Nonclinical Safety Assessment of a Long-Acting Recombinant PEGylated Factor Eight (BAY 94-9027) With a 60 kDa PEG.

BAY 94-9027 (Jivi) is a site-specifically PEGylated human B-domain-deleted (BDD) recombinant factor VIII (FVIII), with a 60 kDa branched PEG molecule attached. The nonclinical safety of BAY 94-9027 was evaluated in a toxicology program that included 2 weeks intravenous (IV) toxicity studies in rats and rabbits, a juvenile toxicity study in rats as well as a 26-week chronic study in rats. Doses of 75, 750, or 2250 IU/kg given every other day for 2 weeks did not elicit any findings related to BAY 94-9027. Specifically, no thrombus formation or histological changes such as cellular vacuolation were seen. In the chronic toxicity study, 40, 400, and 1200 IU/kg of BAY 94-9027 given twice weekly did not induce adverse effects related to BAY 94-9027, and no tissue vacuolation was observed. There was no PEG detected in choroid plexus or other areas of the brain, cerebrospinal fluid or in spleen or kidneys. These results were supported by toxicity studies in rats and rabbits treated with PEG 60 kDa attached to the maleimide linker (PEG-60-Mal-Cys). No findings related to PEG-60-Mal-Cys were seen. These results demonstrate the safety of BAY 94-9027 for long-term use.

Skin microbiota-associated inflammation precedes autoantibody induced tissue damage in experimental epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7). Immunization of SJL/J mice with recombinant murine COL7 results in break of tolerance and skin blisters. Strikingly, despite circulating autoantibodies, the same genetic background and identical environmental conditions, 20% of mice remain healthy. To elucidate the regulation of the transition from the presence of autoantibodies to overt autoimmune disease, we characterized the innate and adaptive immune response of mice that remain healthy after immunization and compared it to mice that developed skin disease. Both clinically healthy and diseased SJL/J mice showed circulating autoantibodies and deposition of complement-fixing IgG2c autoantibodies and C3 at the dermal-epidermal junction. However, only in diseased animals significant neutrophil infiltration and increase in FcgRIV expression were observed in the skin. In contrast, the expression of T cell signature cytokines in the T cell zone of the draining lymph node was comparable between clinically healthy and diseased animals after immunization. Surprisingly, health was associated with a decreased expression of CD11c, TNFA and KC (CXCL1) in the skin prior to immunization and could be predicted with a negative predictive value of >80%. Furthermore, mice that did not develop clinical disease showed a significantly higher richness and distinctly clustered diversity of their skin microbiota before immunization. Our data indicate that the decision whether blisters develop in the presence of autoantibodies is governed in the skin rather than in the lymph node, and that a greater richness of cutaneous bacterial species appears to be protective.

Activated CD4+ T cells enter the splenic T-cell zone and induce autoantibody-producing germinal centers through bystander activation.

CD4(+) T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4(+) T-cell subsets within different organ compartments. Such information is important because there are indications that CD4(+) T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4(+) T cells through the different compartments of the spleen. Resting and recently activated CD4(+) T cells were separated from thoracic duct lymph and activated CD4(+) T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4(+) T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependent T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner.

Complement-fixing anti-type VII collagen antibodies are induced in Th1-polarized lymph nodes of epidermolysis bullosa acquisita-susceptible mice.

The environment encountered in secondary lymphoid organs (e.g., lymph nodes) influences the outcome of immune responses. Immunization of mice with type VII collagen, an adhesion protein expressed at the cutaneous basement membrane, induces experimental epidermolysis bullosa acquisita (EBA). In this model, clinical disease is associated with the H2s haplotype of the MHC found in SJL/J mice. Most other strains (e.g., BALB/c, C57BL/6, NZM2410/J) are resistant to clinical disease, despite autoantibody production. Comparison of autoantibody response in EBA-resistant and -susceptible mice showed an IgG2-dominated response in the latter. We hypothesized that EBA susceptibility is due to specific cytokine gene expression in draining lymph nodes (dLN). To challenge this hypothesis, EBA-susceptible (SJL/J) and -resistant (BALB/c, C57BL/6) mice were immunized with type VII collagen, followed by analysis of clinical phenotype, subclasses of circulating and tissue-bound autoantibodies, complement activation, and cytokine gene expression in dLN. Disease manifestation was associated with induction of complement-fixing autoantibodies, confirming previous observations. Furthermore, however, IFN-γ/IL-4 ratio in dLN of EBA-susceptible mice was significantly increased compared with EBA-resistant strains, suggesting a Th1 polarization. Immunization of H2s-congenic C57BL/6 mice (B6.SJL-H2s) led to Th1 polarization in dLN and clinical disease. In addition to their cytokine milieu, EBA-susceptible and -resistant mice also differed regarding the expression of FcγR on peripheral leukocytes, in which a higher FcγRIV expression in SJL/J and B6.SJL-H2s mice, compared with C57BL/6, was associated with skin lesions. In summary, blistering in experimental EBA is regulated by both adaptive (divergent class switch recombination due to polarized cytokine expression) and innate (FcγR expression) immune mechanisms.

Epidermal Damage Induces Th1 Polarization and Defines the Site of Inflammation in Murine Epidermolysis Bullosa Acquisita.

Epidermolysis bullosa acquisita is an autoimmune skin disease characterized by subepidermal blisters. The pathogenesis is mediated by deposits of autoantibodies directed against type VII collagen in the skin, but the sequence of events regulating the localization of skin blisters is not fully understood. In this study, using the immunization-induced mouse model of epidermolysis bullosa acquisita, we demonstrate that epidermal disruption induces not only an infiltration of CD4+ T cells but also a T helper type 1 phenotype as it has been described for delayed-type hypersensitivity reactions. This T helper type 1 reaction was not found when different antigens were applied. Deep T-cell receptor ß profiling revealed shifts in the V/J gene usage only in epidermolysis bullosa acquisita, suggesting an infiltration of autoantigen-specific T cells. To target these autoantigen-specific T cells, we established an approach with which skin inflammation could be prevented without impairing the functionality of autoantibodies. We conclude that T-cell involvement in skin blistering diseases such as epidermolysis bullosa acquisita relates not only to T-cell help for B cells that produce pathogenic autoantibodies but also to autoreactive T helper type 1 effector cells that migrate into injured skin sites, exacerbate inflammation through production of inflammatory cytokines such as IFNγ, and prevent wound healing.

Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein-Barr virus replication and the simple tetracycline repressor.

We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100:1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24:1 and 34:1, respectively. With enhanced green fluorescent protein, a 23:1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable.

Generation of antibodies of distinct subclasses and specificity is linked to H2s in an active mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease, characterized by antibodies to type VII collagen (COL7). EBA can be induced in mice by immunization with a fragment of the non-collagenous 1 domain of murine COL7. Contrary to other autoimmune diseases, e.g., rheumatoid arthritis, little is known about the genetic susceptibility for EBA. We therefore used the EBA mouse model to address the hypothesis that disease induction depends on the major histocompatibility complex (MHC) haplotype. Mice from different inbred strains were immunized with recombinant murine COL7. Five distinct responses were observed: induction of (i) severe disease in SJL/J (H2s) and female MRL/MpJ (H2k), (ii) mild and transient disease in C57Bl/10.s (H2s), (iii) microscopic blistering in DBA/1J (H2q), (iv) only presence of non-pathogenic autoantibodies in C57Bl/6J (H2b), NZM2410/J (H2z), BXD2 (H2b), and male MRL/MpJ, and (v) complete resistance to EBA in NOD/ShiLtJ (H2g7) and C57Bl/10.q (H2q) mice. Overall, susceptibility to EBA was strongly associated with H2s. In addition, the diseased phenotype was associated with autoantibodies to specific regions of COL7. Our findings show that induction of antibodies with a distinct specificity is linked to the MHC haplotype in experimental EBA. Furthermore, our data are the basis for future studies with the goal of identifying non-MHC EBA susceptibility genes.