For largely unknown reasons, biopharmaceuticals evoke potentially harmful antibody formation. Such antibodies can inhibit drug efficacy and, when directed against endogenous proteins, cause life-threatening complications. Insight into the mechanisms by which biopharmaceuticals break tolerance and induce an immune response will contribute to finding solutions to prevent this adverse effect. Using a transgenic mouse model, we here demonstrate that protein misfolding, detected with the use of tissue-type plasminogen activator and thioflavin T, markers of amyloid-like properties, results in breaking of tolerance. In wild-type mice, misfolding enhances protein immunogenicity. Several commercially available biopharmaceutical products were found to contain misfolded proteins. In some cases, the level of misfolded protein was found to increase upon storage under conditions prescribed by the manufacturer. Our results indicate that misfolding of therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity. These findings offer novel possibilities to detect immunogenic protein entities with tPA and reduce immunogenicity of biopharmaceuticals.Over the past decades, the use of therapeutic proteins has become common practice in medicine and as their use is very promising, many more biopharmaceuticals are under development (1, 2). Unfortunately, a major drawback of protein therapeutics is the risk of antibody formation (3-7). These immunogenicity problems are of concern regarding therapeutic efficacy and patient safety (5,8). For example, drug-induced neutralizing antibodies to erythropoietin (EPO) 3 result in pure red cell aplasia (9), whereas drug-induced acquired anti-factor VIII (fVIII) antibodies worsen the pathology associated with hemophilia (10). As more and more recombinant therapeutic proteins become licensed for marketing, the incidence of immunogenicity problems is expected to rise.Initially, when mainly proteins from animal origin were used for therapy, it was thought that their foreign (non-self) nature was the main cause of immunogenicity. Unexpectedly, however, both human plasma derived as well as recombinant human protein therapeutics such as EPO (11) and fVIII (12) also elicit immune responses. This suggests that the molecular characteristic evoking antibody responses is at least more complex than being self or non-self to the human immune system. Several additional factors contributing to immunogenicity have been proposed, including contaminants or impurities, protein aggregation (13), chemical degradation and protein modification, such as differences in glycosylation or oxidation (14,15) to explain the induction of antibodies.Protein misfolding is an intrinsic and problematic property of proteins, which underlies a variety of degenerative diseases, such as Alzheimer disease. These diseases are characterized by the occurrence of fibrillar deposits, classically termed amyloid, containing aggregates of misfolded proteins. Whereas the term amyloid is classically used to classify these fi...