Christoph Rösli

A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo

The alternatively spliced extra‐domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra‐domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody‐based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (KD = 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody‐based targeted anti‐cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over‐expression is detectable not only in solid tumors, but also in hematological malignancies.

A comparative immunofluorescence analysis of three clinical-stage antibodies in head and neck cancer

BackgroundThe antibody-based targeted delivery of bioactive molecules to tumour vasculature is an attractive avenue to concentrate therapeutic agents at cancer sites, while sparing normal organs. L19, F8 and F16 are three fully human monoclonal antibodies, specific to splice isoforms of fibronectin and tenascin-C, which bind to sites of active tissue remodeling and which are currently in Phase I and II clinical trials as radio-immunoconjugates and immunocytokines in patients with cancer and arthritis.In this article, we report the first comparative analysis of expression patterns for the extra domains EDB and EDA of fibronectin and A1 of tenascin-C in both primary and metastatic head and neck cancer lesions.MethodsWe performed a comparative immunofluorescence analysis with the L19, F8 and F16 antibodies in 40 freshly frozen human head and neck cancer specimens.ResultsOn average, F8 and F16 exhibited similar staining intensities, which were typically stronger than L19. Interestingly, some specimens exhibited striking differences in staining by the three antibodies.ConclusionsThese results suggests that an individualized treatment procedure (e.g., choice of L19, F8 or F16 based on immuno-PET or immunofluorescence procedure) may represent the most logical avenue for offering the best possible antibody to any given patient.

Identification of Protective B Cell Antigens of Legionella pneumophila

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires’ disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

Quantitative Recovery of Biotinylated Proteins from Streptavidin-Based Affinity Chromatography Resins

The strong interaction between streptavidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for the facile derivatization of a variety of molecules (in particular, proteins) with biotin have been introduced, in order to allow their efficient recovery, immobilization and detection with streptavidin-based reagents. However, when desired, the release of biotinylated proteins from the streptavidin-based reagents remains a major problem, due to the extraordinary stability of this complex. This chapter presents a protocol developed in our laboratory for the quantitative elution of biotinylated proteins from streptavidin sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method is shown by the recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin.