Loss-of-function mutations in the gene encoding progranulin (PGRN) are a common cause of frontotemporal dementia, leading to markedly decreased PGRN levels. A potential therapeutic strategy is therefore to increase extracellular PGRN by blocking sortilin-mediated PGRN clearance. Here, we describe the systematic design and optimization of small biparatopic sortilin-binding proteins based on the non-immunoglobulin affibody scaffold. Two anti-sortilin affibody molecules were genetically fused into a panel of heterodimeric constructs exploring multiple domain orientations, linker lengths, and helix truncations. In total, ten distinct dimer variants were generated and evaluated for binding and functional activity. Optimization of domain arrangement and truncations yielded constructs exhibiting subnanomolar sortilin affinities, corresponding to up to an approximately 45-fold improvement over the strongest monomeric affibody and pronounced avidity effects. In a PGRN clearance assay, the lead 18.6-kDa dimer efficiently increased extracellular PGRN levels with an EC50 value of 0.32 nM and produced substantially greater PGRN fold changes than monomeric constructs. This PGRN elevation was accompanied by marked reductions in both cell surface and total sortilin levels, consistent with effective receptor blockade and modulation. Together, these results demonstrate how systematic combination and geometric optimization of two individually moderate-affinity binders can generate a highly potent biparatopic inhibitor, as well as illustrating the versatility of compact and modular affibody molecules as building blocks in therapeutic protein design. Given the growing amount of evidence implicating sortilin in neurodegeneration, inflammation, and cancer, biparatopic affibody-based inhibitors may enable exploration of this pathway in diverse biological contexts, both as research tools and as starting points for drug development.

BACKGROUND: Heterozygous loss-of-function mutations in the gene encoding progranulin (GRN) are causative in around 5-10% of frontotemporal dementia (FTD) cases. These mutations lead to a more than 50% reduction of progranulin levels in the plasma and cerebrospinal fluid of mutation carriers compared to healthy controls. Increasing the progranulin levels in FTD-GRN patients via inhibition of sortilin-mediated progranulin degradation has shown promise as a therapeutic strategy, as exemplified by Alector's latozinemab, currently in phase 3 clinical trials. Here we detail the development of an anti-sortilin affibody-based miniprotein as a non-immunoglobulin alternative, offering potential advantages due to its small size and cost-effective production.

METHOD: Sortilin-binding affibodies were selected by phage display and subsequently genetically fused to short peptides derived from the progranulin C-terminus. The resulting miniproteins were characterized in terms of affinity, structure, stability, and their ability to increase extracellular progranulin levels in a clearance assay using progranulin-secreting, sortilin-expressing U-251 MG cells.

RESULT: A set of moderate-affinity sortilin-binding affibodies were obtained from phage display selections. Following genetic fusion with short peptides derived from the progranulin C-terminus and optimization of the fusion constructs, a lead candidate with 185 pM affinity for sortilin was obtained. The affibody-peptide fusion, but not its parental affibody or peptide, was capable of elevating extracellular progranulin levels in vitro with similar potency as latozinemab.

CONCLUSION: A sortilin-binding affibody-based miniprotein was developed and optimized, performing on par with latozinemab in in vitro functional studies. Affibody-based miniproteins provide a promising alternative to immunoglobulins in cases such as the present, where Fc functions are undesirable, and long-term high-dose antibody treatments risk becoming prohibitively expensive.

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.

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.

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.