Development of new affinity proteins for neurodegenerative disorders
Time: Fri 2023-03-24 10.00
Location: F3, Lindstedtsvägen 26 & 28, Stockholm
Video link: https://kth-se.zoom.us/j/65440212256
Subject area: Biotechnology
Doctoral student: Linnea C. Hjelm , Proteinvetenskap
Opponent: Professor Ylva Ivarsson, Uppsala Universitet
Supervisor: Professor John Löfblom, Proteinvetenskap; Professor Stefan Ståhl, Proteinvetenskap; Doktor Hanna Lindberg, Proteinvetenskap
Neurodegenerative disorders include a full spectrum of diagnoses, including dementias and other neuronal diseases, characterised by degradation of neurons in the brain occurring along with disease progression. Amongst the dementias, the most prevalent are Alzheimer’s (AD) and Parkinson’s disease (PD) that affect millions of people worldwide. During the last years, advancements in potential treatments have been made where the first two clinical antibodies have been approved by the US Food and Drug Administration (FDA) for a disease modifying effect on Alzheimer’s disease.
As alternatives to antibodies, other types of affinity reagents that are based on non-immunoglobulin protein scaffolds are also investigated. Such alternative scaffolds often demonstrate distinct and complementary properties compared to antibodies. In this thesis, the development of a new type of affinity protein scaffold called sequestrin is described. Sequestrins are derived from the affibody molecule and comprise two heterogenic subunits with truncated N-terminals fused as a head-to-tail construct. Sequestrins undergo a structural rearrangement upon target binding and forms a stabile complex. The scaffold is designed for interactions with disease-related amyloidogenic peptides e.g. amyloid beta and alpha-synuclein involved in AD and PD, respectively. In the first paper, a sequestrin library was developed and its compatibility with phage display was investigated. Successful panning against the amyloid beta peptide resulted in binders with high affinity. Further on in paper II, the alpha-synuclein peptide was targeted and sequestrins with low nanomolar affinities were obtained. All sequestrins displayed structural rearrangement upon target engagement, which stabilized the interaction to the target peptides and further inhibited toxic aggregation, opening up for future studies of disease modifying effects in vivo.
When targeting the brain, passage through the blood–brain barrier (BBB) is an obstacle that needs to be addressed to reach sufficiently high therapeutic concentrations. To overcome this barrier, brain shuttles have been developed with the capability to transport a cargo over the BBB. One such mechanism of transportation is by receptor-mediated transcytosis, which is utilized by e.g. the transferrin receptor (TfR). In paper III, a TfR-targeting shuttle was investigated for BBB passage when fused to a sequestrin targeting the amyloid beta peptide, resulting in a higher penetration through the BBB, and maintained functionality of the sequestrin.
High-throughput in vitro methods would facilitate development of novel brain shuttles. Thus, in paper IV, a transwell system based on nanofibrillar silkmembranes with murine brain endothelial cells was developed. Evaluation of the method using a TfR-specific antibody demonstrated higher transfer over the barrier compared to an isotype control and the method has potential to facilitate screening of transcytosis capability of brain shuttles.
In paper V, TfR-specific affibody-based brain shuttles were developed and investigated for transcytosis capability using the in vitro transcytosis assay. A panel of affibody molecules were evaluated, demonstrating both cross-species reactivity to murine and human TfR and active receptor-mediated transcytosis. These candidates could thus potentially be used in further development of CNS-targeting therapeutics.
In conclusion, a new sequestrin scaffold was developed that can be utilised for targeting amyloidogenic peptides found in neurodegenerative disorders. An affibody-based brain shuttle was also developed, which showed transcytosis capability. In the future, the new brain shuttle might be combined with sequestrins to create multifunctional fusion proteins for facilitated delivery over the BBB, which hopefully can result in therapeutic concentrations in the brain even when administered with a lower dosage.