| 1. |
- Leandersson Bogefors, Karolina, et al.
(author)
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Androgen receptor gene CAG and GGN repeat lengths as predictors of recovery of spermatogenesis following testicular germ cell cancer treatment
- 2017
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In: Asian Journal of Andrology. - 1008-682X .- 1745-7262. ; 19:5, s. 538-542
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Journal article (peer-reviewed)abstract
- Spermatogenesis is an androgen-regulated process that depends on the action of androgen receptor (AR). Sperm production may be affected in men treated for testicular cancer (TC), and it is important to identify the factors influencing the timing of spermatogenesis recovery following cancer treatment. It is known that the CAG and GGN repeat numbers affect the activity of the AR; therefore, the aim of this study is to investigate if the CAG and GGN polymorphisms in the AR gene predict recovery of sperm production after TC treatment. TC patients (n = 130) delivered ejaculates at the following time points: postorchiectomy and at 6, 12, 24, 36, and 60 months posttherapy (T0, T6, T12, T24, T36, and T60). The CAG lengths were categorized into three groups, <22 CAG, 22-23 CAG, and >23 CAG, and the GGN tracts were also categorized into three groups, <23 GGN, 23 GGN, and >23 GGN. At T12, men with 22-23 CAG presented with a statistically significantly (P = 0.045) lower sperm concentration than those with other CAG numbers (8.4 × 10 6 ml-1 vs 16 × 10 6 ml-1 ; 95% CI: 1.01-2.65). This association was robust to omitting adjustment for treatment type and sperm concentration at T0 (P = 0.021; 3.7 × 10 6 ml-1 vs 10 × 10 6 ml-1 ; 95% CI: 1.13-4.90). The same trends were observed for total sperm number. The least active AR variant seems to be associated with a more rapid recovery of spermatogenesis. This finding adds to our understanding of the biology of postcancer therapy recovery of fertility in males and has clinical implications.
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| 2. |
- Magnusson, Maria K, 1972, et al.
(author)
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Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu.
- 2007
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In: Cancer gene therapy. - : Springer Science and Business Media LLC. - 0929-1903 .- 1476-5500. ; 14:5, s. 468-79
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Journal article (peer-reviewed)abstract
- In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.
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| 3. |
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| 4. |
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| 5. |
- Agaton, C., et al.
(author)
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Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues
- 2003
-
In: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 2, s. 405-
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Journal article (peer-reviewed)abstract
- Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.
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| 6. |
- Altai, Mohamed, et al.
(author)
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188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors : preclinical assessment
- 2014
-
In: Journal of nuclear medicine : official publication, Society of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 55:11, s. 8-1842
-
Journal article (peer-reviewed)abstract
- UNLABELLED: Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors.METHODS: ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment.RESULTS: Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible.CONCLUSION: (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.
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| 7. |
- Altai, Mohamed, et al.
(author)
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Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
- 2018
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In: Cells. - : MDPI AG. - 2073-4409. ; 7:10
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Journal article (peer-reviewed)abstract
- Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.
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| 8. |
- Altai, Mohamed, et al.
(author)
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Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of Tc-99m-labeled recombinant Affibody molecules
- 2012
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In: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 42:5, s. 1975-1985
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Journal article (peer-reviewed)abstract
- Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide Tc-99m. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of Tc-99m-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied Tc-99m-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of Tc-99m. The biodistribution of all Tc-99m-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of Tc-99m was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.
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| 9. |
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| 10. |
- Altai, Mohamed, et al.
(author)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.
- 2014
-
In: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 87, s. 519-28
-
Journal article (peer-reviewed)abstract
- Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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| 11. |
- Altai, Mohamed, et al.
(author)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re
- 2013
-
In: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 40:Suppl. 2, s. S219-S220
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Journal article (other academic/artistic)abstract
- Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172±32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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| 12. |
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| 13. |
- Andersson, C., et al.
(author)
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Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation
- 2000
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In: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 238:02-jan, s. 181-193
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Journal article (peer-reviewed)abstract
- We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-MS-M, ABP-MS-MI, ZZ-MS-M and ZZ-MS-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce MS-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.
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| 14. |
- Andersson, Christin, et al.
(author)
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In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation
- 2001
-
In: Journal of Immunological Methods. - 0022-1759. ; 255:1-2, s. 135-148
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Journal article (peer-reviewed)abstract
- We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment ΔSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-ΔSAG1 and His6-ABP-ΔSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-ΔSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.
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| 15. |
- Andersson, C., et al.
(author)
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Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column
- 2001
-
In: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 34, s. 25-32
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Journal article (peer-reviewed)abstract
- The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.
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| 16. |
- Andersson, C., et al.
(author)
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Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen
- 2000
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In: FEMS Immunology and Medical Microbiology. - 0928-8244 .- 1574-695X. ; 29:4, s. 247-253
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Journal article (peer-reviewed)abstract
- Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.
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| 17. |
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| 18. |
- Andersson, Ken G., et al.
(author)
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Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coli
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Other publication (other academic/artistic)abstract
- Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is the most frequently used method, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli have several properties that are valuable for library applications and then in particular the high transformation efficiency. Although the first studies on display of recombinant peptides and proteins on E. coli were reported over 25 years ago, the method is still not fully established for directed evolution of affinity proteins. More recently, the use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and in particular for directed evolution of different enzymes. Here, we report on display of a large naïve Affibody library on the outer membrane of E. coli using the autotransporter AIDA-I. The expression cassette was first engineered by removing non-essential sequences, followed by introduction of an Affibody library, comprising more than 109 variants, into the new display vector. Selections by FACS against five different target molecules resulted in a panel of binders with down to nanomolar affinities.
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| 19. |
- Andersson, Ken G., et al.
(author)
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Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli
- 2019
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In: Biotechnology Journal. - : WILEY-V C H VERLAG GMBH. - 1860-6768 .- 1860-7314. ; 14:4
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Journal article (peer-reviewed)abstract
- Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.
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| 20. |
- Andersson, Ken G., 1987-
(author)
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Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
- 2017
-
Doctoral thesis (other academic/artistic)abstract
- Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future.
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| 21. |
- Andersson, Ken G, et al.
(author)
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Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors
- 2015
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In: Oncology Reports. - : Spandidos Publications. - 1021-335X .- 1791-2431. ; 34:2, s. 1042-8
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Journal article (peer-reviewed)abstract
- Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed invitro and invivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with 111In provided stable conjugates. Invitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT‑474 breast carcinoma cells. In mice bearing BT‑474 xenografts, the tumor uptake of the two conjugates was receptor‑specific. Direct invivo comparison of 111In-HEHEHE-Z08698-NOTA and 111In-HEHEHE-Z08699‑NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for 111In-HEHEHE-Z08698-NOTA [12±3 vs. 8±1, 4h post injection (p.i.)] due to more efficient blood clearance. 111In-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.
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| 22. |
- Andersson, Ken G., et al.
(author)
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Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with Tc-99m using a peptide-based cysteine-containing chelator
- 2016
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In: International Journal of Oncology. - : SPANDIDOS. - 1019-6439 .- 1791-2423. ; 49:6, s. 2285-2293
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Journal article (peer-reviewed)abstract
- The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide Tc-99m should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with Tc-99m using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, Tc-99m-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that Tc-99m-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6 1 and 2.5 0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8 0.4 and 8 3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of Tc-99m-labeled ZEGFR:2377 for imaging of EGFR in vivo.
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| 23. |
- Anukam, Anthony, et al.
(author)
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A review of the mechanism of bonding in densified biomass pellets
- 2021
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In: Renewable & sustainable energy reviews. - : Elsevier. - 1364-0321 .- 1879-0690. ; 148
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Journal article (peer-reviewed)abstract
- The production of durable biomass pellets have always been challenged by several factors including the lack of understanding of the mechanism involved in how particles combine to form pellets under standard conditions of the pellet press. This is because contributing factors span several molecular, microscopic, and even nanoscopic levels as biomass undergoes pelleting. The characteristics of the bonds formed between the combining particles and their relevance to the quality of pellets remains vague, no matter how quality is defined. However, even though few researchers have attempted to explain the mechanism of bonding in densified biomass pellets using different theories, none of their hypotheses supports particle bonding from a structural chemistry perspective. There are still no clear explanations which consider the role of molecular structure and the interactions of substances as milled biomass undergo pelleting. In view of these arguments therefore, this review presents an in-depth analysis of a structural chemistry perspective of the mechanism of bonding and the use of additives in densified biomass pellets and helps identify research areas needed to facilitate better understanding of bonding in densified biomass pellets. The status of current research in biomass pelleting, types of materials suitable as additives and their structural characteristics, as well as the current technical specifications of using additives are also discussed.
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| 24. |
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| 25. |
- Assenhöj, Maria, et al.
(author)
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Protein interaction, monocyte toxicity and immunogenic properties of cerium oxide crystals with 5% or 14% gadolinium, cobalt oxide and iron oxide nanoparticles–an interdisciplinary approach
- 2021
-
In: Nanotoxicology. - : Taylor and Francis Ltd.. - 1743-5390 .- 1743-5404. ; 15:8, s. 1035-1038
-
Journal article (peer-reviewed)abstract
- Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle–protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both ‘danger signals’, verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations. © 2021 The Author(s).
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| 26. |
- Bass, Tarek
(author)
-
Affibody molecules targeting HER3 for cancer therapy
- 2017
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Doctoral thesis (other academic/artistic)abstract
- The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3.
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| 27. |
- Bass, Tarek, et al.
(author)
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In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct
- 2017
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- Overexpression of human epidermal growth factor receptor 3 (HER3) is involved in resistance to several therapies for malignant tumours. Currently, several anti-HER3 monoclonal antibodies are under clinical development. We introduce an alternative approach to HER3-targeted therapy based on engineered scaffold proteins, i.e. affibody molecules. We designed a small construct (22.5 kDa, denoted 3A3), consisting of two high-affinity anti-HER3 affibody molecules flanking an albumin-binding domain ABD, which was introduced for prolonged residence in circulation. In vitro, 3A3 efficiently inhibited growth of HER3-expressing BxPC-3 cells. Biodistribution in mice was measured using 3A3 that was site-specifically labelled with In-111 via a DOTA chelator. The residence time of In-111-DOTA-3A3 in blood was extended when compared with the monomeric affibody molecule. In-111-DOTA-3A3 accumulated specifically in HER3-expressing BxPC-3 xenografts in mice. However, In-111-DOTA-3A3 cleared more rapidly from blood than a size-matched control construct In-111-DOTA-TAT, most likely due to sequestering of 3A3 by mErbB3, the murine counterpart of HER3. Repeated dosing and increase of injected protein dose decreased uptake of In-111-DOTA-3A3 in mErbB3-expressing tissues. Encouragingly, growth of BxPC-3 xenografts in mice was delayed in an experimental (pilot-scale) therapy study using 3A3. We conclude that the 3A3 affibody format seems promising for treatment of HER3-overexpressing tumours.
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| 28. |
- Berghel, Jonas, 1966-, et al.
(author)
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A comparison of relevant data and results from single pellet press research is Mission Impossible : A review
- 2022
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In: Bioresource Technology Reports. - : Elsevier. - 2589-014X. ; 18
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Journal article (peer-reviewed)abstract
- A global increase in the wood fuel pellet market requires knowledge of new biomasses pelleting abilities. As large-scale industrial tests of new materials are costly, tests in e.g., a single pellet press (SPP) are desirable. SPPs have many different configurations and it typically produces one pellet at a time and can give results of its pelletability. This review has surveyed the research that has been carried out of SPPs to ascertain the feasibility of comparing their obtained data and the results. The results show that it is almost impossible to compare the data and results of the various different SPP studies, e.g., some information from the data used was missing, resulting in that only 27 out of 70 papers were comparable. One solution could be the introduction of a common SPP testing method using a determined set of data that enables a reference pellet to be produced in every study.
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| 29. |
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| 30. |
- Berghel, Jonas, 1966-, et al.
(author)
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Lösningarna finns! Är pelletsproducenterna medvetna om problemen?
- 2011
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In: Bioenergi. - Gävle : Region Gävleborg. - 9789163391262 ; , s. 25-29
-
Book chapter (other academic/artistic)abstract
- Sverige är inte längre världsledande som pelletsproducent. USA producerar allra mest pellets i världen. Kanada och Ryssland producerar också allt mer pellets. Ingen av dessa länder har någon omfattande inhemsk konsumtion. I stort sett all pellets exporteras och det sker huvudsakligen till Europa. Sannolikt kommer det att leda till att priset på pellets i Europa sjunker, med följd att lönsamheten för svenska pelletsproducenter minskar.
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| 31. |
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| 32. |
- Berghel, Jonas, 1966-, et al.
(author)
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The effects of kraft lignin additives on wood fuel pellet quality, energy use and shelf life
- 2013
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In: Fuel processing technology. - : Elsevier BV. - 0378-3820 .- 1873-7188. ; 112:0, s. 64-69
-
Journal article (peer-reviewed)abstract
- In 2011, the total consumption of pellets in Sweden amounted to 1.9 million tons, which represents an energy value of 9 TWh. The pellets are used in large-scale as well as in small-scale applications, and increased demands on pellet quality are likely to force pellet producers to improve on the pellet properties. One way of increasing pellet quality is by using additives. The purpose of this article, therefore, is to examine kraft lignin as an additive. Pelletswere produced in a small industrial pellet press located at KarlstadUniversity, Karlstad, Sweden, and 1–4% of kraft lignin was added to the pellets. The results indicate that the addition of an increased amount of kraft lignin to the pellets increases their mechanical durability and their lengths. The results also indicate that dry kraft lignin yields pellets with higher durability as compared to wet kraft lignin. The energy demand was unaffected by the increased use of kraft lignin. The general results presented in this paper are useful for producers of lignin, pellet producers and end-users of pellets, who are interested in developing their products and/or improving the production processes.
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| 33. |
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| 34. |
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| 35. |
- Bhalla, Nayanika, et al.
(author)
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Spatial transcriptomics of human placentas reveal distinct RNA patterns associated with morphology and preeclampsia
- 2023
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In: Placenta. - : Elsevier BV. - 0143-4004 .- 1532-3102. ; 139, s. 213-216
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Journal article (peer-reviewed)abstract
- Spatial transcriptomics (ST) maps RNA level patterns within a tissue. This technology has not been previously applied to human placental tissue. We demonstrate analysis of human placental samples with ST. Unsupervised clustering revealed that distinct RNA patterns were found corresponding to different morphological structures. Additionally, when focusing upon terminal villi and hemoglobin associated structures, RNA levels differed between placentas from full term healthy pregnancies and those complicated by preeclampsia. The results from this study can provide a benchmark for future ST studies in placenta.
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| 36. |
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| 37. |
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| 38. |
- Binz, Hans, et al.
(author)
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Method for enhancing the immunogenicity of an immunogenic compound or hapten, and use thereof for preparing vaccines
- 1994
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Patent (pop. science, debate, etc.)abstract
- The present invention relates to a process for improving the immunogenicity of an immunogen, an antigen or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said antigen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to mammalian serum albumin. The invention also relates to the use, as a medicament, of the product which can be obtained in this way.
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| 39. |
- Binz, Hans, et al.
(author)
-
Respiratory syncytial virus protein g expressed on bacterial membrane
- 1994
-
Patent (pop. science, debate, etc.)abstract
- A method for preparing a peptide or protein, wherein (a) a DNA sequence coding for a heterologous polypeptide on a peptide sequence between amino acid residues 130 and 230 of respiratory syncytial virus protein G, sub-groups A and B, or a peptide sequence at least 80 % homologous thereto, and (b) means enabling the expression of the polypeptide on the bacterial membrane surface, are inserted into a bacterium which is not pathogenic for mammals. The resulting conjugate polypeptide and a live bacterium expressing same, pharmaceutical compositions containing them and their use for preparing a vaccine, as well as a DNA sequence coding for said polypeptide, are also disclosed.
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| 40. |
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| 41. |
- Boutajangout, Allal, et al.
(author)
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Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model
- 2019
-
In: Frontiers in Aging Neuroscience. - : Frontiers Media S.A.. - 1663-4365. ; 11
-
Journal article (peer-reviewed)abstract
- Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-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 A beta, demonstrating a therapeutic potential for prevention of AD.
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| 42. |
- Brokken, Leon, et al.
(author)
-
Association between polymorphisms in the aryl hydrocarbon receptor repressor gene and disseminated testicular germ cell cancer.
- 2013
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In: Frontiers in Endocrinology. - : Frontiers Media SA. - 1664-2392. ; 4:Feb.,14
-
Journal article (peer-reviewed)abstract
- In the Western world, testicular germ cell cancer (TGCC) is the most common malignancy of young men. The malignant transformation of germ cells is thought to be caused by developmental and hormonal disturbances, probably related to environmental and lifestyle factors because of rapidly increasing incidence of TGCC in some countries. Additionally, there is a strong genetic component that affects susceptibility. However, genetic polymorphisms that have been identified so far only partially explain the risk of TGCC. Many of the persistent environmental pollutants act through the aryl hydrocarbon receptor (AHR). AHR signaling pathway is known to interfere with reproductive hormone signaling, which is supposed to play a role in the pathogenesis and invasive progression of TGCC. The aim of the present study was to identify whether AHR-related polymorphisms were associated with risk as well as histological and clinical features of TGCC in 367 patients and 537 controls. Haplotype-tagging single-nucleotide polymorphisms (SNPs) were genotyped in genes encoding AHR and AHR repressor (AHRR). Binary logistic regression was used to calculate the risk of TGCC, non-seminoma versus seminoma, and metastasis versus localized disease. Four SNPs in AHRR demonstrated a significant allele association with risk to develop metastases (rs2466287: OR = 0.43, 95% CI 0.21-0.90; rs2672725: OR = 0.49, 95% CI: 0.25-0.94; rs6879758: OR = 0.27, 95% CI: 0.08-0.92; rs6896163: OR = 0.34, 95% CI: 0.12-0.98). This finding supports the hypothesis that compounds acting through AHR may play a role in the invasive progression of TGCC, either directly or through modification of reproductive hormone action.
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| 43. |
- Brokken, Leon, et al.
(author)
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Association of polymorphisms in genes encoding hormone receptors ESR1, ESR2 and LHCGR with the risk and clinical features of testicular germ cell cancer.
- 2012
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In: Molecular and Cellular Endocrinology. - : Elsevier BV. - 1872-8057 .- 0303-7207. ; 351:2, s. 279-285
-
Journal article (peer-reviewed)abstract
- Testicular germ cell cancer (TGCC) is the most common malignancy in young men. Genetic variants known to be associated with risk of TGCC only partially account for the observed familial risks. We aimed to identify additional polymorphisms associated with risk as well as histological and clinical features of TGCC in 367 patients and 214 controls. Polymorphisms in ESR2 (rs1256063; OR=0.53, 95% CI: 0.35-0.79) and LHCGR (rs4597581; OR=0.68, 95% CI: 0.51-0.89, and rs4953617; OR=1.88, 95% CI: 1.21-2.94) associated with risk of TGCC. Polymorphisms in ESR1 (rs9397080; OR=1.85, 95% CI: 1.18-2.91) and LHCGR (rs7371084; OR=2.37, 95% CI: 1.26-4.49) associated with risk of seminoma and metastasis, respectively. SNPs in ESR1 (rs9397080) and LHCGR (rs7371084) were predictors of higher LH levels and higher androgen sensitivity index in healthy subjects. The results suggest that polymorphisms in ESR1, ESR2 and LHCGR contribute to the risk of developing TGCC, histological subtype, and risk to metastasis.
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| 44. |
- Cano, F., et al.
(author)
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Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides
- 2000
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In: Vaccine. - 0264-410X .- 1873-2518. ; 18:24, s. 2743-2752
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Journal article (peer-reviewed)abstract
- A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID50), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.
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| 45. |
- Carlsson, Jörgen, et al.
(author)
-
Polypeptides having binding affinity for HER2
- 2003
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Patent (pop. science, debate, etc.)abstract
- A polypeptide is provided, which has a binding affinity for HER2 and which is related to a domain of staphylococcal protein A (SPA) in that the sequence of the polypeptide corresponds to the sequence of the SPA domain having from 1 to about 20 substitution mutations. Nucleic acid encoding the polypeptide, as well as expression vector and host cell for expressing the nucleic acid, are also provided. Also provided is the use of such a polypeptide as a medicament, and as a targeting agent for directing substances conjugated thereto to cells overexpressing HER2. Methods, and kits for performing the methods, are also provided, which methods and kits rely on the binding of the polypeptide to HER2.
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| 46. |
- Cheng, Qing, et al.
(author)
-
Preclinical PET imaging of EGFR levels : pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
- 2016
-
In: EJNMMI Research. - : Springer. - 2191-219X. ; 6
-
Journal article (peer-reviewed)abstract
- Background: Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. Methods: The EGFR-binding Affibody molecule Z(EGFR:2377) and its size-matched non-binding control Z(Taq:3638) were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (Z(EGFR:2377)-ST and Z(Taq:3638)-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with C-11 for in vivo PET studies. Kinetic scans with the C-11-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. Results: Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZE(GFR:2377)-ST-DyLight488. [Methyl-C-11]-labeled Z(EGFR:2377)-ST-CH3 and Z(Taq:3638)-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-C-11]-Z(EGFR:2377)-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since Z(EGFR:2377) binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. Conclusions: Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and C-11-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.
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| 47. |
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| 48. |
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| 49. |
- Cheung, Pierre, et al.
(author)
-
Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells
- 2023
-
In: EJNMMI Research. - : Springer Nature. - 2191-219X. ; 13:1
-
Journal article (peer-reviewed)abstract
- Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.
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| 50. |
- Dahlsson Leitao, Charles
(author)
-
Affibody-mediated targeting of HER-family receptors for cancer imaging and therapy
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Proteins are remarkable molecules with diverse and specialized functions playing essential roles in most biological processes. One such function is protecting us from diseases by the action of antibodies in our immune system that can recognize and mediate the destruction of invading pathogens by binding to foreign epitopes found on non-self proteins. The concept of utilizing specific protein-protein interactions to achieve a therapeutic effect has for several decades been a cornerstone for the development of cancer-directed treatments. While antibodies have formed a basis for the development of such drugs, other protein alternatives may be engineered to complement current antibody-based treatments, and may even prove to possess superior features. This thesis focuses on the engineering of affibody molecules, a small alternative scaffold protein, for design and development of novel cancer-targeting therapeutic and diagnostic drugs. There are many different strategies that have been investigated for inhibiting cancer progression and tumour growth with perhaps one of the most straightforward involving disruption of dysregulated growth-promoting signalling pathways. Members of the human epidermal growth factor receptor (HER) family is prominently expressed in various cancer types and have been shown to be intricately involved in tumorigenesis. One of the members (HER3) often becomes upregulated in cancer and have been shown to mediate acquired resistance to targeted therapies by the mechanism of ligand-induced activation. We have designed five novel affibody-based HER3-targeting molecules able to prevent ligand-binding and consequently activation of HER3. We investigated the targeting properties and biodistribution profiles of these molecules in vivo and subsequently evaluated the anti-tumour efficacy for the most promising variants in direct comparison to a HER3-targeting antibody with a similar inhibitory mechanism. We observed a large influence of design on both the biodistribution properties and the in vivo efficacy of different affibody molecules. Moreover, we demonstrated that two of the affibody-formats were equally effective as the antibody in inhibiting tumour growth and prolonging survival of mice bearing HER3-positive xenografts. The effectiveness of cancer treatments depends on efficient diagnostic approaches that can reliably stratify patients based on these targetable biomarkers, which is possible using radionuclide molecular imaging. We have performed a direct comparison of the diagnostic potential for visualizing HER3-expressing tumours of affibody- and antibody-based imaging probes. We concluded that affibody molecules provide superior imaging quality with higher diagnostic potential and enable early visualization of HER3-expression in tumours. Another member of the HER family that is of interest for cancer therapy is HER1 (or EGFR) but due to substantial expression in healthy tissues, targeted therapies may lead to severe side-effects. One possible solution to this is taking advantage of the distinct milieu of the tumour microenvironment to design EGFR-targeting drugs that become conditionally activated at the tumour site, but not in normal tissues, with the aim of drastically reducing systemic toxicity. We have generated an affibody molecule with anti-idiotypic binding specificity for a previously generated EGFR-binding affibody molecule, which we used to construct an affibody-based prodrug. We were able to show that, in a proof-of-concept format, this anti-idiotypic masking domain was able to block the binding to EGFR until removed by protease-mediated cleavage. We subsequently developed and characterized a more refined version of this prodrug, which we call a pro-affibody, and could show that activation by cancer-associated proteases confers binding to EGFR-expressing cancer cells and enables conditional cytotoxic payload delivery in vitro. The pro-affibody was further evaluated in vivo using tumour-bearing mice to investigate the feasibility for masked uptake in healthy tissues while retaining binding-activity in tumours. We observed a substantial reduction in EGFR-specific liver uptake compared to a control construct without a masking domain, and a strong indication of protease-mediated EGFR-binding in tumours. In conclusion, the experimental work presented in this thesis provides a rationale for designing effective affibody-based cancer therapeutics and diagnostics with different targeting strategies and demonstrates the potential of such drugs from preclinical in vivo data.
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