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Studies of the interactions between the anticytokeratin 8 monoclonal antibody TS1, its antigen and its anti-idiotypic antibody alphaTS1
Umeå University, Faculty of Medicine, Clinical Microbiology.
Umeå University, Faculty of Medicine, Clinical Microbiology.
Umeå University, Faculty of Medicine, Clinical Microbiology.
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2003 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 16, no 3, 157-163 p.Article in journal (Refereed) Published
Abstract [en]

The monoclonal antibody TS1 against cytokeratin 8 and its antiidiotype alphaTS1 have been used for immunotargeting and therapy of carcinomas in experimental tumor model systems. The interaction surfaces between mab TS1, the cytokeratin 8 epitope, and its anti-idiotypic antibody, alphaTS1, were studied in detail in order to make future veneering of the interactions possible. The V-genes of TS1 and alphaTS1 were cloned and sequenced and the CDRs and the framework residues were identified. Amino acids participating in the interactions were identified following chemical modifications of residues in non-protected and protected molecules of cytokeratin 8, alphaTS1 and TS1. From the sequences, the three-dimensional structures were generated using computer modelling of the antibody variable regions. Several charged amino acid, histidine and tyrosine residues were displayed in the antibody surfaces implicated in the interactions and chemical modification confirmed the importance of these amino acids. The cytokeratin 8 epitope has previously been identified by Johansson et al. and it displays negatively charged amino acid residues which could be identified in the chemical modification. It was also revealed that the TS1 binding to cytokeratin 8 and alphaTS1 respectively are partly overlapping; a histidine identified in TS1 is probably involved only in the interaction with alphaTS1. Furthermore, the chemical modification demonstrated that exchanging aspartic-glutamic acids to asparagine-glutamine residues in TS1 increased the binding of TS1 to cytokeratin 8, indicating that there is at least one acidic amino acid that is an obstacle in the TS1-CK8 binding. The detailed assembly of the interaction surfaces will facilitate the future use of site directed mutagenesis to improve the TS1-CK8 association rate and the clearing of TS1 with alphaTS1 in vivo. Copyright 2003 John Wiley & Sons, Ltd.

Place, publisher, year, edition, pages
2003. Vol. 16, no 3, 157-163 p.
Identifiers
URN: urn:nbn:se:umu:diva-4666DOI: 10.1002/jmr.617PubMedID: 12833571OAI: oai:DiVA.org:umu-4666DiVA: diva2:143863
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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