| 1. |
- Altai, Mohamed, et al.
(författare)
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188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors : preclinical assessment
- 2014
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Ingår i: 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
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Altai, Mohamed, et al.
(författare)
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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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Ingår i: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 42:5, s. 1975-1985
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Tidskriftsartikel (refereegranskat)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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| 3. |
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| 4. |
- Altai, Mohamed, et al.
(författare)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.
- 2014
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 87, s. 519-28
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Altai, Mohamed, et al.
(författare)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re
- 2013
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Ingår i: 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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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)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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| 6. |
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| 7. |
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| 8. |
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| 9. |
- Berghel, Jonas, 1966-, et al.
(författare)
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Lösningarna finns! Är pelletsproducenterna medvetna om problemen?
- 2011
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Ingår i: Bioenergi. - Gävle : Region Gävleborg. - 9789163391262 ; , s. 25-29
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Bokkapitel (övrigt vetenskapligt/konstnärligt)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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| 10. |
- Berghel, Jonas, 1966-, et al.
(författare)
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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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Ingår i: Fuel processing technology. - : Elsevier BV. - 0378-3820 .- 1873-7188. ; 112:0, s. 64-69
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Tidskriftsartikel (refereegranskat)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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| 11. |
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| 12. |
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| 13. |
- Brokken, Leon, et al.
(författare)
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Association between polymorphisms in the aryl hydrocarbon receptor repressor gene and disseminated testicular germ cell cancer.
- 2013
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Ingår i: Frontiers in Endocrinology. - : Frontiers Media SA. - 1664-2392. ; 4:Feb.,14
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Tidskriftsartikel (refereegranskat)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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| 14. |
- Brokken, Leon, et al.
(författare)
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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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Ingår i: Molecular and Cellular Endocrinology. - : Elsevier BV. - 1872-8057 .- 0303-7207. ; 351:2, s. 279-285
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Tidskriftsartikel (refereegranskat)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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| 15. |
- Dishisha, Tarek, et al.
(författare)
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An economical biorefinery process for propionic acid production from glycerol and potato juice using high cell density fermentation.
- 2013
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Ingår i: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 135, s. 504-512
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Tidskriftsartikel (refereegranskat)abstract
- An economically sustainable process was developed for propionic acid production by fermentation of glycerol using Propionibacterium acidipropionici and potato juice, a by-product of starch processing, as a nitrogen/vitamin source. The fermentation was done as high-cell-density sequential batches with cell recycle. Propionic acid production and glycerol consumption rates were dependent on initial biomass concentration, and reached a maximum of 1.42 and 2.30gL(-1)h(-1), respectively, from 50gL(-1) glycerol at initial cell density of 23.7g(CDW)L(-1). Halving the concentration of nitrogen/vitamin source resulted in reduction of acetic and succinic acids yields by ∼39% each. At glycerol concentrations of 85 and 120gL(-1), respectively, 43.8 and 50.8gL(-1) propionic acid were obtained at a rate of 0.88 and 0.29gL(-1)h(-1) and yield of 84 and 78mol%. Succinic acid was 13g% of propionic acid and could represent a potential co-product covering the cost of nitrogen/vitamin source.
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| 16. |
- Ekerljung, Lina, 1980-, et al.
(författare)
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Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2
- 2012
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 23:9, s. 1802-1811
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Tidskriftsartikel (refereegranskat)abstract
- Co-expression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here we have generated six such bispecific targeting proteins, which each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly4Ser)3 or (Ser4Gly)3, and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the monomeric affibody molecules was favorable to promote the binding to respective target. Interestingly, bispecific constructs containing the novel (Ser4Gly)3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly4Ser)3. It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimerization and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.
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| 17. |
- Ekman, Simon, et al.
(författare)
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Clinical Phase I study with an Insulin-like Growth Factor-1 Receptor Inhibitor : Experiences in patients with squamous non-small cell lung carcinoma
- 2011
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Ingår i: Acta Oncologica. - 0284-186X .- 1651-226X. ; 50:3, s. 441-447
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Tidskriftsartikel (refereegranskat)abstract
- Background. Inhibition of the Insulin-like Growth Factor-1 receptor (IGF-1R) has resulted in extensive anti-tumor effects. Picropdophyllin (PPP, AXL1717) is a small-molecule inhibitor of the IGF-1R without inhibition of closely related receptors including the insulin receptor and has shown extensive effects against a wide range of tumors in animals. PPP is currently tested as an orally administrated single agent treatment in an open-label combined Phase I/II clinical study in advanced cancer patients with solid tumors which progress in spite of several lines of treatment. Patients and methods. The first part (Phase IA) consisted of single day BID dosing every three weeks with consecutive dose escalations. The second part (Phase IB) consists of seven days or longer BID dosing every three weeks, dosing range being 520-700 mg BID. Non-progressing patients could continue treatment within a compassionate use setting. Results and discussion. The present report describes our experience with the four patients with progressive squamous non-small cell lung cancer (NSCLC) that have received treatment with PPP. Despite more than seven months of PPP treatment as third or fourth line treatment, the reported patients did not develop any additional metastases. Furthermore, CT scans as well as (18)FDG-Positron Emission Tomography (PET) scans of the patients demonstrated large central necrotic areas, which may suggest tumor response. At the same time, the study drug is so far well tolerated. The phenomenon of necrosis in the tumors suggestive of tumor response has not been reported before in anti-IGF-1R treatment and will be subject to further studies in the present clinical trial.
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| 18. |
- Fleetwood, Filippa, et al.
(författare)
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An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13, s. 179-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. Results: The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection using FACS from a 1: 100,000 background yielded around 20,000-fold enrichment in a single round and high viability of the isolated bacteria after sorting. Conclusions: The new expression vectors are promising for combinatorial engineering of Affibody molecules and the strategy for small-scale production of soluble recombinant proteins has the potential to increase throughput of the entire discovery process.
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| 19. |
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| 20. |
- 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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| 21. |
- Fleetwood, Filippa, et al.
(författare)
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Surface display of a single-domain antibody library on Gram-positive bacteria
- 2013
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Nature. - 1420-682X .- 1420-9071. ; 70:6, s. 1081-1093
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Tidskriftsartikel (refereegranskat)abstract
- Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.
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| 22. |
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| 23. |
- Göstring, Lovisa, et al.
(författare)
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Cellular Effects of HER3-Specific Affibody Molecules
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:6, s. e40023-
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.
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| 24. |
- Hjelm, Barbara, et al.
(författare)
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Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784. ; 27:2, s. 129-137
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Tidskriftsartikel (refereegranskat)abstract
- There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.
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| 25. |
- Huttner, Hagen B, et al.
(författare)
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The age and genomic integrity of neurons after cortical stroke in humans
- 2014
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Ingår i: Nature Neuroscience. - : Springer Science and Business Media LLC. - 1097-6256 .- 1546-1726. ; 17:6, s. 801-803
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Tidskriftsartikel (refereegranskat)abstract
- It has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA rearrangements in the human neocortex. Using immunohistochemistry; transcriptome, genome and ploidy analyses; and determination of nuclear bomb test-derived (14)C concentration in neuronal DNA, we found neither to be the case. A large proportion of cortical neurons displayed DNA fragmentation and DNA repair a short time after stroke, whereas neurons at chronic stages after stroke showed DNA integrity, demonstrating the relevance of an intact genome for survival.
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| 26. |
- Hörndahl, Mikael, et al.
(författare)
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EFFECTS OF COOLING RATE AND SILICON CONTENT ON AL/SICp MMC
- 2014
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Konferensbidrag (refereegranskat)abstract
- This paper reports the results of the study ofthe microstructural and mechanical properties ofmetal matrix composites (MMC) based on Al-Si matrix alloyandsilicon carbideparticulates. The scope of the studyis to investigate the effects of cooling rate and silicon content on the microstructure and the mechanical propertiesof MMC.Samples were cast in mouldsof different temperature in order to identifythe effects of the cooling rate while silicon content was varied from 7 to 12.5 %in the matrix material.The conclusion is that the cooling rate has little effect on the properties unless it is taken to the extreme and the effects of the silicon content is no more pronounced than for non-reinforced aluminium.
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| 27. |
- Johnson, Mark D., 1954, et al.
(författare)
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New exposures of Baltic Ice Lake drainage sediments, Götene, Sweden
- 2010
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Ingår i: GFF. - 1103-5897. ; 132:1, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- New exposures created during the construction of highway E20 near Götene, Sweden, reveal poorly sorted gravelly sand overlain and underlain by varved clay. The stratigraphy at Pellagården consists of, from the bottom up, striated gneiss, till, varved marine clay, the gravelly sand unit, and varved marine clay. The varves represent deglacial marine sediment deposited in 40–50 m deep water. The gravelly sand unit contains graded bedding, indistinct horizontal bedding, mud clasts and interstitial mud. It is poorly sorted and poorly organised. The unit has a pebble fabric indicating flow to the northwest. These characteristics and the great water depth suggest that the gravelly sand was deposited from a hyperconcentrated traction current or from concentrated to hyperconcentrated density flows. We interpret the gravelly sand bed to be sediment deposited during the Baltic Ice Lake drainage at around 10,000 14C years BP. The unit likely represents rapidly deposited sediment at the very start of the drainage and does not indicate the duration of the drainage event. The bed was deposited during a single drainage event rather than as a series of events over a few weeks or months. Based on the number of varves and regional ice retreat rates, the ice-margin was 0.2 to 5 km north of Götene at the time of the drainage. These sites represent the first reported occurrences of the drainage sediment in a stratigraphic sequence since the work of Simon Johansson (1926, 1937, and 1941).
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| 28. |
- Kostallas, George
(författare)
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Intracellular systems for characterization and engineering of proteases and their substrates
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention. With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media. By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III). We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest.
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| 29. |
- 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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| 30. |
- Kronqvist, Nina, et al.
(författare)
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Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies
- 2010
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Ingår i: Recent Patents on Biotechnology. - : Bentham eBooks. - 1872-2083. ; 4:3, s. 171-182
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Tidskriftsartikel (refereegranskat)abstract
- The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.
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| 31. |
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| 32. |
- Lindberg, Hanna, et al.
(författare)
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Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus
- 2012
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Ingår i: Tumor Biology. - : Springer. - 1010-4283 .- 1423-0380. ; 33:3, s. 641-651
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.
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| 33. |
- Lindberg, Hanna, et al.
(författare)
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Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
- 2013
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 8:1, s. 139-145
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Tidskriftsartikel (refereegranskat)abstract
- We have previously generated an affibody molecule for the disease-associated amyloid beta (A beta) peptide, which has been shown to inhibit the formation of various A beta aggregates and revert the neurotoxicity of A beta in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the A beta-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and A beta binding. Overall, the detailed engineering and characterization of this promising A beta-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.
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| 34. |
- Luheshi, Leila M., et al.
(författare)
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Sequestration of the A beta Peptide Prevents Toxicity and Promotes Degradation In Vivo
- 2010
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 8:3, s. e1000334-
-
Tidskriftsartikel (refereegranskat)abstract
- Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (A beta) peptide by using a small engineered binding protein (Z(A beta 3)) that binds with nanomolar affinity to A beta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(A beta 3) in the brains of Drosophila melanogaster expressing either A beta(42) or the aggressive familial Alzheimer's disease (AD) associated E22G variant of A beta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer A beta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of A beta aggregation and reveal that Z(A beta 3) not only inhibits the initial association of A beta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.
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| 35. |
- Löfblom, John, et al.
(författare)
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Affibody molecules : Engineered proteins for therapeutic, diagnostic and biotechnological applications
- 2010
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 584:12, s. 2670-2680
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Forskningsöversikt (refereegranskat)abstract
- Affibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.
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| 36. |
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| 37. |
- Löfblom, John, et al.
(författare)
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Non-immunoglobulin based protein scaffolds
- 2011
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Ingår i: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 22:6, s. 843-848
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Forskningsöversikt (refereegranskat)abstract
- Non-immunoglobulin based protein scaffolds have been reported as promising alternatives to traditional monoclonal antibodies for over a decade and are often mentioned as part of the next-generation immunotherapeutics. Today, this class of biologics is beginning to demonstrate its potential for therapeutic applications and several are currently in preclinical or clinical development. A common denominator for most of these new scaffolds is the attractive properties that differentiate them from monoclonal antibodies including small size, cysteine-free sequence, flexible pharmacokinetic properties, and ease of generating multispecific molecules. In addition to therapeutic applications, substantial evidence point to superior performance of several of these scaffolds in molecular imaging compared to full-length antibodies. Here we review the most recent progress using alternative protein scaffolds for therapy and medical imaging.
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| 38. |
- Malm, Magdalena, et al.
(författare)
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Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension
- 2014
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 9:9, s. 1215-1222
-
Tidskriftsartikel (refereegranskat)abstract
- Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.
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| 39. |
- Malm, Magdalena, 1983-
(författare)
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Generation and characterization of Affibody molecules targeting HER3
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the field of oncology, the ability to target specific tumor cells using highly selective targeting molecules is an attractive and emerging concept. In this context, the epidermal growth factor receptor HER3 has proven central to the biology behind many different human cancers and inhibition of the signaling mediated by this receptor could provide antitumoral effects. Consequently, this receptor has emerged as a suitable target for imaging, functional blocking or delivery of toxic payloads. A promising targeting-molecule for such applications is the small non-immunoglobulin derived Affibody molecule. The work upon which this thesis is based, revolves around HER3 with the aim to generate and characterize Affibody molecules targeting this receptor. In the first study, HER3-specific Affibody molecules were generated by combinatorial protein engineering using a combined approach where first generation binders were isolated from a phage-displayed naive library, followed by affinity maturation of these binders using a focused staphylococcal surface-displayed library and flow-cytometric cell sorting. This engineering strategy enabled the successful isolation of HER3-specific Affibody molecules with subnanomolar affinities for the receptor and the ability to compete with the natural ligand heregulin (HRG) for binding to HER3. In the second study, the cellular effects of these Affibody molecules were characterization in vitro. The results demonstrated that the ability to inhibit HRG-binding to the receptor translated into inhibition of ligand-induced phosphorylation of HER3, HER2 as well as the downstream signaling molecules Akt and Erk. As a result, the HER3-specific Affibody molecules also inhibited HRG-induced cell growth of two different breast cancer cell lines in vitro. These promising results, suggested that the HER3-targeting Affibody molecules could have a therapeutic effect in tumors that are dependent on ligand-induced signaling of HER3. However, due to the relatively low expression level of HER3 on tumor cells, we explored two different engineering approaches of the HER3-specific Affibody molecules in order to potentially improve its tumor targeting ability. One approach was to construct bispecific Affibody molecules where a HER3- and a HER2-specific Affibody molecule were fused on each side of an albumin-binding domain (ABD). In the third study, one such bispecific construct was shown to have increased ability to inhibit ligand-induced phosphorylation of HER2 and retained ability to inhibit HRG-induced activation of HER3, as compared to the monomeric anti-HER3 Affibody. Another strategy was to further increase the affinity of the HER3-specific Affibody molecules towards the receptor through a semi-rational affinity maturation approach. In the fourth study, a staphylococcal displayed affinity maturation library was screened by FACS using an off-rate selection procedure. This approach resulted in the successful isolation of picomolar HER3-binders with improved potency of inhibiting HRG-induced cell growth as compared to a first generation binder. Moreover, in the fifth study, in vivo characterization of these HER3-specific Affibody molecules was performed in both normal and xenograft mice. The results suggested specific targeting of HER3 in vivo and provided the first evidence of successful tumor imaging using a HER3-specific Affibody. Taken together, the work included in this thesis describes (to our knowledge) the first non-immunoglobulin derived affinity protein targeting HER3, with promising features for both therapeutic and imaging applications.
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| 40. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification
- 2013
-
Ingår i: PLOS ONE. - : Public Library Science, USA. - 1932-6203. ; 8:5, s. e62791-
-
Tidskriftsartikel (refereegranskat)abstract
- The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.
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| 41. |
- Nguyen, T. N., et al.
(författare)
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Subunit vaccine candidates engineered from the central conserved region of the RSV G protein aimed for parenteral or mucosal delivery
- 2013
-
Ingår i: Molecular Vaccines: From Prophylaxis to Therapy-Volume 1. - Vienna : Springer. - 9783709114193 ; , s. 103-118
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Respiratory syncytial virus (RSV) is a major pathogen causing severe upper and lower respiratory disease in infants and in elderly worldwide. ccording to WHO, an RSV vaccine is urgently needed. Here, we describe the design of various types of subunit vaccine concepts based on molecular engineering aimed to deliver RSV antigens. Gene segment encoding parts of the conserved central region of the RSV G protein (G2Na) were prepared for various expression and delivery formats: (1) prokaryotically expressed and purifi ed G2Na alone or fused to different carrier proteins, one of them, namely, BBG2Na (Alum), has reached clinical trials in the elderly; (2) G protein-derived antigens surface displayed on lived vectors (non pathogenic bacteria) and (3) nucleic acid vectors. These subunit vaccines were administered with or without adjuvant in rodents and in non-human primates by different routes (parenteral or mucosal). We summarise and compare immunogenicity, protective effi cacy and safety conferred by each immunisation format in RSV experimental animal models. Among these, G2Na proved to be the most promising component for an RSV subunit vaccine.
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| 42. |
- Olovsjö, Stefan, 1982, et al.
(författare)
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Methodology for evaluating effects of material characteristics on machinability-theory and statistics-based modelling applied on Alloy 718
- 2012
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Ingår i: International Journal of Advanced Manufacturing Technology. - : Springer Science and Business Media LLC. - 0268-3768 .- 1433-3015. ; 59:1-4, s. 55-66
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Tidskriftsartikel (refereegranskat)abstract
- The potential machinability for Alloy 718 (Inconel 718) is examined in terms of five material characteristics considered to play a key role in the machinability: ductility (elongation to fracture), strain hardening (ultimate tensile strength over yield strength), thermal conductivity, yield strength and abrasiveness (amount of carbides). The material characteristics are simulated with the software JMatPro from Sente software. The effects of composition, grain size, hardness (size of the precipitated intermetallic particles for given volume fraction), heat treatment, temperature and strain rate have been modelled and statistically evaluated. Combining thermodynamics-based modelling (JMatPro), design of experiments and statistical analysis (Minitab), and machinability polar diagram, a concept on methodology to assess variations in material specifications and to optimise these specifications with respect to potential machinability has been developed. The mechanical properties, predicted from the meta-modelling are found to be affected by the same parameters: hardness (intermetallic particles characteristics), grain size, amount of aluminium, strain rate and temperature. The abrasiveness should only be affected by the amount of carbon. Simulated material characteristics for two different types of turbine discs were compared with measured tool wear from production environment machining experiments. Variations in material characteristics between the discs were small as well as the critical tool wear, revealing a robust metal cutting process.
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| 43. |
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| 44. |
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| 45. |
- Rockberg, Johan, et al.
(författare)
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Epitope mapping using gram-positive surface display
- 2010
-
Ingår i: Current Protocols in Immunology. - : Wiley-Blackwell. - 1934-3671 .- 1934-368X. ; :SUPPL. 90, s. 9.9.1-9.9.17
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Forskningsöversikt (refereegranskat)abstract
- Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.
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| 46. |
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| 47. |
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| 48. |
- Ståhl, Karl, et al.
(författare)
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Atypical 'HoBi'-like pestiviruses-Recent findings and implications thereof
- 2010
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Ingår i: Veterinary Microbiology. - : Elsevier BV. - 0378-1135 .- 1873-2542. ; 142, s. 90-93
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Tidskriftsartikel (refereegranskat)abstract
- In 2004, an atypical pestivirus named D32/00_'HoBi', isolated from foetal calf serum (FCS) originating from Brazil, was described (Schirrmeier et al., 2004). A few years later, a closely related virus (Th/04_KhonKaen) was detected in serum from a calf in Thailand, indicating that this group of atypical pestiviruses already is spread in cattle populations in various regions of the world. At the Friedrich-Loeffler-Institute, Insel Riems, Germany, FCS batches are regularly tested for pestivirus contamination, in general with positive PCR results, and in some cases the contaminants have been typed as 'HoBi'-like. At the National Veterinary Institute (SVA) in Uppsala, Sweden, a recent event with contaminated FCS ruined much of the ongoing cell culture work. From the FCS and the contaminated cells we were able to amplify and sequence nucleic acid from three different pestivirus strains, including BVDV-1, -2 and 'HoBi'-like; this in a commercial FCS that had been tested free from pestivirus by the manufacturer.In this short communication we review the current status of atypical 'HoBi'-like pestiviruses, describe recent findings and discuss the implications thereof. (C) 2009 Elsevier B.V. All rights reserved.
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| 49. |
- Ståhl, Karl, et al.
(författare)
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BVDV control and eradication in Europe -an update
- 2012
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Ingår i: Japanese Journal of Veterinary Research. - 0047-1917. ; 60, s. S31-S39
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Forskningsöversikt (refereegranskat)abstract
- Infections with bovine viral diarrhoea virus (BVDV) are endemic in cattle populations worldwide and result in major economic losses. For long, attempts to control BVDV were limited to prophylactic vaccination practices, implemented primarily to reduce or prevent clinical disease on a herd basis. However, the benefit of preventing clinical disease in transiently infected animals is negligible when considering the overall losses of the disease. Another more systematic strategy to control evolved during the 1990s within eradication programmes in the Scandinavian countries. This was based on an initial determination of herd BVDV status, followed by implementation of systematic zoo-sanitary measures at a regional or national scale (without the use of vaccines) to prevent introduction of BVDV in non-infected herds, and to reduce the prevalence of infected herds by identification and elimination of PI animals. These programmes have been very successful, and all of the Scandinavian countries are currently either free, or almost free from BVDV. Today control programmes are underway in several European countries. This short review discusses the general model of BVDV control, and gives an overview of strategies used within, and the current status of, the ongoing control programmes in Europe.
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| 50. |
- Ståhl, Magnus, 1973-, et al.
(författare)
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Effects on Pellet Properties and Energy Use When Starch Is Added in the Wood-Fuel Pelletizing Process
- 2012
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Ingår i: Energy & Fuels. - Washington DC, Oxford UK : American Chemical Society (ACS). - 0887-0624 .- 1520-5029. ; 26:3, s. 1937-1945
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Tidskriftsartikel (refereegranskat)abstract
- The production and use of wood-fuel pellets have increased significantly worldwide in recent years. The increased use of biomaterials has resulted in higher raw material prices, and there are no signs that indicate a decrease in raw material competition. Additives can be used for different purposes. Partly, they are used to facilitate the use of new raw materials to increase the raw material base, and partly, they are used to decrease the energy use in the pelletizing process. They are also used to increase durability or shelf life. Consequently, it is necessary to do research that systematically investigates the consequences of using additives. In this work, it is investigated how various percentages of different kinds of starch influence pellet properties, including shelf life and energy use in the pelletizing process. Four different starch grades were used: native wheat starch, oxidized corn starch, native potato starch, and oxidized potato starch. The pellets were produced in a small industrial pellet press located at Karlstad University, Karlstad, Sweden. The result shows that starch increases the durability of the pellets. Oxidized starches increase the durability more than native starches, and the best results were obtained by adding oxidized corn starch. The durability did not decrease with storage time when the pellets were stored indoors during 7 months. The oxidation process was not consistently altered by the addition of starch. The energy consumption of the pellet press decreases when starch is added. Again, the oxidized corn starch showed the best result; when 2.8% of corn starch was added, the average energy consumption was reduced by 14%
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