A computationally pre-biased, dual-deselected picomolar DARPin targeting the conformational pore-forming epitope of A...

IF a computationally pre-biased DARPin library — with surface-randomized residues designed via Rosetta energy-function modeling to complement the transient hydrophobic beta-sheet edge epitope and antiparallel-sheet geometry unique to A11-reactive, pore-forming Aβ oligomers — is subjected to ribosome display selection using dual competitive deselection (simultaneous solution-phase competition against saturating concentrations of both Aβ monomers and OC-antibody-reactive fibrillar oligomers), yielding a picomolar-affinity pore-forming oligomer-selective DARPin (designated PFO-DARPin), and this PFO-DARPin is then formatted as a bispecific tandem-DARPin fusion incorporating a second, independently selected DARPin module binding transferrin receptor 1 (TfR1) at low-to-moderate affinity (~100–500 nM KD, tuned to permit transcytotic exocytosis rather than lysosomal trapping), and the resulting bispecific construct is administered intravenously (5 mg/kg, weekly, 8 weeks) to aged (18-month-old) male and female 5xFAD mice carrying high accumulated Aβ oligomer burden,

THEN selective clearance and functional neutralization of A11-reactive, pore-forming Aβ oligomers in the brain parenchyma — without significant depletion of Aβ monomers or alteration of plaque burden — will be observed, measured as: (1) ≥70% reduction in A11-immunoreactive species in cortical and hippocampal homogenates by conformation-specific sandwich ELISA; (2) restoration of hippocampal neuronal membrane integrity measured by calcein-AM/propidium iodide live-dead ratio in acute slice preparations; (3) ≥30% improvement in synaptic density assessed by synaptophysin/PSD-95 co-puncta immunofluorescence; and (4) improved spatial memory performance in Morris Water Maze relative to vehicle-treated 5xFAD controls — with Aβ monomer levels and total plaque burden by ThioS staining remaining statistically unchanged,

BECAUSE the following causal chain operates:

1. A11-reactive pore-forming oligomers display a structurally distinct conformational epitope — specifically, exposed hydrophobic patches and antiparallel or out-of-register beta-sheet edges — that is absent from both intrinsically disordered monomers and OC-reactive fibrillar oligomers whose parallel in-register beta-sheets are shielded within the fibril core. The Evidence Set (Section: "Structural Characterization of Toxic Amyloid Oligomers") establishes that A11 and OC antibodies recognise fundamentally non-overlapping conformational architectures, confirming these as physically separable epitope classes suitable for selective targeting.

2. Rosetta-guided pre-biasing of the DARPin library enriches starting diversity toward the pore-forming epitope before selection, reducing the effective library space that must be searched to find conformationally selective clones. Rather than relying entirely on unbiased random diversity, computational docking models of the antiparallel beta-sheet edge onto DARPin repeat surfaces (us...

SENS category: LysoSENS