Autoantibodies targeting a variety of neuronal surface proteins have recently been discovered in a multitude of neurological disorders and continuously define novel autoimmune entities such as NMDA-receptor encephalitis. However, first characterizations of these antibodies were limited to experiments that used bio-samples containing disease-specific but also further antibodies that were present in these patients. Thereby, these studies could not link the observed effects directly to single antibodies. To overcome that limitation and allow the characterization of functional effects for each antibody individually, we have developed a methodology to isolate single (monoclonal) antibodies from patients’ cerebrospinal fluid (Figure). This core technology uses optimized molecular biological techniques together with custom bioinformatic analytics to encode genetic information from large sets of antibody-producing immune cells to then separately reassemble single antibodies in theoretical unlimited amounts. Using this approach, we have successfully isolated disease-defining monoclonal antibodies in multiple forms of autoimmune encephalitis (such as NMDAR, LGI1, Caspr2, IgLON5, mGluRs) and provided proof of their pathogenicity using in vitro and in vivo assays. Besides studying their biological functions, each of these recombinant antibodies can also be used as a tool in scientific applications (e.g., high-resolution microscopy), for optimization of antibody diagnostics or as a starting point for novel antibody-depleting therapies.

In the face of the COVID-19 pandemic, we used the potential of our pipeline to generate SARS-CoV-2 neutralizing monoclonal antibodies to support the global efforts to understand the immune response against the novel coronavirus.

Autoantibodies targeting neuronal antigens are one of the diagnostic hallmarks of autoimmune encephalitis. Disease phenotype and severity depend on the underlying antigen. To understand whether such autoantibodies are directly pathogenic and cause disease, our research group uses patient-derived monoclonal antibodies in different in vitro and in vivo models.

Using primary neurons, transfected cells and rodent brain slices, we study the molecular interaction between autoantibodies and their target antigens (Figure A). The specific binding region of the recombinant human autoantibodies can be determined using epitope mapping, where human cell lines are transfected with different target domains (B). Moreover, application of autoantibodies to neuronal cultures provides insight into the effector mechanisms in disease pathogenesis (C).

Autoantibody pathogenicity can be further characterized in vivo, for instance, through intrathecal (cerebroventricular) delivery of antibodies using osmotic pumps (D). Autoantibodies infused into the CNS target their cognate antigen and can reproduce certain clinical features observed in the human disease. Animal MR imaging and behavioral tests allow quantification of the clinical phenotype. Taking brain slices of treated animals, electrophysiology measurements can help to understand how autoantibody-driven alterations of synaptic currents disturb neuronal function (E). Finally, using flow cytometry, western blot and ELISA, analysis of cells and brain proteins provides mechanistic understand of antibody-mediated down-regulation of CNS proteins.

Anti-neuronal and anti-glial autoantibodies are increasingly recognized as the autoimmune cause of encephalitis, psychosis and dementia. Some autoantibodies are well-established, their direct pathogenicity has been proven in experimental work, and their routine diagnostics has become clinical standard in neurological operating procedures. The repertoire of diseases-defining autoantibodies is still continuously expanding. To identify yet unknown autoantibodies, indirect immunofluorescence can be applied. For this, unfixed rodent brain sections are incubated with human cerebrospinal fluid or serum. If the samples contain novel autoantibodies targeting neuronal epitopes, the antibodies stick to the section and are visualized with fluorescently labelled secondary antibodies (Figure). In the past, many of these new findings resulted in recognition of an underlying autoimmune etiology, treatment of patients and marked clinical improvement. On a collaborative research basis, this service is available to clinical centers and laboratories (please use the request form for shipment of patient sample).