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Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
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2006 (English)In: Hybridoma, ISSN 0272-457X, E-ISSN 2168-7897, Vol. 25, no 4, 181-192 p.Article in journal (Refereed) Published
Abstract [en]

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.

Place, publisher, year, edition, pages
2006. Vol. 25, no 4, 181-192 p.
Identifiers
URN: urn:nbn:se:umu:diva-21198DOI: 10.1089/hyb.2006.25.181PubMedID: 16934014OAI: oai:DiVA.org:umu-21198DiVA: diva2:210957
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2017-12-13
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.

Publisher
62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1051
Research subject
Immunology
Identifiers
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)
Opponent
Supervisors
Available from: 2006-09-25 Created: 2006-09-25 Last updated: 2009-10-29Bibliographically approved

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