Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

Abstract

The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.Alzheimer | antibody | inhibitor | drug development | self-assembly

Apolipoprotein E impairs amyloid‐β fibril elongation and maturation

“…This is of potential interest since inhibiting the process of elongation also will prevent fibril maturation and instead populate intermediate assemblies. Interference with elongation has for this reason previously been suggested as a mechanistic target to be avoided in the quest to develop therapeutic agents [46]. This finding provides a mechanistic explanation to the observed formation of more amorphous assemblies here shown by TEM and also a possible route of formation to the circulating complexes between ApoE and Ab found in vivo [25,26,47,48].…”

“…Although speculative, the ability of ApoE to impair maturation of Ab amyloid fibrils could possibly populate more cytotoxic assemblies. Interfering with the elongation process has for this reason been suggested as a mechanism that should be avoided in the quest to develop inhibitory agents for Ab [46]. A potentiated cytotoxic effect on cells as a function of ApoE has recently been shown in support of the hypothesis [51].…”

Label‐free distribution of anti‐amyloid D‐AIP in Drosophila melanogaster: prevention of Aβ42‐induced toxicity without side effects in transgenic flies

“…); k d = 0.008 s −1 for single‐chain antibody fragment (‘I2’ anti‐amyloid candidate) binding to immobilized Aβ42 fibrils (Munke et al . ); k d = 0.026 s −1 for 7 amino acid cyclic peptide (‘CP2’ anti‐infective candidate) binding to immobilized hSPSB2 (Sadek et al . ).…”

Kinetic fingerprints differentiate anti-Aβ therapies

“…S1D,G). This route for therapeutic intervention may significantly reduce toxicity (10), and requires inhibitors that block the catalytic surface of fibrils, or bind to on-pathway oligomers to prevent their conversion to fibrils (10)(11)(12)25,28,29). Finally, inhibition of elongation (with rate constant k+) causes both a lag-phase extension and a reduction in transition slope (30), an effect that may increase toxicity over time (10, Fig.…”