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Epitope specificity of the monoclonal anticytokeratin antibody TS1
Umeå University, Faculty of Medicine, Clinical Microbiology.
Umeå University, Faculty of Medicine, Clinical Microbiology.
Umeå University, Faculty of Medicine, Clinical Microbiology.
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
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1999 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 59, no 1, 48-51 p.Article in journal (Refereed) Published
Abstract [en]

Due to their abundance in epithelial cells and deposition in necrotic regions intratumorally, cytokeratins (CKs) have been established as valuable targets for both radioimmunolocalization and radioimmunotherapy. The target epitope for the monoclonal anti-CK8 antibody, TS1, used for both experimental radioimmunolocalization and radioimmunotherapy, was determined by means of synthesis of 96 overlapping peptides that covered the entire CK8 molecule. A highly conserved peptide sequence, spanning amino acids (aa) 343-357 and covering the discontinuous epitope in the helical 2B domain, was identified. The epitope retains its helical structure, as shown with circular dichroism spectroscopy, although the length of the peptide (ie., >20 aa) is crucial for maintenance of immunoreactivity. To determine which aa residues are crucial for binding to the monoclonal antibody, alanine scanning was performed on a 26-mer covering aa 340-365, with the sequence QRGELAIKDANAKLSELEAALQRAKQ. The 26 modified peptides were evaluated using ELISA and BIAcore technology. The uniqueness of this epitope has been established by data base sequence comparisons.

Place, publisher, year, edition, pages
1999. Vol. 59, no 1, 48-51 p.
Identifiers
URN: urn:nbn:se:umu:diva-4665PubMedID: 9892182OAI: oai:DiVA.org:umu-4665DiVA: diva2:143862
Available from: 2005-09-05 Created: 2005-09-05 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Interaction studies of idiotypic and antiidiotypic antibodies at experimental tumor targeting
Open this publication in new window or tab >>Interaction studies of idiotypic and antiidiotypic antibodies at experimental tumor targeting
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

An increasing number of antibodies are being labeled with radionuclides for immunotargeting, but the negative side effects generated on normal tissues by the nuclide remains a significant problem. The clearing of non-tumor-targeted radiolabeled antibodies from the circulation contributes significantly to improved targeting; it has been demonstrated that anti-idiotypic antibodies can be used for this purpose. This thesis describes a study of the interactions between the idiotypic antibody TS1, its intracellular tumor-associated antigen CK8 and anti-idiotypic antibody aTS1 as well as the clearing and metabolism of the complexes formed between TS1 and aTS1.

The TS1 epitope on CK8, located at amino acid positions 347–348, 350, 354–355, and 357 in helix 2B, was identified through alanine scanning. Upon chemical modification of TS1 using protective and unprotective approaches, it became apparent that the TS1 interaction sites for aTS1 and CK8 were partially overlapping. Aspartic acid, glutamic acid, and tyrosine residues and primary amino groups are important for the interactions of TS1 with both CK8 and aTS1. One modification of acidic amino acids in TS1 increased the affinity for CK8. Furthermore, TS1 contains one histidine residue that is located in a groove of the interaction surface and, from chemical modification studies; this histidine residue appears to play an important role for the interaction only with aTS1. Using phage display technology, specifically binding peptides having homologies with aTS1 were selected against TS1; site-directed mutagenesis verified the importance of two histidines, one valine, and one tyrosine residue in the homologous region of the light chain of aTS1. Site-directed mutagenesis also demonstrated that Y32 and K50 in the light chain and K33 and Y52 in the heavy chain positioned centrally on the antibody combining site of aTS1 were crucial for the binding to TS1. Two aTS1 mutants having affinities for TS1 similar to that of the wild type, but having faster association and dissociation rate constants, were identified.

The clearing capacity of the monoclonal aTS1IgG wild type was studied in vivo in non-tumor-bearing mice. At a molar ratio of TS1:aTS1 of 1:1.2, the whole-body radioactivity decreased by 85%; this value compares to the 25% reduction observed for the control, which received no aTS1. The metabolism of the complex formed between TS1 and aTS1 in vivo was also investigated; most of the degradation occurred in the liver. The complexes formed between TS1 and aTS1 were studied using electron microscopy, which indicated that ring structures of four, six, or eight antibodies were preferred; dimers were rare. ScFv, Fab, and Fab´2 fragments of aTS1 were generated and their abilities to clear the circulation from radiolabeled TS1 in vivo in non-tumor-bearing mice were studied. IgG was the most efficient clearing agent; Fab´2 and Fab displayed lower, but significant, clearing abilities. The scFv:s provided a slight degree of clearing if preincubated with TS1.

The results presented in this Thesis demonstrate that it is possible to engineer the properties of antibodies to make them suitable for use in immunotherapy.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi, 2005. 78 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 974
Keyword
anti-idiotypic antibody, clearing, cytokeratin 8, epitope, idiotypic antibody, immunotherapy
Identifiers
urn:nbn:se:umu:diva-583 (URN)91-7305-905-6 (ISBN)
Public defence
2005-09-19, Sal A103, Astrid Fagraeussalen, By 6, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Available from: 2005-09-05 Created: 2005-09-05 Last updated: 2009-11-12Bibliographically approved

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