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

• 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.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)

Nyckelord

single-chain antibody

functional mapping

site-directed mutagenesis

immune complex

metabolism

Molecular biology

Molekylärbiologi

Biomedical Laboratory Science

Biomedicinsk laboratorievetenskap

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