Misfolding and aggregation of the neuronal protein alpha-synuclein (aSyn) has been identified as a hallmark of Parkinson’s disease (PD) pathology and other synucleinopathies. Preventing formation of intracellular aSyn accumulations constitutes a therapeutic strategy against disease development. We recently reported on a new type of affinity protein, denoted Sequestrin, aimed for efficient and stable interactions with aggregation-prone amyloidogenic proteins and peptides. Upon binding, sequestrins interact with the aggregation-prone peptide and form a stabilizing four-stranded beta sheet with similarities to the beta sheet rich structures seen in amyloid fibrils. Here, high-affinity aSyn-binding sequestrins were isolated from a large naïve sequestrin library using phage display technology. The best binders demonstrated dissociation constant, KD, values in the 10 nM-range, and structural rearrangements in both the sequestrin and aSyn protein upon binding. Modelling using AlphaFold, followed by NMR spectroscopy suggested that the sequestrins bind an N-terminal region of aSyn that is critical for amyloidogenic aggregation. In an in vitro aggregation study, the sequestrins demonstrated complete inhibition of aSyn aggregation at equimolar concentrations, including the three familial mutants A30P, E46K, and A53T that are associated with Parkinson’s disease and Lewy body dementia.

Affibody molecules are small (6-kDa) affinity proteins folded in a three-helical bundle and generated by directed evolution for specific binding to various target molecules. The most advanced affibody molecules are currently tested in the clinic, and data from more than 300 subjects show excellent activity and safety profiles. The generation of affibody molecules against a particular target starts with the generation of an affibody library, which can then be used for panning using multiple methods and selection systems. This protocol describes the molecular cloning of DNA-encoded affibody libraries to a display vector of choice, for either phage, Escherichia coli, or Staphylococcus carnosus display. The DNA library can come from different sources, such as error-prone polymerase chain reaction (PCR), molecular shuffling of mutations from previous selections, or, more commonly, from DNA synthesis using various methods. Restriction enzyme-based subcloning is the most common strategy for affibody libraries of higher diversity (e.g., >10⁷ variants) and is described here.

Affibody molecules are small, robust, and versatile affinity proteins currently being explored for therapeutic, diagnostic, and biotechnological applications. Surface-exposed residues on the affibody scaffold are randomized to create large affibody libraries from which novel binding specificities to virtually any protein target can be generated using combinatorial protein engineering. Affibody molecules have the potential to complement—or even surpass—current antibody-based technologies, exhibiting multiple desirable properties, such as high stability, affinity, and specificity, efficient tissue penetration, and straightforward modular extension of functional domains. It has been shown in both preclinical and clinical studies that affibody molecules are safe, efficacious, and valuable alternatives to antibodies for specific targeting in the context of in vivo diagnostics and therapy. Here, we provide a general background of affibody molecules, give examples of reported applications, and briefly summarize the methodology for affibody generation.

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for selection via multiple display methods. This protocol describes selection from affibody libraries by Escherichia coli cell surface display. With this method, high-diversity libraries of 1011 can be displayed on the cell surface. The method involves two steps for selection of binders from high-diversity libraries: magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS is used first to enrich the library in target-binding clones and to decrease diversity to a size that can be effectively screened and sorted in the flow cytometer in a reasonable time (typically <107 cells). The protocol is based on methodology using an AIDA-I autotransporter for display on the outer membrane, but the general procedures can also be adjusted and used for other types of autotransporters or alternative E. coli display methods.

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library. This is then followed by amplification of the library, which can then be used for biopanning using multiple methods. This protocol describes amplification of affibody libraries, followed by biopanning using phage display and analysis of the selection output. The general procedure is mainly for selection of first-generation affibody molecules from large naive (unbiased) libraries, typically yielding affibody hits with affinities in the low nanomolar range. For selection from affinity maturation libraries with the aim of isolating variants of even higher affinities, the procedure is similar, but parameters such as target concentration and washing are adjusted to achieve the proper stringency.

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus. Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed.

The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.

Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation.

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.

Neurodegenerativa sjukdomar är en samlingsterm för olika tillstånd som bland annat inkluderar demenssjukdomar som Alzheimers och Parkinsons sjukdom, där hjärnans neuroner degraderas med sjukdomsutvecklingen. Miljontals personer är drabbade av dessa sjukdomar och tyvärr finns det få tillgängliga behandlingar. Nyligen har två antikroppar godkänts av amerikanska läkemedelsverket (the US Food and Drug Administration, FDA) som läkemedel för behandling av Alzheimers sjukdom, och dessa två är de första i sitt slag som påverkar sjukdomsförloppet.

I denna avhandling beskrivs utvecklingen av en ny typ av proteiner som kallas sequestriner, vilka är speciellt lämpade för bindning till aggregeringsbenägna amyloida proteiner som ofta kopplas till neurodegenerativa sjukdomar. Strukturen hos sequestrinerna undersöktes för möjligheten att binda samt blockera aggregering av amyloid beta och alfa-synuklein, proteiner som förekommer i Alzheimers samt Parkinsons sjukdom. Utvecklingen av ett nytt sequestrinbiblioteket och dess kompabilitet för användning i selektion med fag-display mot amyloid beta beskrivs i artikel I och mot alfa-synuklein i artikel II. Sequestrinerna som togs fram uppvisade hög bindningsstyrka samt inhiberade aggregering av målproteinerna. Transport av molekyler över blod–hjärnbarriären är strikt reglerad, varmed det behövs speciella transportörer för att nå hjärnan. Essentiella molekyler som inte når hjärnan via adsorption eller diffusion kan transporteras via receptor-medierad transcytos, till exempel via transferrinreceptorn (TfR) som normalt tar upp transferrin som binder till järn. I artikel III undersöktes en tidigare version av en sequestrin som binder amyloid beta för dess möjlighet att länkas till en TfR- medierad transportör för att komma över blod–hjärnbarriären. Fusionsproteinet visade en bibehållen funktionalitet och ett ökat upptag till cerebrospinalvätskan i hjärnan, sett för den variant med länkad TfR-transportör jämfört med sequestrin utan transportör.

För att kunna utvärdera aktiv transcytos utvecklades en metod för att utvärdera och ranka denna egenskap in vitro. Detta kan göras genom en ”transwell”-metod där ett nanofiber-membran av rekombinant spindelsilke används för att stödja tillväxten av hjärn-endotelceller. Med hjälp av fluorescens kan därav transcytos studeras och utvärderas. I artikel IV kunde en tidigare validerad TfR-transportör urskiljas från negativa kontrollen i denna metod.

I artikel V beskrivs arbetet med att utveckla ett antal varianter av affibodymolekyler med målet att fungera som transportörer via TfR för transport in till hjärnan. Dessa affibodymolekyler karakteriserades för sin funktion att binda till både humant och murint cellulärt uttryckt TfR. Vidare visades det att även andra generationens affibodymolekyler för TfR kunde genomgå transcytos över barriärmodellen.

Sammanfattningsvis presenteras det i denna avhandling en utveckling av sequestrinstrukturen och framtagande av nya bindare för inhibering av aggregationsprocessen hos proteiner som frekvent återfinns inom neurodegenerativa sjukdomar, samt utvecklingen utav affibody-transportörer över blod–hjärnbarriären.

The blood-brain barrier (BBB) limits the uptake of central nervous system (CNS)-targeting drugs into the brain. Engineering molecular shuttles for active transportation across the barrier has thus potential for improving the efficacy of such drugs. In vitro assessment of potential transcytosis capability for engi-neered shuttle proteins facilitates ranking and the selection of promising candidates during develop-ment. Herein, the development of an assay based on brain endothelial cells cultured on permeable recombinant silk nanomembranes for screening of transcytosis capability of biomolecules is described. The silk nanomembranes supported growth of brain endothelial cells to form confluent monolayers with relevant cell morphology, and induced expression of tight-junction proteins. Evaluation of the assay using an established BBB shuttle antibody showed transcytosis over the membranes with an apparent permeability that significantly differed from the isotype control antibody.