| 1. |
- Al-Amin, Rasel Abdullah, Researcher, 1983-, et al.
(författare)
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Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 98:28, s. 10054-10061
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Tidskriftsartikel (refereegranskat)abstract
- High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.
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| 2. |
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| 3. |
- Drews, Anna, et al.
(författare)
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Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations.
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| 4. |
- Dumoulin, Mireille, et al.
(författare)
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A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme.
- 2003
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 424:6950, s. 783-8
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues, often with serious pathological consequences. Non-neuropathic systemic amyloidosis is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H. Our structural studies reveal that the epitope includes neither the site of mutation nor most residues in the region of the protein structure that is destabilized by the mutation. Instead, the binding of the antibody fragment achieves its effect by restoring the structural cooperativity characteristic of the wild-type protein. This appears to occur at least in part through the transmission of long-range conformational effects to the interface between the two structural domains of the protein. Thus, reducing the ability of an amyloidogenic protein to form partly unfolded species can be an effective method of preventing its aggregation, suggesting approaches to the rational design of therapeutic agents directed against protein deposition diseases.
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| 5. |
- Fleetwood, Filippa, et al.
(författare)
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Surface display of a single-domain antibody library on Gram-positive bacteria
- 2013
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Nature. - 1420-682X .- 1420-9071. ; 70:6, s. 1081-1093
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Tidskriftsartikel (refereegranskat)abstract
- Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.
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| 6. |
- Hofström, Camilla, 1979-
(författare)
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Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.
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| 7. |
- Moonens, Kristof, et al.
(författare)
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Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori
- 2016
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Ingår i: Cell Host and Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 19:1, s. 55-66
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Tidskriftsartikel (refereegranskat)abstract
- The Helicobacter pylori adhesin BabA binds mucosal ABO/Le b blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.
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| 8. |
- Subedi, Suresh, et al.
(författare)
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Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA
- 2014
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Ingår i: Acta Crystallographica Section F. - 2053-230X. ; 70, s. 1631-1635
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Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA(25-460)) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA 25-460 were collected to 2.25 angstrom resolution from a crystal that belonged to space group P2(1), with unit-cell parameters a = 50.96, b = 131.41, c = 123.40 angstrom, alpha = 90.0, beta = 94.8, gamma = 90.0 degrees, and which was predicted to contain two BabA(25-460)-nanobody complexes per asymmetric unit.
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| 9. |
- Taussig, Michael J., et al.
(författare)
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ProteomeBinders : planning a European resource of affinity reagents for analysis of the human proteome
- 2007
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 4:1, s. 13-17
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Tidskriftsartikel (refereegranskat)abstract
- ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.
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| 10. |
- Yin, Wen, 1993-
(författare)
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Engineering of alternative scaffold derived drug conjugates and fusion-toxins for targeted cancer therapy
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a major health problem, with premature death for many patients, and with a high monetary cost to society. The disease is complex to treat due to the many different types, but also because the disease-causing cells, that needs to be removed, are very similar to the normal cells. Traditional chemotherapy and radiotherapy therefore have a narrow therapeutic window. Targeted cancer therapy aims to precisely deliver a drug to the tumor tissue by recognizing biomarkers that are only, or at least mostly, associated with the cancer cells. Drug conjugates and fusion-toxins are two types of targeted drugs. They both consist of a cancer-targeting protein, a linker and a cytotoxic payload. The efficacy of drug conjugates and fusion toxins are determined by their targeting accuracy and cytotoxic potency. Optimization of drug conjugates and fusion-toxins concerns selecting the best combination of the three components for the best efficacy. In my studies I have worked with targeting the human epidermal growth factor receptor 2 and 3, HER2 and HER3, respectively. I have used affibody molecules: ZHER2 and ZHER3, and an albumin binding domain?derived affinity protein targeting HER2, ADAPT6. For in vivo half-life extension, I have used an albumin binding domain, ABD. As payloads, I have used the cytotoxic drugs DM1, MMAE, and MMAF, as well as a truncated version of Pseudomonas exotoxin A, PE25. In paper I, the goal was to investigate the architecture of the tumor targeting part of affibody-derived drug conjugates. Seven different affibody constructs with different number and position of the affibody and ABD-domains were designed and evaluated, in vitro and in vivo. They differed in their effect on cell proliferation, where particularly the constructs with two affibody domains increased the rate of proliferation for the SKOV-3 cell line with high expression of HER2. In vivo, the constructs with one affibody domain had a longer blood retention and lower hepatic uptake compared to the constructs with two affibody domains. Two constructs were selected for characterization as drug conjugates, one that promoted cell proliferation strongly, and one that had only a minor effect on cell proliferation. The conjugates were investigated as drugs for treatment of mice carrying HER2- overexpressing SKOV-3 tumors. The results showed that the affibody drug conjugate ZHER2-ABD-mcDM1 was the most efficient drug. ZHER2-ABD- mcDM1 suppressed tumor growth and extended the median survival time of the mice from 37 to 63 days. In paper II, the goal was to investigate the properties of affibody or ADAPT- based fusion-toxins, targeting the HER2 receptor. The results showed that utilizing ZHER2 as targeting domain, to carry the toxic peptide PE25 to HER2 overexpressing cells, was better than using a dual-HER2 binder, consisting of ZHER2 and ADAPT6. Furthermore, the results showed that PE25?based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxic effect beyond a certain point in affinity for the receptor. In paper III, the first HER3-targeting affibody drug conjugate was produced and studied. It was designed using the optimal format from paper I, i.e. affibody-ABD-mcDM1. The results showed a drug conjugate with a potent cytotoxic effect on the pancreatic cancer cell line, BxPC3, with an IC50 value of 6 nM. In mice, injection of a radiolabeled version of ZHER3-ABD-mcDM1, showed uptake in implanted BxPC3 tumors peaking at 6.3 ± 0.4 %ID/g at 6 h post-injection. The general biodistribution showed uptake in liver, lung, salivary gland, stomach, and small intestine, organs known to express HER3 naturally. Collectively, the results show that ZHER3-ABD-mcDM1 is a highly potent and specific drug conjugate, that motivates further development, and possibly investigation of its functionality in experimental therapy of HER3 overexpressing tumors in mice, and later humans. In paper IV, the goal was to investigate different cytotoxic drugs as part of an affibody-based drug conjugate targeting HER2. The optimal architecture from paper I, was employed also here, ZHER2-ABD-drug. The drugs tested were the tubulin polymerization inhibitors DM1, MMAE, and MMAF. ZHER2-ABD-mcMMAF had the most potent cytotoxic effect, with an IC50 value of 0.18 nM for SKBR3 cells with high HER2 expression. ZHER2-ABD- mcMMAE did not perform well in the cytotoxicity experiment. A better linker connecting the drug to the protein is probably required. ZHER2-ABD- mcMMAF was the best performing drug conjugate with the highest potency, and lowest uptake in liver; slightly outperforming ZHER2-ABD-mcDM1. In conclusion, the four papers cover generation and optimization of drug conjugates and fusion-toxin based on affibody molecules and ADAPTs. In the papers, new variants with desirable properties were identified and characterized.
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