Design and optimization of binding and solubility of anti-amyloid antibodies

Abstract

We identified specific types of mutations that enable grafted Aᵦ antibodies to specifically recognize Aᵦ fibrils relative to soluble monomers as well as other mutations that cause antibody binding to both conformers. Moreover, we found that the conditions used to prepare Aᵦ fibrils significantly affect the detection sensitivity of the grafted antibodies, as sonication of such fibrils (which reduces their size and generates new fibril ends) significantly increases antibody binding. This observation is consistent with the grafted Aᵦ antibodies mimicking the natural process of amyloid formation and binding to the ends of fibrils. Finally, we extended these approaches to generate grafted antibodies specific for amyloid aggregates of islet amyloid polypeptide that are associated with type 2 diabetes. We expect that our findings will be useful for generating even higher affinity grafted antibodies as well as using them for sensitive detection of amyloid aggregates and designing grafted antibodies specific for different types of amyloid aggregates.

A number of human disorders are associated with the formation of protein aggregates such as amyloid deposits. Antibodies that recognize amyloid aggregates with both conformational and sequence specificity have a number of potential diagnostic and therapeutic applications, but are challenging to generate using conventional antibody generation methods. We have developed methods for designing antibodies that bind specifically to amyloid-forming proteins (such as the Alzheimer’s Aᵦ peptide) via interactions that mimic those involved in natural amyloid aggregation. This approach involves grafting peptide segments that mediate amyloid formation into the complementarity-determining regions of antibody fragments, including single-domain antibodies. We have investigated the effects of inserting charged and uncharged polar mutations at the edges of loops containing hydrophobic Aᵦ peptide segments that mediate binding to Aᵦ aggregates. Interestingly, residues inserted at the edges of these binding loops strongly affect the solubility of the antibody domains as well as their ability to bind specifically to different aggregated and soluble Aᵦ conformers.