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Functional mapping and in vivo metabolism of the monoclonal antibody TS1 and its single-chain fragment: Its interaction with the antigen and the anti-idiotype
Karlstad University, Faculty of Technology and Science.
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Antibodies are proteins capable of specific interactions to a wide range of molecules. These interactions are facilitated by the complementary determining regions (CDR).

Carcinomas are the most common of human cancers and they release significant amount of cytokeratins (CK) in the necrotic areas of the tumors. The CKs stay in the tumor, since they have low solubility. The antibody studied in this thesis, the anti-CK 8 antibody TS1, has shown to be effective in tumor targeting and is proposed to be useful in therapy.

Single-chain antibodies (scFv) are recombinant antibodies which are much smaller than the intact IgG. This is an advantage when used in tumor therapy, since they can penetrate the tumors more easily than the larger IgG. Moreover, they are expressed by one single gene which make them easy to modify, for example by site-directed mutagenesis.

The anti-idiotypic antibody αTS1 can be used to clear the TS1 form the circulation and thereby clear the body from non-tumor bound TS1 in therapy. To be able to modify the binding of an antibody to its antigen and or anti-idiotype, these interactions must be studied. In this study this is accomplished by chemical modifications of the IgGs TS1 and αTS1 and the antigen CK 8. Guided by these results, amino acid residues were mutated by using site-directed mutagenesis in the TS1-218 scFv and the effects were studied. From mutational study results, the functional epitope could be mapped and it was found that there are mainly tyrosines, but also charged residues, serine and a tryptophan that are important for both interactions. The binding of TS1-218 to both αTS1 and CK 8 could be improved by changing the negatively charged side-chains by mutations to their corresponding amide or alanine.

Both the IgG and scFv versions of TS1 were administered in vivo. The IgG αTS1 was used to clear the TS1 from the circulation by forming immune complexes. The immune complexes, consisting of four or more antibodies, were mainly metabolized by the liver. The scFv TS1-218 could localize to the tumor in a tumor xenograft mouse model, although a higher uptake would be desired in a therapeutic strategy. The scFv was cleared rapidly by the kidneys, but the clearance could be slowed by pre-formed immune complexes with anti-TS1 scFv in vitro, prior to administration in vivo.

Place, publisher, year, edition, pages
Fakulteten för teknik- och naturvetenskap , 2006.
Series
Karlstad University Studies, ISSN 1403-8099 ; 2006:62
Keywords [en]
single-chain antibody, functional mapping, site-directed mutagenesis, immune complex, metabolism
National Category
Cell and Molecular Biology
Research subject
Biomedical Laboratory Science
Identifiers
URN: urn:nbn:se:kau:diva-727ISBN: 91-7063-093-3 (print)OAI: oai:DiVA.org:kau-727DiVA, id: diva2:6425
Public defence
2006-12-18, Fryxellsalen, 1B 306, Karlstad University, Karlstad, 10:15
Opponent
Supervisors
Available from: 2007-03-14 Created: 2007-03-14 Last updated: 2018-01-11
List of papers
1. Idiotypic-anti-idiotypic complexes and their in vivo metabolism
Open this publication in new window or tab >>Idiotypic-anti-idiotypic complexes and their in vivo metabolism
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2002 (English)In: Cancer, ISSN 0008-543X, Vol. 94, no 4 suppl, p. 1306-1313Article in journal (Refereed) Published
Identifiers
urn:nbn:se:kau:diva-2081 (URN)11877761 (PubMedID)
Available from: 2007-03-14 Created: 2007-03-14 Last updated: 2013-10-08Bibliographically approved
2. Studies of the interactions between the anticytokeratin 8 monoclonal antibody TS1, its antigen and its anti-idiotypic antibody αTS1
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 αTS1
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2003 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 16, no 3, p. 157-163Article in journal (Refereed) Published
Abstract [en]

The monoclonal antibody TS1 against cytokeratin 8 and its antiidiotype αTS1 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, αTS1, were studied in detail in order to make future veneering of the interactions possible. The V-genes of TS1 and αTS1 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, αTS1 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 αTS1 respectively are partly overlapping; a histidine identified in TS1 is probably involved only in the interaction with αTS1. 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 αTS1 in vivo.

Keywords
CDR, canonical classes, molecular modelling, chemical modification
National Category
Cell and Molecular Biology
Research subject
Biomedical Sciences
Identifiers
urn:nbn:se:kau:diva-2324 (URN)10.1002/jmr.617 (DOI)
Available from: 2010-08-31 Created: 2010-08-31 Last updated: 2018-01-13Bibliographically approved
3. Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1
Open this publication in new window or tab >>Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1
2007 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 44, no 6, p. 1075-1084Article in journal (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:kau:diva-2083 (URN)10.1016/j.molimm.2006.08.001 (DOI)
Available from: 2007-03-14 Created: 2007-03-14 Last updated: 2017-12-06Bibliographically approved
4. The biodistribution of the anti-cytokeratin 8 scFv TS1-218 in a tumor xenograft mouse model
Open this publication in new window or tab >>The biodistribution of the anti-cytokeratin 8 scFv TS1-218 in a tumor xenograft mouse model
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:kau:diva-2084 (URN)
Available from: 2007-03-14 Created: 2007-03-14 Last updated: 2013-03-20Bibliographically approved

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