AIM:To confirm the observation of IgM interference in the anti-adeno-associated virus (AAV) IgG immune complex (IC) assay format and to verify that IgM-specific digestion can improve anti-AAV IgG detection in IC assays. METHODS:Treatment-emergent anti-AAV2 and anti-AAV9 IgG signals were measured in IC assays with and without IgM-specific digestion. Anti-AAV2 and anti-AAV9 IgM signals were measured in parallel. RESULTS:IgM digestion increased anti-AAV2 and anti-AAV9 IgG signals when anti-AAV2 or anti-AAV9 IgM were present in the matrix. CONCLUSIONS:Co-existing anti-AAV IgM cause interference in the anti-AAV IC assay format. Selective IgM digestion improves the detection of anti-AAV IgG in the IC assay.

Antibodies are generated with great diversity in nature resulting in a set of molecules, each optimized to bind a specific target. Taking advantage of their diversity and specificity, antibodies make up for a large part of recently developed biologic drugs. For therapeutic use antibodies need to fulfill several criteria to be safe and efficient. Polyspecific antibodies can bind structurally unrelated molecules in addition to their main target, which can lead to side effects and decreased efficacy in a therapeutic setting, for example via reduction of effective drug levels. Therefore, we created a neural-network-based model to predict polyspecificity of antibodies using the heavy chain variable region sequence as input. We devised a strategy for enriching antibodies from an immunization campaign either for antigen-specific or polyspecific binding properties, followed by generation of a large sequencing data set for training and cross-validation of the model. We identified important physico-chemical features influencing polyspecificity by investigating the behaviour of this model. This work is a machine-learning-based approach to polyspecificity prediction and, besides increasing our understanding of polyspecificity, it might contribute to therapeutic antibody development.

Transgenic animals incorporating human antibody genes are extremely attractive for drug development because they obviate subsequent antibody humanization procedures required for therapeutic translation. Transgenic platforms have previously been established using mice, but also more recently rats, chickens, and cows and are now in abundant use for drug development. However, rabbit-based antibody generation, with a strong track record for specificity and affinity, is able to include gene conversion mediated sequence diversification, thereby enhancing binder maturation and improving the variance/selection of output antibodies in a different way than in rodents. Since it additionally frequently permits good binder generation against antigens that are only weakly immunogenic in other organisms, it is a highly interesting species for therapeutic antibody generation. We report here on the generation, utilization, and analysis of the first transgenic rabbit strain for human antibody production. Through the knockout of endogenous IgM genes and the introduction of human immunoglobulin sequences, this rabbit strain has been engineered to generate a highly diverse human IgG antibody repertoire. We further incorporated human CD79a/b and Bcl2 (B-cell lymphoma 2) genes, which enhance B-cell receptor expression and B-cell survival. Following immunization against the angiogenic factor BMP9 (Bone Morphogenetic Proteins 9), we were able to isolate a set of exquisitely affine and specific neutralizing antibodies from these rabbits. Sequence analysis of these binders revealed that both somatic hypermutation and gene conversion are fully operational in this strain, without compromising the very high degree of humanness. This powerful new transgenic strategy will allow further expansion of the use of endogenous immune mechanisms in drug development.

MOTIVATION:Canonical forms of the antibody complementarity-determining regions (CDRs) were first described in 1987 and have been redefined on multiple occasions since. The canonical forms are often used to approximate the antibody binding site shape as they can be predicted from sequence. A rapid predictor would facilitate the annotation of CDR structures in the large amounts of repertoire data now becoming available from next generation sequencing experiments.RESULTS:SCALOP annotates CDR canonical forms for antibody sequences, supported by an auto-updating database to capture the latest cluster information. Its accuracy is comparable to that of a standard structural predictor but it is 800 times faster. The auto-updating nature of SCALOP ensures that it always attains the best possible coverage.AVAILABILITY AND IMPLEMENTATION:SCALOP is available as a web application and for download under a GPLv3 license at opig.stats.ox.ac.uk/webapps/scalop.SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

Event Abstract Back to Event High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits Sonja Offner1*, Stefan Seeber1, Francesca Ros1, Irmgard Thorey1, Georg Tiefenthaler1, Klaus Kaluza1, Valeria Lifke1, Jens Fischer1, Stefan Klostermann1, Josef Platzer1 and Brigitte Kaluza1 1 Roche Diagnostics GmbH, Germany We have developed a robust high throughput B-cell cultivation method using peripheral rabbit B cells. This rapid procedure generates a diverse set of antibodies using blood of immunized rabbits. The entire workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (v) the recombinant expression and purification of IgG antibodies. Rabbits immunized with the human IL1RL1 antigen (also known as ST2, DER4, FIT-1, IL33R, ST2L, ST2V, T1) were used for the proof-of-concept study. The IL33-IL1RL1 ligand-receptor system plays an important role in autoinflammatory diseases such as asthma, ulcerative colitis or arthritis. The fully integrated and to a large degree automated platform yielded clonal and very diverse IL1RL1-specific antibodies. Due to the high IgG productivity of the cultivated B-cell clones the IL1RL1-inhibiting IgGs from individual animals could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. In summary, this powerful B-cell cultivation method has proven to be an efficient and robust tool for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal. Acknowledgements We would like to express our sincere gratitude and thanks for expert technical assistance to Sabrina Kettenbach and Marco Friedrich (immunization of rabbits), Daniela Buntefuss (preparation of rbTSN), Basile Siewe and Ireneus Jonetzko (B-Cell Cloning), Manuela Paul and Sven Kroemmelbein (screening, biochemical assays), Dominique Ostler, Simone Hoege (B-Cell PCR), Peter Kern (in vitro NK-cell IL1RL1 inhibition assay), and Florian Lipsmeier (statistical analyses). Keywords: B-cell cultivation, antibody production, high throughput, rabbit, Peripheral Blood, B lymphocyte, costimulation, B-cell PCR, IL1RL1, peripheral B cell Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Offner S, Seeber S, Ros F, Thorey I, Tiefenthaler G, Kaluza K, Lifke V, Fischer J, Klostermann S, Platzer J and Kaluza B (2013). High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01170 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sonja Offner, Roche Diagnostics GmbH, Penzberg, Germany, sonja.offner@roche.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza Google Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza Google Scholar Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza PubMed Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

Two rabbit germline bacterial artificial chromosome (BAC) libraries from animals with the b5 and b4 allotype were screened with probes specific for the immunoglobulin kappa1 light chain locus. Two partially overlapping BAC clones containing Vkappa elements of b5 allotype were isolated from the b5 library and one BAC clone containing Jkappa1, Ckappa and Vkappa was isolated from the b4 library. These three BAC clones were sequenced. They span about 0.4 MB of the rabbit Ig kappa1 light chain locus including 36 Vkappa elements, five J elements and the coding region of Ckappa1. The organization of the locus and the potential function of newly identified functional and structural elements are discussed.

A bacterial artificial chromosome (BAC) library was created using partially digested rabbit chromosomal DNA. Four BAC clones spanning about 0.5 Mb of the rabbit immunoglobulin (Ig) heavy chain locus were isolated and sequenced. Three of the BAC clones were partially overlapping. Thirty-four V elements, 11 D elements, DQ52, six J elements and the coding regions of Cmicro, Cgamma, C and four Calpha genes were identified and characterized. Additionally, the sequence of a fosmid clone spanning Calpha13 and 30 kb 3'enhancer region was determined. The organization of the locus and the potential function of newly identified functional and structural elements are discussed.

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids (“chromatid selectivity”). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.