We showed that the degree of FHR4-multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH-mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady-state towards activation on tumour cell surface

Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future

Optical and mechanical design of a straylight rejection baffle for CoRoT

The COROT mission is part of the program "Petites Missions" of CNES (French space agency). It implies international cooperation between France, Belgium, Germany, Austria, Spain and the European Space Agency (ESA). COROT aims to perform astroseismology measurements and to detect exoplanets. Long duration observations of stars will be used to detect periodic variations with an afocal telescope followed by a dioptric objective and 4 CCDs. Due to the orbit of the spacecraft (low altitude polar orbit) and even if the observation are performed in a direction perpendicular to orbit plane, the measurements can be disturbed by the straylight reflected on the earth (albedo) that can generate periodic perturbation. CSL is in charge of the design and procurement, with the help of Belgian industries, of a baffle and its protective cover that will be mounted on top of the afocal entrance telescope. The requirements are very stringent from the optical point of view as well as from the mechanical point of view. The rejection of the baffle must be of the order of 10[SUP]13[/SUP] for field angles above 20 degrees while the allocated mass is 19 kilograms

SM98-146/413 Effective Modal Parameters to Evaluate Structural Stresses

peer reviewe

Straylight analysis of the external baffle of COROT

The COROT mission is part of the program "mini-satellite" of CNES (French space agency). It implies international cooperation between European institutes and research centres. COROT aims to perform astroseismology observations and to detect exoplanets. Long duration observations of stars will be used to detect periodic variations with an afocal telescope followed by a dioptric objective and 4 CCDs. These very small variations can be caused by star seismic activities (about 10-6 variation of signal) or transits of planets (few 10-4 variation of signal). Due to the orbit of the spacecraft (low altitude polar orbit) and even if the observations are performed in a direction perpendicular to orbit plane, the measurements can be disturbed by the straylight reflected by the earth (albedo) that can generate a periodic perturbation. The paper details the overall optical design of the baffle. The baffle modelling and straylight computation methods are described and the expected performances are discussed. © 2017 SPIE

A Test Cryostat for Herschel-PACS Mechanism

The PACS Grating Assembly consists of a flat ruled grating of 320x80 mm²; its positioning mechanism; and launch-locking system. The Grating Assembly is part of the PACS Focal Plane Unit (FPU) and is operated at liquid helium temperature (4K). It is remote-controlled from the Detector & Mechanism Controller (DEC/MEC), which is located on the Herschel Service Module. The grating shall be capable of accurate positioning (4 arcsec) within a large angular throw (40 arcdeg) and restricted dissipation to the 4K heat sink (< 5 mW). The development of the Grating mechanism controller has urged the need for a cryogenic simulator of the Grating Assembly with regard to the large change in value of mechanical and electrical characteristics between room temperature and liquid helium temperature. The simulator consists of a functional prototype of the Grating Assembly and a dedicated helium cryostat with appropriate interfaces. The test cryostat is a homemade facility (CSL) designed to cool down the prototype (and subsequent models) to liquid helium temperature. The test set-up also provides relevant instrumentation such as cryogenic temperature read-out, electrical feedthroughs toward the payload and optical viewport

Manufacturing and verification of ZnS and Ge prisms for the JWST MIRI imager

The JWST Mid-Infrared Instrument (MIRI) is designed to meet the JWST science requirements for mid-IR capabilities and includes an Imager MIRIM provided by CEA (France). A double-prism assembly (DPA) allows MIRIM to perform low-resolution spectroscopy. The MIRIM DPA shall meet a number of challenging requirements in terms of optical and mechanical constraints, especially severe optical tolerances, limited envelope and very high vibration loads. The University of Cologne (Germany) and the Centre Spatial de Liege (Belgium) are responsible for design, manufacturing, integration, and testing of the prism assembly. A companion paper (Fischer et al. 2008) is presenting the science drivers and mechanical design of the DPA, while this paper is focusing on optical manufacturing and overall verification processes. The first part of this paper describes the manufacturing of Zinc-sulphide and Germanium prisms and techniques to ensure an accurate positioning of the prisms in their holder. (1) The delicate manufacturing of Ge and ZnS materials and (2) the severe specifications on the bearing and optical surfaces flatness and the tolerance on the prism optical angles make this process innovating. The specifications verification is carried out using mechanical and optical measurements; the implemented techniques are described in this paper. The second part concerns the qualification program of the double-prism assembly, including the prisms, the holder and the prisms anti-reflective coatings qualification. Both predictions and actual test results are shown.MIRI for JWS