Managing unwanted immunogenicity of biologicals.

All protein drugs (biologicals) have an immunogenic potential and we are armed with multiple guidelines, regulatory documents and white papers to assist us in assessing the level of risk for unwanted immunogenicity of new biologicals. However, for certain biologicals, significant immunogenicity becomes only apparent after their use in patients. Causes of immunogenicity are multifactorial but not yet fully understood. Within the pharmaceutical industry there are only a few opportunities to openly discuss the causes and consequences of immunogenicity with regard to the development of new biologicals. The annual Open Scientific Symposium of the European Immunogenicity Platform (EIP) is one such meeting that brings together scientists and clinicians from academia and industry to build know-how and expertise in the field of immunogenicity. The critical topics discussed at the last EIP meeting (February 2014) will be reviewed here. The current opinion of this expert group is that the assessment of unwanted immunogenicity can be improved by using prediction tools, optimizing the performance of immunogenicity assays and learning from the clinical impact of other biologicals that have already been administered to patients. A multidisciplinary approach is warranted to better understand and minimize drug immunogenicity and its clinical consequences.

Synthetic dimeric Aß(28-40) mimics the complex epitope of human anti-Aß autoantibodies against toxic Aß oligomers.

Covalently linked carboxyl-terminal segments of the ß-amyloid peptide (Aß) were tested for their qualification as minimal conformational epitopes of the naturally occurring human autoantibodies against ß-amyloid (nAbs-Aß). nAbs-Aß specifically recognize the toxic oligomers of Aß and not the monomeric or the fibrillar forms of Aß. The synthetic dimers of Aß(28-40) described herein mimic the toxic Aß oligomers but are not kinetic intermediates with uncertain compositions. CD spectra identified a surprisingly rich conformational behavior of selected miniamyloids. We observed a highly cooperative conformational transition of ß-sheet to α-helix upon the addition of the helix enforcing co-solvent hexafluoroisopropanol. The CD curves of dimer 9 resembled, in a completely reversible manner, the CD spectra measured during the irreversible fibrillation of the parent Aß(1-40). Synthetic peptide epitopes with high affinities for nAbs-Aß are needed to identify the physiological roles of nAbs-Aß and are promising epitopes for vaccination experiments.

Prion peptide uptake in microglial cells--the effect of naturally occurring autoantibodies against prion protein.

In prion disease, a profound microglial activation that precedes neurodegeneration has been observed in the CNS. It is still not fully elucidated whether microglial activation has beneficial effects in terms of prion clearance or whether microglial cells have a mainly detrimental function through the release of pro-inflammatory cytokines. To date, no disease-modifying therapy exists. Several immunization attempts have been performed as one therapeutic approach. Recently, naturally occurring autoantibodies against the prion protein (nAbs-PrP) have been detected. These autoantibodies are able to break down fibrils of the most commonly used mutant prion variant PrP106-126 A117V and prevent PrP106-126 A117V-induced toxicity in primary neurons. In this study, we examined the phagocytosis of the prion peptide PrP106-126 A117V by primary microglial cells and the effect of nAbs-PrP on microglia. nAbs-PrP considerably enhanced the uptake of PrP106-126 A117V without inducing an inflammatory response in microglial cells. PrP106-126 A117V uptake was at least partially mediated through scavenger receptors. Phagocytosis of PrP106-126 A117V with nAbs-PrP was inhibited by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, indicating a separate uptake mechanism for nAbs-PrP mediated phagocytosis. These data suggest the possible mechanisms of action of nAbs-PrP in prion disease.

Immunotherapy in prion disease.

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, describe a group of fatal neurodegenerative disorders affecting both humans and animals. Accumulation of misfolded prion proteins is the pathological hallmark of these disorders; such accumulation occurs in lymphoreticular tissue prior to CNS involvement in scrapie, experimental models and human variant Creutzfeldt-Jakob disease. Lymphoreticular accumulation of misfolded prion protein has not been demonstrated in human sporadic or genetic forms of TSE. Once clinical symptoms develop, all prion disorders have a rapidly progressive and lethal course, and no effective therapy exists. In the past 10 years, antibody-based immunotherapy has been considered for other neurodegenerative disorders associated with protein misfolding and, therefore, might also be an effective approach to prevention or treatment of prion disease. Self-tolerance to endogenous prion protein is, however, a major challenge to the development of effective immunotherapy, as is the risk of adverse effects from active immunization. This Review summarizes the evidence that immunization could slow disease progression or increase lifespan in animal models of prion diseases. The therapeutic potential of these strategies in treating patients with prion diseases is also discussed.

Cytomegalovirus upregulates vascular endothelial growth factor and its second cellular kinase domain receptor in human fibroblasts.

Human cytomegalovirus (HCMV) activation and elevated levels of vascular endothelial growth factor (VEGF) have been found to be associated with transplant rejection. However, information is lacking about whether elevated levels of this multifunctional factor are directly due to viral activation, or if they derive from impurities within the culture supernatant of infected cell cultures. We used purified as well as unpurified viral stocks to infect human fibroblasts in vitro and applied PCR, Western blot, ELISA, and immunofluorescence staining to investigate the expression of VEGF and its receptors. Our data suggest that HCMV infection triggers an early and sustained induction of VEGF and kinase insert domain receptor (KDR) mRNAs, whereas transcript levels of FLT-1 remain unchanged by viral infection. Analysis of the extracellular VEGF and cellular KDR protein expression after infection with purified and unpurified HCMV strains AD169 and TOLEDO showed, in clear contrast to UV-inactivated virus preparations, an increased release of VEGF and KDR proteins. In addition, immunofluorescence revealed that HCMV infection was also accompanied by a profound increase in intracellular VEGF and KDR levels. These results confirm that active HCMV infection is required to induce VEGF and the most important VEGF receptor KDR, and that the upregulation of VEGF and KDR are a direct viral effect and not a secondary effect mediated by inflammatory cytokines within the supernatant. The HCMV-dependent upregulation of VEGF and KDR contributes to the theory that viral-induced immune mediators play a key role in transplant rejection.

Human anti-prion antibodies block prion peptide fibril formation and neurotoxicity.

Prion diseases are a group of rare, fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K-resistant conformer, termed scrapie PrP (PrP(Sc)). Aggregates of PrP(Sc) deposited around neurons lead to neuropathological alterations. Currently, there is no effective treatment for these fatal illnesses; thus, the development of an effective therapy is a priority. PrP peptide-based ELISA assay methods were developed for detection and immunoaffinity chromatography capture was developed for purification of naturally occurring PrP peptide autoantibodies present in human CSF, individual donor serum, and commercial preparations of pooled intravenous immunoglobulin (IVIg). The ratio of anti-PrP autoantibodies (PrP-AA) to total IgG was ∼1:1200. The binding epitope of purified PrP-AA was mapped to an N-terminal region comprising the PrP amino acid sequence KTNMK. Purified PrP-AA potently blocked fibril formation by a toxic 21-amino acid fragment of the PrP peptide containing the amino acid alanine to valine substitution corresponding to position 117 of the full-length peptide (A117V). Furthermore, PrP-AA attenuated the neurotoxicity of PrP(A117V) and wild-type peptides in rat cerebellar granule neuron (CGN) cultures. In contrast, IgG preparations depleted of PrP-AA had little effect on PrP fibril formation or PrP neurotoxicity. The specificity of PrP-AA was demonstrated by immunoprecipitating PrP protein in brain tissues of transgenic mice expressing the human PrP(A117V) epitope and Sc237 hamster. Based on these intriguing findings, it is suggested that human PrP-AA may be useful for interfering with the pathogenic effects of pathogenic prion proteins and, thereby has the potential to be an effective means for preventing or attenuating human prion disease progression.