Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor.

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.

Cloning of Affibody Libraries for Display Methods.

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.

Selection of Affibody Molecules Using Escherichia coli Display.

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.

Selection of Affibody Molecules Using Phage Display.

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.

Selection of Affibody Molecules Using Staphylococcal Display.

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.

Engineering of Affibody Molecules.

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.

Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting.

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived proteins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naïve affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recognition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide technology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.

Construction and Validation of a New Naïve Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides.

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.

Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aß-Affibodies.

Affibodies targeting amyloid-beta (Aß) could potentially be used as therapeutic and diagnostic agents in Alzheimer's disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aß protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood-brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aß deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aß and mTfR1 affinity resulted in low intrabrain retention around Aß deposits.

Are Farming and Animal Exposure Risk Factors for the Development of Granulomatosis With Polyangiitis? Environmental Risk Factors Revisited: A Case-control Study.

OBJECTIVE: To investigate the possible association between animal exposure and risk for granulomatosis with polyangiitis (GPA). METHODS: Patients with GPA at the Department of Rheumatology, Uppsala University Hospital, between January 1, 2011, and December 31, 2018, were consecutively included. All patients filled in a questionnaire on possible environmental exposures: occupation, hobbies, and animal contact. As controls we included 128 patients with rheumatoid arthritis (RA) and 248 population controls collected from the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study, matched for age, sex, and geographical area of residence. The controls filled out a questionnaire on current and past contact with farming and animals, at the time of the RA patient's diagnosis. RESULTS: A total of 62 patients with GPA, 128 patients with RA, and 248 population controls were included in the study. GPA was significantly associated with horse exposure, with a 2- to 3-fold increased risk compared with RA (OR 3.08, 95% CI 1.34-7.08) and population controls (OR 2.61, 95% CI 1.29-5.29). Borderline increased risks were found for any animal contact, but no association was found when analyzing contact with cats/dogs only. A significant association was found between GPA and farming compared to the population controls (OR 7.60, 95% CI 3.21-17.93). CONCLUSION: This study has identified for the first time, to our knowledge, a significant association between exposure to specific animals, namely horses, and the development of GPA. The results also support previous studies reporting an association between farming and GPA, underscoring the possibility of exogenous factors as initiators in the development of GPA.

Pulmonary toxicity of synthetic amorphous silica - effects of porosity and copper oxide doping.

Materials can be modified for improved functionality. Our aim was to test whether pulmonary toxicity of silica nanomaterials is increased by the introduction of: a) porosity; and b) surface doping with CuO; and whether c) these modifications act synergistically. Mice were exposed by intratracheal instillation and for some doses also oropharyngeal aspiration to: 1) solid silica 100 nm; 2) porous silica 100 nm; 3) porous silica 100 nm with CuO doping; 4) solid silica 300 nm; 5) porous silica 300 nm; 6) solid silica 300 nm with CuO doping; 7) porous silica 300 nm with CuO doping; 8) CuO nanoparticles 9.8 nm; or 9) carbon black Printex 90 as benchmark. Based on a pilot study, dose levels were between 0.5 and 162 µg/mouse (0.2 and 8.1 mg/kg bw). Endpoints included pulmonary inflammation (neutrophil numbers in bronchoalveolar fluid), acute phase response, histopathology, and genotoxicity assessed by the comet assay, micronucleus test, and the gamma-H2AX assay. The porous silica materials induced greater pulmonary inflammation than their solid counterparts. A similar pattern was seen for acute phase response induction and histologic changes. This could be explained by a higher specific surface area per mass unit for the most toxic particles. CuO doping further increased the acute phase response normalized according to the deposited surface area. We identified no consistent evidence of synergism between surface area and CuO doping. In conclusion, porosity and CuO doping each increased the toxicity of silica nanomaterials and there was no indication of synergy when the modifications co-occurred.

Short-term oral administration of non-porous and mesoporous silica did not induce local or systemic toxicity in mice.

In this study, two sets of methyl-coated non-porous and mesoporous amorphous silica materials of two target sizes (100 and 300 nm; 10-844 m2/g) were used to investigate the potential role of specific surface area (SSA) and porosity on the oral toxicity in mice. Female Swiss mice were administered by oral gavage for 5 consecutive days. Two silica dose levels (100 and 1000 mg/kg b.w.) were tested for all four materials. All dispersions were characterized by transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). Batch dispersions of porous silica were rather unstable due to agglomeration. Animals were sacrificed one day after the last administration or after a three-week recovery period. No relevant toxicological effects were induced by any of the silica materials tested, as evaluated by body weight, gross pathology, relative organ weights (liver, spleen, kidneys), hematology, blood biochemistry, genotoxicity (Comet assay in jejunum cells and micronucleus test in peripheral blood erythrocytes), liver and small intestine histopathology, and intestinal inflammation. The presence of silica particles in the intestine was evaluated by a hyperspectral imaging microscopy system (CytoViva) using histological samples of jejunum tissue. Silica spectral signatures were found in jejunum samples with all the treatments, but only statistically significant in one of the treatment groups.

An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor.

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 (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 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 ZSYM73. 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.

Affibody-Mediated Sequestration of Amyloid ß Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model.

Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid ß (anti-Aß) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of Aß-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted ZSYM73-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric ZSYM73 affibody for sequestering of monomeric Aß-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with ZSYM73-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric Aß, demonstrating a therapeutic potential for prevention of AD.

Nanofibrillated cellulose causes acute pulmonary inflammation that subsides within a month.

Nanofibrillated cellulose (NFC) is a renewable nanomaterial that has beneficial uses in various applications such as packaging materials and paper. Like carbon nanotubes (CNT), NFCs have high aspect ratio and favorable mechanical properties. The aspect ratio also rises a concern whether NFC could pose a health risk and induce pathologies, similar to those triggered by multi-walled CNT. In this study, we explored the immunomodulatory properties of four NFCs in vitro and in vivo, and compared the results with data on bulk-sized cellulose fibrils and rigid multi-walled CNT (rCNT). Two of the NFCs were non-functionalized and two were carboxymethylated or carboxylated. We investigated the production of pro-inflammatory cytokines in differentiated THP-1 cells, and studied the pulmonary effects and biopersistence of the materials in mice. Our results demonstrate that one of the non-functionalized NFCs tested reduced cell viability and triggered pro-inflammatory reactions in vitro. In contrast, all cellulose materials induced innate immunity response in vivo 24 h after oropharyngeal aspiration, and the non-functionalized NFCs additionally caused features of Th2-type inflammation. Modest immune reactions were also seen after 28 days, however, the effects were markedly attenuated as compared with the ones after 24 h. Cellulose materials were not cleared within 1 month, as demonstrated by their presence in the exposed lungs. All effects of NFC were modest as compared with those induced by rCNT. NFC-induced responses were similar or exceeded those triggered by bulk-sized cellulose. These data provide new information about the biodurability and pulmonary effects of different NFCs; this knowledge can be useful in the risk assessment of cellulose materials.

Identification of proteins that specifically recognize and bind protofibrillar aggregates of amyloid-ß.

Protofibrils of the 42 amino acids long amyloid-ß peptide are transient pre-fibrillar intermediates in the process of peptide aggregation into amyloid plaques and are thought to play a critical role in the pathology of Alzheimer's disease. Hence, there is a need for research reagents and potential diagnostic reagents for detection and imaging of such aggregates. Here we describe an in vitro selection of Affibody molecules that bind to protofibrils of Aß42cc, which is a stable engineered mimic of wild type Aß42 protofibrils. Several binders were identified that bind Aß42cc protofibrils with low nanomolar affinities, and which also recognize wild type Aß42 protofibrils. Dimeric head-to-tail fusion proteins with subnanomolar binding affinities, and very slow dissociation off-rates, were also constructed. A mapping of the chemical properties of the side chains onto the Affibody scaffold surface reveals three distinct adjacent surface areas of positively charged surface, nonpolar surface and a polar surface, which presumably match a corresponding surface epitope on the protofibrils. The results demonstrate that the engineered Aß42cc is a suitable antigen for directed evolution of affinity reagents with specificity for wild type Aß42 protofibrils.

Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method.

Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-ß (Aß) peptide in fusion to green-fluorescent protein (GFP), and an Aß aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aß aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.

Effect of particle size and dispersion status on cytotoxicity and genotoxicity of zinc oxide in human bronchial epithelial cells.

Data available on the genotoxicity of zinc oxide (ZnO) nanoparticles (NPs) are controversial. Here, we examined the effects of particle size and dispersion status on the cytotoxicity and genotoxicity of nanosized and fine ZnO, in the presence and absence of bovine serum albumin (BSA; 0.06%) in human bronchial epithelial BEAS-2B cells. Dynamic light scattering analysis showed the most homogenous dispersions in water alone for nanosized ZnO and in water with BSA for fine ZnO. After a 48-h treatment, both types of ZnO were cytotoxic within a similar, narrow dose range (1.5-3.0µg/cm(2)) and induced micronuclei at a near toxic dose range (1.25-1.75µg/cm(2)), both with and without BSA. In the comet assay, nanosized ZnO (1.25-1.5µg/cm(2)), in the absence of BSA, caused a statistically significant increase in DNA damage after 3-h and 6-h treatments, while fine ZnO did not. Our findings may be explained by better uptake or faster intracellular dissolution of nanosized ZnO without BSA during short treatments (3-6h; the comet assay), with less differences between the two ZnO forms after longer treatments (>48h; the in vitro micronucleus test). As ZnO is genotoxic within a narrow dose range partly overlapping with cytotoxic doses, small experimental differences e.g. in the dispersion of ZnO particles may have a substantial effect on the genotoxicity of the nominal doses added to the cell culture.

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes.

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1-200 µg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2-10.8 mg/m(3)) for 4 d, 4 h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1-200 µg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m(3)). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 µg/cm(2)), while MWCNT-T increased strand breakage only at 200 µg/cm(2). Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.

The amyloid hypothesis suggests that accumulation of amyloid ß (Aß) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aß aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aß peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aß peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aß-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aß with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aß1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aß peptide.