Bio-Rad (Germany)

ABSTRACT Antibody phage display, a biotechnological method for selecting fully human antibodies from diverse libraries, is firmly established in antibody drug discovery due to its speed and flexibility. In this study, we introduce Pioneer, one of the largest synthetic human antibody libraries developed to date, with approximately 2.2 × 1011 functional members. Pioneer utilizes SpyDisplay, a phage display selection system that relies on SpyTag-SpyCatcher protein ligation technology and enables rapid selection of high-affinity antibodies. The library’s antibodies are designed for favorable biophysical properties and developability and they can be rapidly labeled or converted into various formats for screening and functional assays. We validated the performance of Pioneer in a series of selection campaigns against diverse antigens. For antibodies against four targets, TIGIT, IL-6RA, C5aR and CXCR4, we performed in-depth functional and developability characterization. For all four targets, including the two challenging G-protein coupled receptors, we demonstrated that antibodies with parameters comparable to late-stage clinical candidates can be selected directly from the library. Our results highlight the effectiveness of the Pioneer library combined with SpyDisplay in generating therapeutic lead candidates with excellent affinity, specificity, and developability, offering a robust platform for rapid antibody discovery and development.

As innovators who made non-animal-derived antibodies available to researchers starting 20 years ago, we have generated more than 60,000 antibodies during this time span. Here, we review and highlight the key challenges that hampered the adoption of research antibodies generated by phage display. We describe the advantages of this method over traditional immunisation-based methods, and provide examples of specific projects. As active participants in attesting the scientific validity of non-animal-derived antibodies, we have contributed and supported key advancements in phage display for the generation of research antibodies. Such advancements have paved the way for the scientific community to switch to animal-free antibodies at a faster pace, as a goal for the near future.

This study aims to evaluate the performance of the BeO-based myOSLchip system (RadPro International GmbH, Remscheid, Germany) for dosimetry of proton and electron radiotherapy beams. Although beryllium oxide (BeO) has been recognised as a promising material for luminescence dosimetry in radiotherapy, this research extends beyond material properties and examines the entire BeO-based dosimetry system, including the detector, holder, and readout components.Packages of myOSLchip dosimeters were irradiated either in a (70−230) MeV proton beam or in a 16 MeV electron beam. The readouts were carried out using the portable myOSLchip reader. In the electron beam, tests on the precision, dose response up to 100 Gy and dose-rate effects of the system were carried out. In the proton beam, the system was tested for its dose response (up to 10 Gy), fading, and linear energy transfer (LET) response.For proton irradiations, the myOSLchip BeO OSLDs exhibited stability within 2% over 135 days, as well as a linear dose response in the tested range, (0.1−10) Gy. The efficiency showed a reduction for proton beams with LET values (for water) above 0.6 keV/μm, with up to 40% loss in efficiency at 4 keV/μm. For the electron irradiations, they showed a linear dose–response up to 20 Gy and dose-rate independence, with a constant response at least up to 2.99 × 105 Gy s−1. Using individual dosimeter sensitivity correction, the precision for a single dosimeter was around 3.5% (standard deviation of the data of all dosimeters) and for a package comprising four dosimeters was 1.7% (standard deviation of the mean of the four dosimeters).These findings suggest the myOSLchip system’s potential as a reliable alternative to existing dosimetry systems in clinical applications.

ABSTRACTAnti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

Abstract The validation of analytical procedures in laboratory medicine has been discussed by many authors and several recommendations have been elaborated by working groups and standardization bodies. However, some problems have not sufficiently been solved, for example, permissible limits for the comparability between methods (permissible equivalence), what is the best statistical procedure for estimating the function of the fitting line in scatter plots, and permissible limits for the spread around the fitting line. The present article attempts to summarize the present state-of-the-art and to provide answers to some of the open problems.

Photoacoustic FT-IR spectra recorded using a new photoacoustic cell are described. The study of the photoacoustic spectra of cast films of Plexiglas of different thicknesses shows that the depth of penetration of the FT-IR photoacoustic experiments is of the order of a few micrometers and that photoacoustic saturation effects play a key role in the qualitative appearance of the spectra. These differences can be misinterpreted as due to structural changes in different layers of the sample.© (1981) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.