The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (Z_SYM73) via binding to the transferrin receptor (TfR). Z_SYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule Z_SYM73. Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.

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.

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 engineered shuttle proteins facilitates ranking and the selection of promising candidates during development. 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.

Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.

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.

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes, due to limited access to donor islets as well as low efficacy and poor reproducibility of the current procedure. Herein we describe a method to create islets-like composite clusters (co-clusters) from dispersed endocrine cells and supportive cells, with an attempt to more efficiently make use of the donor-derived material and improve compatibility with the recipient. Recombinant spider silk functionalized with cell binding motifs are used as 3D support mimicking the extracellular matrix. A cell binding motif derived from fibronectin, FN, was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein, 2R, increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like co-clusters. Both xenogenic and allogenic versions of these co-clusters were found viable and able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found co-localized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like co-clusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.