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Idiotyppic-anti-idiotypic complexes and their in vivo metabolism.
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, Clinical Microbiology.
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2002 (English)In: Cancer, ISSN 0008-543X, Vol. 94, no S4, 1306-1313 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Different strategies can be used to improve the tumor:non-tumor ratio of radiolabeled antibodies in immunotargeting. One approach is to use secondary antibodies to clear out redundant, circulating primary antibodies. In the current study, the in vitro complex formation and in vivo clearing capabilities and metabolism of the monoclonal antibody TS1 and its monoclonal anti-idiotype, alphaTS1, were studied. METHODS: Complex formation studies were performed using polyacrylamide gel electrophoresis (PAGE), gel permeation chromatography, and electron microscopy. The clearance and metabolism of the complexes were studied in nude mice. RESULTS: PAGE and gel permeation chromatography showed that more than 70% of the antibodies formed complexes. The electron microscopy studies revealed that the complexes formed between TS1 and alphaTS1 are mainly ring-shaped (66.6-73.4%), comprising 4 to > 8 antibodies. These rings consist of equal numbers of idiotype and anti-idiotype. The most commonly observed complexes were tetrameric rings (26.8-40.5%), hexameric rings (10.7-11.9%), and rings containing more than eight monoclonal antibodies (6.6-14-4%). The in vivo study illustrated that within 24 hours 80% of the total nuclide content had been degraded and excreted via the urine, compared with 25% for similarly treated mice that did not receive any anti-idiotype. CONCLUSIONS: Interestingly, the electron microscopy study demonstrated that dimers were rare (0.4-1.2%), probably reflecting a location of epitopes incompatible with tight, sterically constrained dimeric interactions; insufficient flexibility of the immunoglobulin G1 subtype hinge regions; or both. The anti-idiotypic clearing mechanisms proved efficient in nude mice. In vivo metabolic studies indicate that the accumulation and degradation of TS1/alphaTS1 immune complexes, to a large extent, take place in the liver, where a substantial amount was detected as soon as 1 hour after anti-idiotype injection. Copyright 2002 American Cancer Society.

Place, publisher, year, edition, pages
2002. Vol. 94, no S4, 1306-1313 p.
Keyword [en]
anti-idiotype, idiotype, immune complex, metabolism, TS1
URN: urn:nbn:se:umu:diva-4668DOI: 10.1002/cncr.10301PubMedID: 11877761OAI: diva2:143865
Available from: 2005-09-05 Created: 2005-09-05Bibliographically 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.
Umeå University medical dissertations, ISSN 0346-6612 ; 974
anti-idiotypic antibody, clearing, cytokeratin 8, epitope, idiotypic antibody, immunotherapy
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)
Available from: 2005-09-05 Created: 2005-09-05 Last updated: 2009-11-12Bibliographically approved

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