== To evaluate our hypothesis that antibodies grafted with A amyloidogenic motifs would selectively recognize aggregated A conformers, we first sought to identify an antibody scaffold that is highly stable and tolerant to grafting diverse peptide segments into its CDR loops. neutralize the toxicity of each A conformer. We expect that our antibody design strategy is not limited to A and can be LEQ506 used to readily generate gammabodies against other toxic misfolded proteins. Keywords:misfolding, beta-amyloid, protein engineering A hallmark of protein misfolding disorders is that polypeptides of unrelated sequence fold into similar oligomeric and fibrillar assemblies that are cytotoxic (1). The structures of these enigmatic conformers have captured the imagination of many investigators who have sought to explain the molecular basis of proteotoxicity in conformational disorders such as Alzheimers disease. Because misfolded proteins are typically refractory to structural methods such as X-ray crystallography and solution NMR, few high-resolution structures of full-length misfolded proteins have been reported (ref.2and references therein). The structures of oligomeric intermediates have proven especially difficult to characterize because these conformers are labile, transient, and, in many cases, heterogeneous. Given the complexity of high-resolution structural analysis of misfolded proteins, alternative biochemical approaches are critical for understanding structurefunction relationships of aggregated proteins. A breakthrough in this area has been the development of conformation-specific antibodies that selectively recognize uniquely folded conformers of amyloidogenic proteins (313). Indeed, multiple conformation-specific antibodies have been reported that recognize structural features within amyloidogenic oligomers (5) and fibrils (4,6,8) in a sequence-independent manner. These and related antibodies have proven invaluable for identifying oligomeric and fibrillar conformers of several disease-linked proteins both in vitro and in vivo (313). The next important step LEQ506 in using antibodies to characterize misfolded proteins is to develop systematic approaches for generating conformation-specific antibodies that recognize sequence-specific epitopes within amyloidogenic proteins. The utility of such antibodies would be even greater if they recognized linear sequence epitopes (instead of discontinuous epitopes) within misfolded proteins because continuous epitopes are easier to identify and provide more direct structural information. To develop conformation-specific antibodies that target linear sequence epitopes, we sought to mimic the natural process of amyloid assembly that is commonly mediated via homotypic interactions between small amyloidogenic peptide segments within misfolded proteins (1417). We posited that grafting amyloidogenic motifs from the A42 peptide associated with Alzheimers disease into the complementarity determining regions (CDRs) of antibodies would generate antibody variants that selectively recognize aggregated A conformers but not A monomers. Moreover, we hypothesized that these antibodies would employ homotypic interactions between the grafted A motifs and the corresponding peptide segments within aggregated A conformers to mediate conformation-specific antibody recognition. Our hypotheses are motivated by the structure of A fibrils in which amyloidogenic peptide motifs stack in-register with identical motifs from other A molecules (1821), as well as the ability of A amyloidogenic motifs (by LEQ506 themselves or conjugated to other molecules) to inhibit A aggregation via homotypic interactions (2225). Our hypotheses are also motivated by the conformation-specific antibodies developed by Williamson and coworkers against the mammalian prion protein (PrP) (26). Although these full-length monoclonal antibodies displaying PrP peptide fragments recognize aggregated PrP conformers, it is Tlr2 unknown whether the LEQ506 antibodies use homotypic interactions to mediate PrP recognition because their binding sites were not determined. Moreover, the PrP-specific recognition of these antibodies is mediated primarily by electrostatic interactions (26,27). In contrast, we seek to exploit amyloidogenic (nonelectrostatic), homotypic interactions between grafted motifs.