This project uses a novel approach to deal with alpha-synuclein, which accumulates abnormally in Parkinson’s disease (PD). Cutting-edge recombinant antibody technologies will yield anti-alpha-synuclein “nanobody” molecules that have the potential to serve as direct therapeutics and/or drug discovery tools for PD. The main target is the hydrophobic interaction region that is critical to abnormal alpha-synuclein aggregation. These nanobodies can then be used to manipulate localization, turnover and folding of this key protein in Parkinson’s disease.
In order to identify stable, high-affinity nanobodies against critical targets on the alpha-synuclein protein, we will begin with immunization of alpacas, which naturally produce stable heavy-chain only antibodies. Peptides that are specific to the hydrophobic region will be used, to allow the pre-clinical models to respond to a part of the protein that is not conventionally immunodominant. Once we have confirmed that the alpacas have mounted a vigorous immune response, RNA encoding all possible antibody heavy chain variable regions (nanobodies) from their blood cells will be cloned into more than one million individual phage particles. This library of nanobodies can then be biopanned to identify individual nanobody clones that recognize internal alpha-synuclein sequences, in some cases as specific conformations. Nanobodies will be validated in cell.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Nanobodies can be selected, engineered, and manipulated as genes, then delivered as either genes or proteins. They are more stable than more conventional intracellular antibody fragments, and more amenable to multiplexing. The target region will also offer specificity to alpha-synuclein. Since similar reagents are already in human clinical trials for other abnormal protein diseases, translation is highly feasible. Nanobodies can also serve as novel drug discovery tools identifying specific forms of alpha-synuclein.
The major output of this project will be a set of stable nanobodies that will have undergone affinity maturation in vivo. These nanobodies will target the hydrophobic interaction region of alpha-synuclein. Since this region is recognized as the critical site for aggregation, these reagents will serve as leads for direct therapeutics (delivered as genes or proteins), as well as for novel drug discovery. The phage display library itself will also serve as a resource.
We have completed immunization of two pre-clinical models with alpha-synuclein proteins plus the hydrophobic interaction region peptide. One of the two alpacas yielded very high titer antibodies against both targets. The process of highly-specific nanobody library construction is almost complete. In parallel, we have worked out a method for increasing both solubility and efficacy for a human VH nanobody that recognizes the critical alpha-synuclein interaction region. This method, which involves an engineered bifunctional fusion, will allow us to pursue multiple lead nanobodies in parallel. We are using the human fusion VH nanobody for a full set of protection assays against both intracellular and extracellular (secreted) alpha-synuclein. Biopanning of the highly immune model VHH phage display library will begin shortly, and we anticipate several additional lead nanobodies with stronger binding and/or binding to potentially even more protective forms of alpha-synuclein. The phage display library will also be shared with Parkinson’s disease investigators worldwide, to be used for target validation and rational drug design.