| 1. |
- Honarvar, Hadis, et al.
(författare)
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Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
- 2013
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Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 40:3, s. 378-386
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). Methods: The HER2-targeting NCL-cyclized Affibody molecule Z(HER2:342min) has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. The binding affinity of DOTA-Z(HER2:342min) was evaluated in vitro. The targeting properties of In-111- and (68) Ga-DOTA-Z(HER2:342min) were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of In-111- and (68) Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. Results: The dissociation constant (K-D) for DOTA-Z(HER2:342min) binding to HER2 was 18 nM according to SPR measurements. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K-D1 in low nanbmolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 +/- 1.3 for In-111-DOTA-Z(HER2:342min) and 4.6 +/- 0.7 for (68) Ga-DOTA-Z(HER2:342min). However, the uptake of DOTA-Z(HER2:342min) in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Conclusions: Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z(HER2:342min)) with low nanomolar target affinity and specific tumor uptake.
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| 2. |
- Järver, Peter, et al.
(författare)
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Chemical synthesis and evaluation of a backbone-cyclized minimized 2-helix Z-domain
- 2011
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 17:6, s. 463-469
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Tidskriftsartikel (refereegranskat)abstract
- The Z-molecule is a small, engineered IgG-binding affinity protein derived from the immunoglobulin-binding domain B of Staphylococcus aureus protein A. The Z-domain consists of 58 amino acids forming a well-defined antiparallel three-helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three-helix bundle. The small size and the stable three-helix structure are two attractive properties comprised in the Z-domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein-based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z-domain and joining the N- and C-termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two-helix Z-domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N- and C-termini. Copyright (C) 2011 European Peptide Society and John Wiley & Sons, Ltd.
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| 3. |
- Järver, Peter, et al.
(författare)
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In vivo biodistribution and efficacy of peptide mediated delivery
- 2010
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Ingår i: TIPS - Trends in Pharmacological Sciences. - : Elsevier BV. - 0165-6147 .- 1873-3735. ; 31:11, s. 528-535
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Forskningsöversikt (refereegranskat)abstract
- To transverse the plasma membrane and gain access to the cellular interior is one of the major obstacles for many novel pharmaceutical molecules. Since the late 1990 s, cell-penetrating peptides (CPPs) have been utilized as transport vectors for a broad spectrum of 'biological cargoes', ranging from inert gold particles to multifaceted macromolecules such as proteins and plasmids. Numerous studies have shown that CPPs are efficient carriers for bioactive cargoes in vitro. However, even though CPPs are versatile transport vectors, this does not guarantee they can be developed into useful pharmaceutical molecules. Nevertheless, recent progress in the field has shown CPPs to be effective for in vivo delivery with retained biological activity of a wide variety of bioactive cargoes into virtually any mammalian tissue. This review will focus on recent developments and applications for CPP delivery and distribution in vivo.
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| 4. |
- Yu, Feifan, et al.
(författare)
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Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1)
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:2, s. e56597-
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Tidskriftsartikel (refereegranskat)abstract
- Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG(1) (mIgG(1)). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG(1) monoclonal antibodies (mAbs). The best variant, denoted Z(F5I) corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I) variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG(1), The best photocoupling efficiency to mIgG(1) Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG(1) mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG(1) antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP) protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG(1) mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly h`igher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP) probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.
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