Structural basis for a PABPN1 aggregation-preventing antibody fragment in OPMD.

Oculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle. 3F5 llama antibody specifically interferes with the PABPN1 aggregation process in vitro and in vivo. To understand the structural basis for its epitope recognition we mapped the binding interface of 3F5 with PABPN1 and provide a structural model of the 3F5-PABPN1 complex. We show that 3F5 complementarity determining regions create a cavity in which PABPN1 alpha-helix domain resides by involving critical residues previously implicated in the aggregation process. These results may increase our understanding of the PABPN1 aggregation mechanism and the therapeutic potential of 3F5.

Castration induces autoantibody and T cell responses that correlate with inferior outcomes in an androgen-dependent murine tumor model.

We recently reported that hormone therapy induces antigen-specific autoantibody responses in prostate cancer patients. However, the contribution of autoantibody responses to clinical outcomes is unknown. We used an animal model to test the hypothesis that hormone therapy-induced immune responses may be associated with delayed tumor recurrence. Male DD/S mice bearing established tumors from the androgen-dependent Shionogi carcinoma line were castrated to induce tumor regression. Tumor-specific autoantibody responses were measured by immunoblot, and the underlying antigen was identified by serological screening of a cDNA expression library. T cell responses were assessed by immunohistochemistry and IFN-gamma ELISPOT. Following castration, 97% of mice underwent complete tumor regression. Of these, 72% experienced tumor recurrence 18-79 days postcastration, whereas the remaining 28% remained tumor-free for the duration of the experiment. In 55% of mice, castration induced autoantibody responses to an antigen identified as poly(A) binding protein nuclear 1 (PABPN1). Castration also induced PABPN1-specific T cell responses, which were highly correlated to autoantibody responses, and this was accompanied by dense infiltration of tumors by CD3+ T cells 1-2 weeks after castration. Unexpectedly, mice that developed autoantibody and T cell responses to PABPN1 showed a higher rate and shorter latency of tumor recurrence. In mice with recurrent tumors, T cell responses to PABPN1 were still detectable; however, T cell infiltrates were restricted to the peripheral stroma of tumors. In conclusion, castration-induced immune responses are associated with inferior outcomes in the Shionogi carcinoma model, raising concerns about the influence of treatment-induced immune responses on clinical outcomes in humans.

Prevention of oculopharyngeal muscular dystrophy by muscular expression of Llama single-chain intrabodies in vivo.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disorder characterized by progressive weakening of specific muscles. It is caused by short expansions of the N-terminal polyalanine tract in the poly(A) binding protein nuclear 1 (PABPN1), and it belongs to the group of protein aggregation diseases, such as Huntington's, Parkinson's and Alzheimer diseases. Mutant PABPN1 forms nuclear aggregates in diseased muscles in both patients and animal models. Intrabodies are antibodies that are modified to be expressed intracellularly and target specific antigens in subcellular locations. They are commonly generated by artificially linking the variable domains of antibody heavy and light chains. However, natural single-chain antibodies are produced in Camelids and, when engineered, combined the advantages of being single-chain, small sized and very stable. Here, we determine the in vivo efficiency of Llama intrabodies against PABPN1, using the established Drosophila model of OPMD. Among six anti-PABPN1 intrabodies expressed in muscle nuclei, we identify one as a strong suppressor of OPMD muscle degeneration in Drosophila, leading to nearly complete rescue. Expression of this intrabody affects PABPN1 aggregation and restores muscle gene expression. This approach promotes the identification of intrabodies with high therapeutic value and highlights the potential of natural single-chain intrabodies in treating protein aggregation diseases.