Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy.

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21Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion 22 diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe 23 scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi 24 combines the complementarity-determining regions of the neurotoxic antibody POM1 and the 25 neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. 26We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even 27 when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of 28 soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD 29and FT was more effective than concomitant treatment with the individual molecules or targeting 30 the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that 31 simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion 32 neurotoxicity and may represent a promising strategy for anti-prion immunotherapy. 33 34 Author summary 35Antibody immunotherapy is considered a viable strategy against prion disease. We previously 36showed that antibodies against the so-called globular domain of Prion Protein (PrP) can cause PrP 37 dependent neurotoxicity; this does not happen for antibodies against the flexible tail of PrP, which 38 therefore ought to be preferred for therapy. 39Here we show that simultaneous targeting of both globular domain and flexible tail by a bispecific, 40 combination of a toxic and a non-toxic antibody, results in stronger protection against prion 41 toxicity, even if the bispecific is administered when prion pathology is already conspicuous. 42We hypothesize that neurotoxicity arises from binding to specific "toxicity triggering sites" in the 43 globular domain. We designed our bispecific with two aims: i) occupying one such site and 44

ObjectiveTo determine whether naturally occurring autoantibodies against the prion protein are present in individuals with genetic prion disease mutations and controls, and if so, whether they are protective against prion disease.MethodsIn this case–control study, we collected 124 blood samples from individuals with a variety of pathogenic PRNP mutations and 78 control individuals with a positive family history of genetic prion disease but lacking disease-associated PRNP mutations. Antibody reactivity was measured using an indirect ELISA for the detection of human immunoglobulin G1–4 antibodies against wild-type human prion protein. Multivariate linear regression models were constructed to analyze differences in autoantibody reactivity between (1) PRNP mutation carriers vs controls and (2) asymptomatic vs symptomatic PRNP mutation carriers. Robustness of results was examined in matched cohorts.ResultsWe found that antibody reactivity was present in a subset of both PRNP mutation carriers and controls. Autoantibody levels were not influenced by PRNP mutation status or clinical manifestation of prion disease. Post hoc analyses showed anti-PrPC autoantibody titers to be independent of personal history of autoimmune disease and other immunologic disorders, as well as PRNP codon 129 polymorphism.ConclusionsPathogenic PRNP variants do not notably stimulate antibody-mediated anti-PrPC immunity. Anti-PrPC immunoglobulin G autoantibodies are not associated with the onset of prion disease. The presence of anti-PrPC autoantibodies in the general population without any disease-specific association suggests that relatively high titers of naturally occurring antibodies are well-tolerated.Clinicaltrials.gov identifierNCT02837705.