| 1. |
- Andersen, Jan Terje, et al.
(författare)
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Extending Half-life by Indirect Targeting of the Neonatal Fc Receptor (FcRn) Using a Minimal Albumin Binding Domain
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:7, s. 5234-5241
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Tidskriftsartikel (refereegranskat)abstract
- The therapeutic and diagnostic efficiency of engineered small proteins, peptides, and chemical drug candidates is hampered by short in vivo serum half-life. Thus, strategies to tailor their biodistribution and serum persistence are highly needed. An attractive approach is to take advantage of the exceptionally long circulation half-life of serum albumin or IgG, which is attributed to a pH-dependent interaction with the neonatal Fc receptor (FcRn) rescuing these proteins from intracellular degradation. Here, we present molecular evidence that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn. We show that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent FcRn-albumin binding kinetics. The same result was obtained in the presence of IgG. An in vivo study performed in rat confirmed that the clinically relevant human epidermal growth factor 2 (HER2)-targeting Affibody molecule fused to ABD has a similar half-life and biodistribution profile as serum albumin. The proof-of-concept described may be broadly applicable to extend the in vivo half-life of short lived biological or chemical drugs ultimately resulting in enhanced therapeutic or diagnostic efficiency, a more favorable dosing regimen, and improved patient compliance.
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| 2. |
- Andersson, Hanna, et al.
(författare)
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Natur på skolgården för lärande, hälsa och hållbarhet
- 2024. - 2024
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Rapport (populärvet., debatt m.m.)abstract
- Gröna och artrika utemiljöer främjar barns och ungas välbefinnande och kunskap, bådegenom hälsofördelar kopplade till biologisk mångfald och genom att skapa förutsättningarför lek och lärande om natur och miljöfrågor. Skolgården skulle kunna bidra till allt detta,men är idag i hög grad en outnyttjad plats för biologisk mångfald och klimatanpassningav städer. I denna policy brief presenteras huvudsakliga motiv och möjliga åtgärder för attutveckla gröna miljöer och biologisk mångfald på skolgårdar och förskolegårdar.
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| 3. |
- Ekblad, Caroline, et al.
(författare)
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Polypeptides based on a scaffold
- 2022
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Patent (populärvet., debatt m.m.)abstract
- The disclosure provides a population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence Xsc1AELDXsc2Xsc3GVG AXXIKXIXsc4XA XXVEXVQXXK QXILAX. The disclosure also provides methods for selecting and identifying polypeptides from the population, as well as such polypeptides themselves.
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| 4. |
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| 5. |
- Lindbo, Sarah, et al.
(författare)
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Design, construction and characterization of an ABD-based library with improved stability
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Recombinant affinity proteins binding specifically to other molecules are important tools for many clinical and industrial applications. Small robust protein scaffolds have proven to be well suited as frameworks for generation of novel affinity binders due to their stability. Here we used the albumin-binding domain (ABD) of protein G from Streptococcus G148 as scaffold to design a new combinatorial library capable of generating stable binders to various target proteins with high affinity and specificity. To create a robust framework able to generate highly stable binders, mutations in the non-binding region were evaluated and residues providing increased stability were introduced into the scaffold. By combining rational design with combinatorial protein engineering we also evaluated the surface exposed amino acids at the albumin-binding interface and identified 11 residues suitable for randomization. The potency of the novel scaffold library was assessed by screening for binders using phage display against three distinct targets; complement factor 4, (C4), insulin and interleukin-6 (IL-6). Binders in the nanomolar range with melting temperature above 57°C were selected for all three targets. Notably, the identified IL-6 binders were characterized by extreme thermal stability with variants demonstrating organized structures even at 90°C. This demonstrates that stable binders with distinct specificities can be generated and thus proves the high potential of the library.
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| 6. |
- Lindgren, Joel, et al.
(författare)
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A Native Chemical Ligation Approach for Combinatorial Assembly of Affibody Molecules
- 2012
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 13:7, s. 1024-1031
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Tidskriftsartikel (refereegranskat)abstract
- Affinity molecules labeled with different reporter groups, such as fluorophores or radionuclides, are valuable research tools used in a variety of applications. One class of engineered affinity proteins is Affibody molecules, which are small (6.5 kDa) proteins that can be produced by solid phase peptide synthesis (SPPS), thereby allowing site-specific incorporation of reporter groups during synthesis. The Affibody molecules are triple-helix proteins composed of a variable part, which gives the protein its binding specificity, and a constant part, which is identical for all Affibody molecules. In the present study, native chemical ligation (NCL) has been applied for combinatorial assembly of Affibody molecules from peptide fragments produced by Fmoc SPPS. The concept is demonstrated for the synthesis of three different Affibody molecules. The cysteine residue introduced at the site of ligation can be used for directed immobilization and does not interfere with the function of the investigated proteins. This strategy combines a high-yield production method with facilitated preparation of proteins with different C-terminal modifications.
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| 7. |
- Lindgren, Joel, et al.
(författare)
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N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
- 2010
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 19:12, s. 2319-2329
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)
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| 8. |
- Lindgren, Joel, et al.
(författare)
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N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
- 2010
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 19:12, s. 2319-2329
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)
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| 9. |
- Liu, Yongsheng, et al.
(författare)
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Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis
- 2022
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 14:11
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Tidskriftsartikel (refereegranskat)abstract
- Targeted molecular radiation therapy is a promising emerging treatment modality in oncology, and peptide synthesis may shorten the time to reach the clinical stage. In this study, we have explored Chemo-Enzymatic Peptide Synthesis, or CEPS, as a new means of producing a therapeutic HER2 targeted Affibody((R)) molecule, comprising a C-terminal albumin binding domain (ABD) for half-life extension and a total length of 108 amino acids. In addition, a DOTA moiety could be incorporated at N-terminus directly during the synthesis step and subsequently utilized for site-specific radiolabeling with the therapeutic radionuclide Lu-177. Retained thermodynamic stability as well as retained binding to both HER2 and albumin was verified. Furthermore, HER2 binding specificity of the radiolabeled Affibody molecule was confirmed by an in vitro saturation assay showing a significantly higher cell-bound activity of SKOV-3 (high HER2 expression) compared with BxPC3 (low HER2 expression), both in the presence and absence of HSA. In vivo evaluation in mice bearing HER2 expressing xenografts also showed specific tumor targeting as well as extended time in circulation and reduced kidney uptake compared with a HER2 targeted Affibody molecule without the ABD moiety. To conclude, we have demonstrated that CEPS can be used for production of Affibody-fusion molecules with retained in vitro and in vivo functionality.
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| 10. |
- Ravi, Naveen, et al.
(författare)
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Identification of targetable lesions in anaplastic thyroid cancer by genome profiling
- 2019
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 11:3
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Tidskriftsartikel (refereegranskat)abstract
- Anaplastic thyroid cancer (ATC) is a rare and extremely malignant tumor with no available cure. The genetic landscape of this malignancy has not yet been fully explored. In this study, we performed whole exome sequencing and the RNA-sequencing of fourteen cases of ATC to delineate copy number changes, fusion gene events, and somatic mutations. A high frequency of genomic amplifications was seen, including 29% of cases having amplification of CCNE1 and 9% of CDK6; these events may be targetable by cyclin dependent kinase (CDK) inhibition. Furthermore, 9% harbored amplification of TWIST1, which is also a potentially targetable lesion. A total of 21 fusion genes in five cases were seen, none of which were recurrent. Frequent mutations included TP53 (55%), the TERT promoter (36%), and ATM (27%). Analyses of mutational signatures showed an involvement of processes that are associated with normal aging, defective DNA mismatch repair, activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity, failure of DNA double-strand break repair, and tobacco exposure. Taken together, our results shed new light on the tumorigenesis of ATC and show that a relatively large proportion (36%) of ATCs harbor genetic events that make them candidates for novel therapeutic approaches. When considering that ATC today has a mortality rate of close to 100%, this is highly relevant from a clinical perspective.
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| 11. |
- Rubins, Daniel J., et al.
(författare)
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In Vivo Evaluation and Dosimetry Estimate for a High Affinity Affibody PET Tracer Targeting PD-L1
- 2021
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Ingår i: Molecular Imaging and Biology. - : Springer. - 1536-1632 .- 1860-2002. ; 23, s. 241-249
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [F-18]AlF-NOTA-Z(PD-L1_1) as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule Z(PD-L1_4), to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1.Procedures: Z(PD-L1_4) was conjugated with NOTA and radiolabeled with either [F-18]AlF or Ga-68. [F-18]AlF-NOTA-Z(PD-L1_4) and [Ga-68]NOTA-Z(PD-L1_4) were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses.Results: Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([F-18]AlF-NOTA-Z(PD-L1_4): LOX: 8.7 +/- 0.7 %ID/g (N = 4) SUDHL6: 0.2 +/- 0.01 %ID/g (N = 6), [Ga-68]NOTA-Z(PD-L1_4): LOX: 15.8 +/- 1.0 %ID/g (N = 6) SUDHL6: 0.6 +/- 0.1 %ID/g (N = 6)), considerably higher than Z(PD-L1_1). In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([F-18]AlF-NOTA-Z(PD-L1_4): 1.26 +/- 0.13 SUV, [Ga-68]NOTA-Z(PD-L1_4): 1.11 +/- 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses.Conclusions: [F-18]AlF-NOTA-Z(PD-L1_4) and [Ga-68]NOTA-Z(PD-L1_4) are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [F-18] or [Ga-68] may expand access to clinical sites.
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| 12. |
- Sand, Elin, et al.
(författare)
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Buserelin treatment to rats causes enteric neurodegeneration with moderate effects on CRF-immunoreactive neurons and Enterobacteriaceae in colon, and in acetylcholine-mediated permeability in ileum
- 2015
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Ingår i: BMC Research Notes. - : Springer Science and Business Media LLC. - 1756-0500. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The gonadotropin-releasing hormone (GnRH) analog buserelin causes enteric neuronal loss. Acute stress or injection of corticotropin-releasing factor (CRF) affects motility, secretion, and barrier function of the gastrointestinal tract. The aim of the study was to characterize the CRF immunoreactivity in enteric neurons after buserelin treatment, and to evaluate possible effects of enteric neuropathy on gut microbiota, intestinal permeability, and stress response behavior. Results: Sixty rats were given buserelin (20 μg) or saline subcutaneously for 5 days, repeated four times with 3 weeks in-between. At the study end, enteric neuronal density, enteric expression of CRF, gut microbial composition, and plasma levels of adrenocorticotropic hormone (ACTH) and CRF were analyzed. Intestinal permeability was examined in Ussing chambers and the reaction to stressful events was measured by behavior tests. Buserelin treatment reduced the number of neurons along the entire gastrointestinal tract, with increased relative numbers of CRF-immunoreactive submucosal and myenteric neurons in colon (p < 0.05 and p < 0.01, respectively). The overall microbial diversity and relative abundance did not differ between groups, but Enterobacteriaceae was decreased in colon in buserelin-treated rats (p = 0.020). Basal intestinal permeability did not differ between groups, whereas carbachol stimulation increased ileum permeability in controls (p < 0.05), but not in buserelin-treated rats. Buserelin did not affect stress behavior. Conclusions: Although buserelin treatment leads to enteric neuronal loss along the gastrointestinal tract with an increased percentage of CRF-immunoreactive neurons in colon, the physiology is well preserved, with modest effects on colon microbiota and absence of carbachol-induced permeability in ileum as the only observed changes. © 2015 Sand et al.
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| 13. |
- Seijsing, Johan, et al.
(författare)
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An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 111:48, s. 17110-17115
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Tidskriftsartikel (refereegranskat)abstract
- Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.
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| 14. |
- Trotter, Dinko E. Gonzalez, et al.
(författare)
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In Vivo Imaging of the Programmed Death Ligand 1 by F-18 PET
- 2017
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 58:11, s. 1852-1857
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Tidskriftsartikel (refereegranskat)abstract
- Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1-binding Affibody molecule NOTA-Z(PD-L1_1) with F-18 and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1-binding Affibody ligand Z(PD-L1_1) was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of F-18-AlF. In vivo studies were performed in PD-L1-positive, PD-L1-negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by gamma-counting biodistribution. Immunohistochemical staining with an antibody specific for anti-PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-Z(PD-L1_1) was labeled, with a radiochemical yield of 15.1% +/- 5.6%, radiochemical purity of 96.7% +/- 2.0%, and specific activity of 14.6 +/- 6.5 GBq/mu mol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1-positive tumor, whereas the PD-L1-negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1-positive (percentage injected dose per gram [%ID/g] = 2.56 +/- 0.33) and -negative (% ID/g = 0.32 +/- 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.
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| 15. |
- Tuisku, Jouni, et al.
(författare)
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Effects of age, BMI and sex on the glial cell marker TSPO : a multicentre [11C]PBR28 HRRT PET study
- 2019
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer. - 1619-7070 .- 1619-7089. ; 46:11, s. 2329-2338
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Tidskriftsartikel (refereegranskat)abstract
- Purpose The purpose of this study was to investigate the effects of ageing, sex and body mass index (BMI) on translocator protein (TSPO) availability in healthy subjects using positron emission tomography (PET) and the radioligand [C-11]PBR28. Methods [C-11]PBR28 data from 140 healthy volunteers (72 males and 68 females; N = 78 with HAB and N = 62 MAB genotype; age range 19-80 years; BMI range 17.6-36.9) were acquired with High Resolution Research Tomograph at three centres: Karolinska Institutet (N = 53), Turku PET centre (N = 62) and Yale University PET Center (N = 25). The total volume of distribution (V-T) was estimated in global grey matter, frontal, temporal, occipital and parietal cortices, hippocampus and thalamus using multilinear analysis 1. The effects of age, BMI and sex on TSPO availability were investigated using linear mixed effects model, with TSPO genotype and PET centre specified as random intercepts. Results There were significant positive correlations between age and V-T in the frontal and temporal cortex. BMI showed a significant negative correlation with V-T in all regions. Additionally, significant differences between males and females were observed in all regions, with females showing higher V-T. A subgroup analysis revealed a positive correlation between V-T and age in all regions in male subjects, whereas age showed no effect on TSPO levels in female subjects. Conclusion These findings provide evidence that individual biological properties may contribute significantly to the high variation shown in TSPO binding estimates, and suggest that age, BMI and sex can be confounding factors in clinical studies.
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