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In vitro evaluation...
In vitro evaluation of avidin antibody pretargeting using 211At-labeled and biotinylated poly-L-lysine as effector molecule.
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- Frost, Sofia, 1981 (författare)
- Gothenburg University,Göteborgs universitet,Institutionen för kliniska vetenskaper, Avdelningen för radiofysik,Institute of Clinical Sciences, Department of Radiation Physics
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Jensen, Holger (författare)
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- Lindegren, Sture, 1960 (författare)
- Gothenburg University,Göteborgs universitet,Institutionen för kliniska vetenskaper, Avdelningen för radiofysik,Institute of Clinical Sciences, Department of Radiation Physics
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(creator_code:org_t)
- 2010-02-02
- 2010
- Engelska.
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Ingår i: Cancer. - : Wiley. - 0008-543X. ; 116:4 Suppl, s. 1101-10
- Relaterad länk:
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https://onlinelibrar...
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https://gup.ub.gu.se...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- BACKGROUND: Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, (211)At-labeled poly-L-lysine was constructed and analyzed in vitro. METHODS: Avidin activated by 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) and thiolated trastuzumab were incubated overnight at 4 degrees C. The monomeric fraction was extracted using size exclusion fast protein liquid chromatography (FPLC) and further purified on an iminobiotin affinity column. Poly-L-lysine was biotinylated with succinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin), followed by direct (211)At-labeling with N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), and succinylation with succinic anhydride. The avidin-trastuzumab conjugate was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and FPLC, together with cell-binding and biotin-binding analyses. The labeled poly-L-lysine conjugate was assessed in terms of radiochemical purity and avidin binding. Furthermore, the full pretargeting system was evaluated in a tumor cell binding assay. RESULTS: The estimated size of the pretargeting molecule was 220 kDa, which corresponds to that of the expected avidin-trastuzumab monomer. Neither cell-binding ability (64%) nor biotin-binding ability (85%-95%) indicated any severe adverse effects from the chemical modifications. The radiochemical purity of the effector molecule was 92%-97%, and the avidin binding capacity was 91%-93%. The complete pretargeting assay resulted in a binding of 75.3 +/- 6.2% of added effector molecules to cells. CONCLUSIONS: The high binding of effector molecules to cells demonstrates a proof of concept for the synthesized molecules and pretargeting system, which will be further evaluated in vivo in future studies.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin -- Radiologi och bildbehandling (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine -- Radiology, Nuclear Medicine and Medical Imaging (hsv//eng)
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