CRYSTALLOGRAPHIC STUDY OF AN ANTI –CARBONIC ANHYDRASE IX MONOCLONAL ANTIBODY M75

Renata Štouračová¹, Jan Závada¹, Zuzana Závadová¹, Silvia Pastoreková², Jiří Brynda¹, Milan Fábry¹, Vlastimil Král¹, Magda Hořejši¹, and Juraj Sedláček¹

¹Institute of Molecular Genetics, Academy of Sciences, 166 37 Prague, Czech Republic

²Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic

Carbonic anhydrase IX (CA IX) is a cell surface protein, strongly associated with certain types of human carcinomas. The predicted protein of cloned CA IX cDNA consists of the signal peptide, proteoglycan-related sequence, carbonic anhydrase domain, trasmembrane segment and a short intracellular tail (1, 2). Until now, molecular basis of involvement of CA IX in carcinogenesis has remained unclear. CA IX is a cell adhesion molecule, its carbonic anhydrase (CA) is enzymaticaly active. Structural study of a CA IX-binding monoclonal antibody (mAb) M75, complexed with its epitope peptide may contribute toward elucidation of the role of CA IX. To achieve this goal, two parallel approaches were chosen: analysis of Fab fragment, or of a smaller scFv fragment, both containing the complete antigen binding site present in mAb M75.

Monoclonal antibody M75 was obtained (3) and proved to react excellently with native and denaturated CA IX. Using synthetic oligopeptides, the epitope of mAb M75 was localized in the proteoglycan domain of CA IX, in the region of a tandem repeat and identified as amino acids PGEEDLP (4). The Fab fragment was obtained by papain cleavage. We obtained crystals of free Fab M75 and Fab M75 complexed with two different epitope peptides. The data set for Fab M75 was collected and the structure solving is underway.

Another approach is to prepare sc Fv fragment of this antibody (described in the contribution of Vlastimil KRAL, Milan Fabry, Magda Horejsi, Jan Zavada, Juraj Sedlacek: Molecular cloning, E. coli expression and purification of scFv antibody fragments
of diagnostic/therapeutic interest.

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2. Opavský R, Pastoreková S, Zelník V, Gibadulinová A, Stanbridge EJ, Závada J, Kettmann R, Pastorek Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. (1996) Genomics. 33:480-7.

3. Pastoreková S, Závadová Z, Kostál M, Babusiková O, Závada J. A novel quasi-viral agent, MaTu, is a two-component system. (1992) Virology. 187:620-6.

4. Závada J, Závadová Z, Pastorek J, Biesová Z, Ježek J, Velek J. Human tumour-associated cell adhesion protein MN/CA IX: identification of M75 epitope and of the region mediating cell adhesion. (2000) Br J Cancer. 82:1808-13