| 1. |
- Fleetwood, Filippa, et al.
(författare)
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Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
- 2014
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4, s. 7518-
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.
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| 2. |
- Kronqvist, Nina, et al.
(författare)
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Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
- 2011
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 24:4, s. 385-396
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Tidskriftsartikel (refereegranskat)abstract
- Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
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| 3. |
- Yu, Feifan, et al.
(författare)
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An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo
- 2014
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Ingår i: mAbs. - : Informa UK Limited. - 1942-0862 .- 1942-0870. ; 6:6, s. 1598-1607
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Tidskriftsartikel (refereegranskat)abstract
- In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor a (sIL-6R alpha) and IL6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z(IL-6_13) with an affinity (K-D) for IL-6 of similar to 500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, Z(IL-6_13) was fused to either the N-or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira (R)). One AffiMab construct with Z(IL-6_13) positioned at the N-terminus of the heavy chain, denoted Z(IL-6_13)-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z(IL-6_13)-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.
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