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Construction and purification of a covalently linked divalent tandem single-chain Fv antibody against placental alkaline phosphatase.
Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Radiation Sciences, Diagnostic Radiology.
Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
2006 (English)In: Hybridoma, ISSN 0272-457X, Vol. 25, no 5, 255-263 p.Article in journal (Refereed) Published
Abstract [en]

Multivalency is a recognized means to increase the functional affinity of single-chain antibody fragments (scFvs) for optimizing tumor uptake at radioimmunotargeting. A unique divalent tandem single-chain Fv antibody [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) H7, has now been generated. The antibody differs from other dimeric single-chain constructs in that a linker sequence (L) is introduced between the repeats of VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed as a His-tagged TrxA fusion protein in the Escherichia coli strain Origami B. Following cleavage with AcTev protease, the antibody was obtained in soluble and active form in E. coli and could be purified by Ni-NTA and cation-exchange chromatography. Purity and immunochemical properties were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blot, and Biacore analyses. The [sc(Fv)2] displayed proper stability and could be purified to homogeneity. This antibody also maintained immunoreactivity at 42 degrees C with only slight decrease at 52 degrees C. The high affinity displayed by the original antibody H7, 6.7 x 10(9) M(-1), was only slightly decreased to 1.2 x 10(9) M(-1) as determined by Biacore. The generation of such a divalent single-chain Fv with a molecular weight around 60 kd would be of value for clinical applications such as radioimmunolocalization and radioimmunotherapy.

Place, publisher, year, edition, pages
2006. Vol. 25, no 5, 255-263 p.
URN: urn:nbn:se:umu:diva-21197DOI: 10.1089/hyb.2006.25.255PubMedID: 17044780OAI: diva2:210956
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2009-12-03
In thesis
1. Recombinant antibodies and tumor targeting
Open this publication in new window or tab >>Recombinant antibodies and tumor targeting
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates.

In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice.

One hybridoma, producing the monoclonal antibody H7, against placental alkaline phosphatase was used to provide CDR-containing regions for new recombinant constructs. Both single-chain variable fragments (scFv) antibodies or divalent tandem monospecific scFv [sc(Fv)2] antibodies with properties suitable for targeting were produced. By site directed mutagenesis four selective sequence substitutions were made in the VL fragment and one in the VH fragment of the antibody to improve solubility. To increase the stability of the antibody an extra -S-S- bond was introduced between the VL fragment and the VH region to create single-chain disulphide stabilized variable fragments (scdsFv) antibodies. Altogether four different recombinant scFv/scdsFv antibody constructs were produced and compared in terms of solubility, stability, affinity and production properties. The overall affinity could be maintained at the same order of magnitude as the wild type antibody (KA >109 (M-1) as determined by Biacore measurements. The antibodies could easily be expressed in the E.Coli strain BL21 Origami B (DE3). The purified recombinant antibodies could be maintained stable in concentrations up to 0.8 mg/ml.

Similarly a bivalent recombinant scFv antibody was generated from the same basic scFv, in form of a divalent tandem single-chain antibody [sc(Fv)2] by introduction of a short linker sequence between the two scFvs. The recombinant antibody was characterized by SDS-PAGE, ELISA, Western blot and Biacore analyses and was found to retain a similar high affinity as the original H7 antibody. All recombinant antibody derivates were subsequently investigated for targeting ability in vivo. Nude mice, inoculated with HeLa Hep2 tumor cells were exposed in vivo to the 125I-labelled recombinant constructs. All recombinant antibodies were found to localize and display characteristic differences in retention time within the tumor, tumor to non-tumor ratios and biodistribution properties. The tandem antibody, with a molecular weight around 60 kDa, was found to be superior compared to the scFv/scdsFv and intact antibodies in terms of discriminating properties at localization.

The immunotherapeutic potential of the intact antibodies was tested both alone with radiolabeled antibody (radioimmunotherapy, RIT) and in combination with external radiotherapy (RT). The combined effects of RT and RIT were found to cause significant growth retardation of the tumor with dramatic changes in morphology within the tumors and appearance of both necrosis and apoptosis. Increase in amount of connective tissue and cysts and decrease in cell density were observed. These result support the notion that antibodies have the potential of becoming significant tools in the arsenal of therapeutics for treatment of malignancies.

62 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1051
Research subject
urn:nbn:se:umu:diva-875 (URN)91-7264-165-7 (ISBN)
Public defence
2006-10-13, Astrid Fagraeussalen (A 103), 6A, NUS, Umeå, 09:00 (English)
Available from: 2006-09-25 Created: 2006-09-25 Last updated: 2009-10-29Bibliographically approved

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