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- Ekerljung, Lina, 1980-
(författare)
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EGFR- and HER2-Binding Affibody Molecules : Cellular studies of monomeric, dimeric and bispecific ligands
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy. Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging. The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors.
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| 2. |
- Göstring, Lovisa, 1970-
(författare)
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Cellular Studies of HER-family Specific Affibody Molecules
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy.Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated.In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules.Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation.The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.
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| 3. |
- Jafari, Rozbeh, 1977-
(författare)
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Construction, expression and evaluation of anti-keratin 8 single-chain antibody fragments
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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