Book of abstract 2008

Monoclonal antibody 2A2 and its association with cathepsin B
Bojana Mirković1, Vesna Hodnik3, Bojan Doljak1, Gregor Anderluh2, Aleš Premzl1, Janko
Kos1 ,3
1University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia; 2University of Ljubljana, Biotechnical
Faculty, Ljubljana, Slovenia; 3Jožef Stefan Institute, Ljubljana, Slovenia
In our previous studies a monoclonal antibody 2A2 was prepared, which specifically
binds cathepsin B and inhibits its enzymatic activity. Significant inhibition of tumour
cell invasion in vitro and in vivo, internalization of the antibody into tumour cells and its
ability to retain binding affinity for cathepsin B at neutral and acid pH suggest possible
application in treatment of cancer and other diseases with increased cathepsin B activity.
Fab fragments and chimeric human/mouse antibody were also prepared in order to
enhance cell internalisation and to reduce immunogenicity during cancer therapy.
Using SPOT analysis we identified the epitope for 2A2 Mab at the occluding loop of the
cathepsin B and we propose that the antibody may regulate endopeptidase/exopeptidase
activity of the enzyme. Kinetics of the interaction between 2A2 Mab and cathepsin B was
determined by Surface Plasmon Resonance. The results show that 2A2 preferentially binds
cathepsin B/cystatin C complex compared to free enzyme. As shown by native PAGE
the binding of the monoclonal antibody 2A2 or its Fab fragment to cathepsin B/cystatin
C complex causes dissociation of cystatin C from the complex. Additionally, synthetic
peptides, mimicking the epitope or adjacent parts of the molecule have been prepared and
tested for binding to the components of the extracellular matrix.
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