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Interaction studies of idiotypic and antiidiotypic antibodies at experimental tumor targeting
Umeå University, Faculty of Medicine, Clinical Microbiology.
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 [en]
anti-idiotypic antibody, clearing, cytokeratin 8, epitope, idiotypic antibody, immunotherapy
Identifiers
URN: urn:nbn:se:umu:diva-583ISBN: 91-7305-905-6 (print)OAI: oai:DiVA.org:umu-583DiVA: diva2:143867
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
List of papers
1. Epitope specificity of the monoclonal anticytokeratin antibody TS1
Open this publication in new window or tab >>Epitope specificity of the monoclonal anticytokeratin antibody TS1
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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.

Identifiers
urn:nbn:se:umu:diva-4665 (URN)9892182 (PubMedID)
Available from: 2005-09-05 Created: 2005-09-05 Last updated: 2017-12-14Bibliographically approved
2. Studies of the interactions between the anticytokeratin 8 monoclonal antibody TS1, its antigen and its anti-idiotypic antibody alphaTS1
Open this publication in new window or tab >>Studies of the interactions between the anticytokeratin 8 monoclonal antibody TS1, its antigen and its anti-idiotypic antibody alphaTS1
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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.

Identifiers
urn:nbn:se:umu:diva-4666 (URN)10.1002/jmr.617 (DOI)12833571 (PubMedID)
Available from: 2005-09-05 Created: 2005-09-05 Last updated: 2017-12-14Bibliographically approved
3. Functional mapping of the antiidiotypic antibody αTS1 by use of site directed mutagenesis, kinetic analysis and phage displayed peptide libraries.
Open this publication in new window or tab >>Functional mapping of the antiidiotypic antibody αTS1 by use of site directed mutagenesis, kinetic analysis and phage displayed peptide libraries.
Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:umu:diva-4667 (URN)
Available from: 2005-09-05 Created: 2005-09-05Bibliographically approved
4. Idiotyppic-anti-idiotypic complexes and their in vivo metabolism.
Open this publication in new window or tab >>Idiotyppic-anti-idiotypic complexes and their in vivo metabolism.
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2002 (English)In: Cancer, ISSN 0008-543X, E-ISSN 1097-0142, 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.

Keyword
anti-idiotype, idiotype, immune complex, metabolism, TS1
Identifiers
urn:nbn:se:umu:diva-4668 (URN)10.1002/cncr.10301 (DOI)11877761 (PubMedID)
Available from: 2005-09-05 Created: 2005-09-05 Last updated: 2017-12-14Bibliographically approved
5. In vivo clearing of idiotypic antibodies with antiidiotypic antibodies and their derivatives
Open this publication in new window or tab >>In vivo clearing of idiotypic antibodies with antiidiotypic antibodies and their derivatives
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2006 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 43, no 6, 599-606 p.Article in journal (Refereed) Published
Abstract [en]

At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.

Identifiers
urn:nbn:se:umu:diva-13533 (URN)10.1016/j.molimm.2005.04.019 (DOI)15978666 (PubMedID)
Available from: 2007-02-23 Created: 2007-02-23 Last updated: 2017-12-14Bibliographically approved

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