A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity

Abstract

The amyloid hypothesis suggests that accumulation of amyloid b (Ab) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Ab aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Ab peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Ab peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Ab-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Ab with a K D value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Ab 1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Ab peptide. Biotechnology JournalBiotechnol. J. 2015, 10, 1707-1718 therapies [8,16,17]. Alternative scaffold-proteins are in general much smaller than full-length antibodies and can usually be engineered into multivalent as well as multispecific units with an overall size that remain smaller than the conventional antibody [17,18]. Such features can potentially improve in vivo biodistribution of the agent [19][20][21][22]. One type alternative binding-protein that has been investigated for various diagnostic and therapeutic applications is the small three-helical bundle Affibody molecule (6.5 kDa) [17,23]. We previously generated an Affibody molecule (denoted Z Ab3 ) targeting monomeric Ab with a 17 nM affinity, as determined by isothermal titration calorimetry (ITC) and confirmed by surface plasmon resonance SPR [24][25][26]. Although this binder is based on the Affibody scaffold, it has evolved to adopt a unique and complex binding mechanism that is potentially more efficient for interactions with aggregation-prone peptides. Structural analysis has shown that two identical disulfide-linked Affibody units encapsulate one Ab peptide, and that both the Affibody domains and the Ab peptide undergo structural rearrangement upon binding. The Ab peptide folds into a b-hairpin structure, allowing the first α-helices in both Affibody molecules to adopt a b-sheet structure with unstructured N-termini [25,27]. In a recent in vivo study using an Ab-transgenic fruit fly model of AD, it was demonstrated that the Z Ab3 Affibody molecule efficiently inhibits the formation of Ab aggregates, thereby abolishing the neurotoxic effects and restoring the life span of the flies [25,28].For further po...

Affibody molecules as engineered protein drugs

“…Improved binders could be isolated and the best had a 50-fold improved affinity with around 300 p M KD. 81 This binder was fused to an ABD and tested in vivo in a double transgenic mouse model of AD and shown to efficiently protect the mice from Abeta-induced pathology. 81 …”

Amyloid-β oligomers are captured by the DNAJB6 chaperone: Direct detection of interactions that can prevent primary nucleation

“…The capture of the Aβ oligomers and their removal from solution, which does not occur to the same extent in the DNAJB6 ST18A-mutant, is obviously dependent on S/T-residues in the chaperone, which we believe form intermolecular hydrogen bonds to Aβ. This is reminiscent of how Aβ peptides are captured by ZAβ3-related affibodies (35,36). In such systems hydrogen bonds are formed between the affibody and the Aβ backbone, forming a complex where a monomeric Aβ peptide is captured in its βhairpin state.…”

Developing therapeutic vaccines against Alzheimer’s disease

“…The mechanistic strategy of using the peripheral sink, can also be achieved by other sAβ binding proteins. An example is the use of Affibodies that specifically bind monomeric Aβ, which are in preclinical development [73,74]. …”