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- Jafari, Rozbeh, 1977-
(author)
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Construction, expression and evaluation of anti-keratin 8 single-chain antibody fragments
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Antibodies are glycoproteins specifically binding to a variety of antigens and today extensively used in prevention, diagnosis and treatment of diseases. Carcinomas originate from epithelial tissues and are the most common forms of human malignancies. In the necrotic areas of carcinomas significant amounts of keratins (K) are found extracellularly. They remain there due to their low solubility and can be used as targets in immuno-targeting and -therapy. Single-chain fragment variable (scFv) may display several advantages over intact IgGs in various applications but their use in immunotherapy of tumors could be limited. Their small size and monovalency may result in low accumulation of the scFv in the tumors. The targeting efficiency of antibodies needs to be evaluated in vitro prior to in vivo studies in order to exclude poor candidates. In vitro models should preferably resemble the in vivo situation as much as possible. In the present study a scFv variant (TS1-218) of the anti-keratin 8 (K 8) monoclonal antibody TS1 (mAb TS1) was constructed and characterized using site-directed mutagenesis. In addition, the valency of the TS1-218 and one of its mutants, HE1-Q, were increased by construction of a covalently linked divalent single-chain fragment variable (sc(Fv)2). To improve the yield of the antibody fragments, the expression host was changed from E. coli to P. pastoris and culture conditions were optimized using Design of Experiments (DoE). Furthermore, a HeLa HEp-2 multicellular tumor spheroid (MCTS) in vitro model was established. The functionality of the radiolabeled TS1-218 alone and in immunocomplex with its anti-idiotype scFv, αTS1 scFv, were evaluated in the MCTS model and compared to a tumor xenograft nude mouse model. The targeting efficiency of the scFv and sc(Fv)2s were also investigated using MCTS. TS1-218 in immunocomplex with αTS1 scFv displayed a significantly higher uptake than the TS1-218 alone in both MCTS and tumor xenografts. The sc(Fv)2s, DiTS1-218 and DiHE1-Q demonstrated a higher functional affinity to K 8 in ELISA and MCTS and were retained to a larger extent in the MCTS than their scFv counterparts, with a 3.9 and 9.4-fold longer half-life, respectively. Furthermore, the yield of the antibody fragments were improved after expression in P. pastoris with an 86-fold improvement for the TS1-218 following optimization using DoE.
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- Watz, Johan, 1977-, et al.
(author)
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Wood addition in the hatchery and river environments affects post-release performance of overwintering brown trout
- 2019
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In: Freshwater Biology. - : Wiley. - 0046-5070 .- 1365-2427. ; 64:1, s. 71-80
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Journal article (peer-reviewed)abstract
- Habitat structural complexity affects the behaviour and physiology of individuals, and responses to the environment can be immediate or influence performance later in life through delayed effects. Here, we investigated how structural enrichment, both pre-release in the hatchery rearing environment and post-release in the wild, influenced winter growth and site fidelity of brown trout stocked into side channels of a regulated river. Experiencing structural enrichment in the rearing environment during 3 months in autumn had no pre-release effect on growth, but a delayed positive effect after release during the subsequent winter. Moreover, trout recaptured in wood-treated sections of the side channels had grown more than trout recaptured in control sections. Wood enrichment in the side channels also increased overwinter site fidelity. These results show that adding structure during a relatively short period may alter growth trajectories, and adding wood to side channels is a cost-effective method to enhance winter habitat carrying capacity for juvenile salmonids in regulated rivers.
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