Molecular Identification of Antigen Recognition Structures in Immune Complexes for Immunotherapeutic Applications by Proteolytic and Mass Spectrometric Methods

Abstract

Recent advances in immunology and molecular biology have lead to the development of therapeutic vaccines which are of potential use in chronic diseases such as cancer, cardiovascular disorders and neurodegenerative diseases, where efficacies of available therapies are poor. Future advances in vaccine development will rely substantially on a more complete understanding of the structural basis of immune response. Mass spectrometry has emerged as a widespread technique for the study of protein structure, function and interaction with other biomolecules. To obtain information on complex protein mixtures and to dissect the structure of the molecular recognition domains diverse applications have been developed in conjunction with mass spectrometry. These methods include chromatographic and electrophoretic separations, proteolytic assays, differential chemical modification of specific amino acid functions and bioinformatic tools for data analysis.One of the hallmarks of Alzheimer´s Disease is the accumulation in the human brain of extracellular plaques containing aggregates of the neurotoxic ß-amyloid peptide. The immunotherapeutical approaches capable of triggering the clearance of amyloid plaques and preventing Aß aggregation have gained increasing interest in recent years. The first two parts of the thesis are focused on the development and application of mass spectrometric and immuno-analytical methods to the identification of epitopes on Aß recognized by anti-Aß antibodies.The first part of the thesis was focused on the detailed characterization of the ß-amyloid (4-10) FRHDSGY interaction with cognate antibodies. The sequence has been previously identified as a structural epitope for two antibodies, a polyclonal anti-Aß(1-42) and a monoclonal anti-Aß(1-17) antibody. In order to determine the functional significance of these residues to the antibodies, site-directed mutagenesis was performed using synthetic ß-amyloid (4-10) mutants as model substrate peptides. Selective identification of the affinity preserving mutant peptides was achieved by comparative ELISA binding studies. While the interaction to the polyclonal antibody was preserved in the D7A, S8A, G8A and Y10A mutants indicating F4, R5 and H6 as essential residues, for the monoclonal antibody all amino acid residues were essential for binding.The second and major part of the thesis was focused on the identification of the epitope recognized by anti-Aß-autoantibodies naturally occurring in human blood. The antibodies were isolated by affinity chromatography from human immunoglobulin preparations, and serum samples of Alzheimer´s disease patients. An affinity column for antibody isolation was prepared by immobilising Cys-Aß(1-40) on a iodoacetyl-support. For mass spectrometric epitope identification, an affinity column was prepared using the purified antibodies. In epitope excision, selective proteolytic cleavage of the intact Aß affinity- bound to the immobilised antibody was performed using trypsin or endoproteinase V8, followed by MALDI-TOF mass spectrometric analysis of the epitope- and non-epitope fractions, and provided direct information that the epitope is located within the sequence Aß(12-40). A consistent result was obtained by epitope extraction-mass spectrometry. The use of pronase provided the identification of Aß(21-37) as the minimal epitope structure for recognition. Comparative binding studies of human Aß-antibodies with Aß(1-16), Aß(1-40), Aß(12-40) and Aß(17-28), each synthesized with a pentaglycine spacer and biotin at the N-terminal end, were performed by indirect ELISA. The results showed that Aß(1-16) and Aß(17-28) do not interact with the antibodies while Aß(12-40) and Aß(1-40) reacted with the autoantibodies in a concentration-dependent manner. Similar results were obtained by analyzing samples of anti-Aß autoantibodies isolated from AD patients.A further part of the dissertation was focused on the serine protease HtrA1 which has been implicated in amyloid precursor protein processing. Astrocytes produce significant levels of HtrA1 and Aß and application of an HtrA1 inhibitor leads to the accumulation of Aß in cell culture supernatants. Proteolytic digestion by HtrA1 was analysed for APP(672-770) in comparison to APP(672-711), APP(672-713), APP(724-770) and APP(661-687), and digestion products were identified directly by high-resolution MALDI-FT-ICR. Digestion of APP(672-770) was established to occur after residues Val-683, Gln-686, Asn-755, and Asp-672 providing degradation products of approximately equal sequence lengths.