mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121-231. Both mAbs were cross-reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137-143 of MoPrP and buried in PrP C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP Sc in cultured scrapie-infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132-217 and this epitope was exposed on the cell surface PrP C . mAb T2 showed an excellent inhibitory effect on PrP Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector.
Coculturing of ScN2a cells with scFv T2-producing N2a58 cells induced a clear inhibitory effect on PrPSc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP Sc accumulation.Key words anti-prion effect, monoclonal antibody, single-chain fragment variable region.Prion diseases, also called transmissible spongiform encephalopathies, are a group of neurodegenerative disorders, which includes BSE in cattle, CWD in deer and elk, scrapie in sheep and goats, and CJD in humans. The recent emergence of a new human prion disease, variant CJD, which is thought to result from consumption of BSEinfected materials by humans (1), is a major public health and safety issue (2, 3). The infectious agent, or prion, is mainly composed of PrP Sc , the detergent-insoluble and partially protease-resistant isoform of the host-encoded Anti-prion effect by scFv PrP Sc accumulation in prion-infected cell cultures, presumably by disrupting the PrP C -PrP Sc interaction (6, 7) and transgenic expression of anti-PrP mAb fragments prevents prion pathogenesis in mice (8). However, administration of mAbs has resulted in prevention of prion pathogenesis only when applied simultaneously, or shortly after, peripheral prion infection (9). This is probably due to poor diffusion of administered antibodies from vessels into tissues, particularly into the CNS. One study has demonstrated apoptosis of hippocampal and cerebellar neurons following intracerebral injection of mAbs reactive with PrP C (10), indicating that cross-linking or clustering PrP C by the anti-PrP antibody may trigger an abnormal signaling pathway. One possible means of delivering antiPrP antibodies efficiently into the CNS, and preventing undesirable adverse effects of such administration, is scFv antibodies, in which the polypeptides of antibody variable regions essential to antigen-binding are genetically linked through a polypeptide linker sequence (11-13). Because of its large size, a whole antibody molecule, with its relatively complex structure consisting of heavy and light polypeptide chains, is unsuitable for efficient gene manipulation with vectors. H...