Year : 2005 Paracrine inhibition of prion propagation by anti-PrP single-chain

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C). A growing body of evidence suggests that antibodies to PrP(C) can antagonize deposition of PrP(Sc). However, host tolerance hampers the induction of immune responses to PrP(C), and cross-linking of PrP(C) by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP(Sc), even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases. JOURNAL OF VIROLOGY, July 2005, p. 8330–8338 Vol. 79, No. 13 0022-538X/05/$08.00 0 doi:10.1128/JVI.79. 13.8330–8338.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Paracrine Inhibition of Prion Propagation by Anti-PrP Single-Chain Fv Miniantibodies Gaetano Donofrio,†‡ Frank L. Heppner,‡ Magdalini Polymenidou, Christine Musahl,§ and Adriano Aguzzi* Institute of Neuropathology, University Hospital Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland Received 5 February 2005/Accepted 3 March 2005 Prion diseases are characterized by the deposition of PrP, an abnormal form of the cellular prion protein PrP. A growing body of evidence suggests that antibodies to PrP can antagonize deposition of PrP. However, host tolerance hampers the induction of immune responses to PrP, and cross-linking of PrP by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP, even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases. Prion diseases, or transmissible spongiform encephalopathies, are invariably lethal neurodegenerative illnesses that affect humans and many animal species. They include bovine spongiform encephalopathy of cattle, chronic wasting disease of mule deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans (3). The causative agent is termed a prion (36) and was proposed to be identical to PrP, a pathological conformer of the cellular protein PrP encoded by the Prnp gene (31). PrP is expressed on the surfaces of almost all cells in the body but at particularly high levels on neurons in the peripheral and central nervous systems. PrP is essential for the development of prion disease (7), and Prnp mice, which lack PrP, are resistant to scrapie (8). The bovine spongiform encephalopathy epidemic (42) can be arguably deemed the direct consequence of flawed technology assessment and is caused by prions that were amplified through the bovine food chain (41). Transmission of bovine prions to humans has then given rise to variant CJD (44). At the time of this writing, Switzerland is experiencing an alarming increase in the incidence of a form of CJD which is clinically and biochemically indistinguishable from sporadic CJD and whose cause has not yet been conclusively determined (11, 12). Also, the recent increase in chronic wasting disease in North America (38) underlines the fact that prion diseases of farm and wildlife animals still represent a threat to public health. For all the reasons enumerated above, there is an urgent and growing need for efficient preventive and/or therapeutic measures against prion diseases (2). Prions use immune cells to gain access to the brain (14, 20, 35). On the other hand, several reports indicate that humoral immune responses against the prion protein can antagonize prion infections. This is true even when such responses are directed primarily against PrP and do not selectively target the disease-associated prion protein, PrP. For example, monoclonal antibodies directed against PrP have been shown to prevent de novo scrapie infection and to abrogate prion infectivity and PrP from chronically scrapie-infected neuroblastoma cells (10, 33). Transgenic expression (16) or peripheral administration of anti-PrP antibodies in mice (43) can arrest peripheral scrapie pathogenesis, and PrP-Fc fusion molecules interfere with prion replication (30). One major obstacle to devising efficacious regimens of active immunization is host tolerance to endogenous PrP that inhibits a host-derived anti-PrP antibody immune response. Active immunization attempts have thus far resulted in the induction of meager anti-PrP titers at best (13, 22, 34, 39). Accordingly, the biological efficacy of these immunization series was limited (34, 39), emphasizing the need for alternative