טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentArvatz Gil
SubjectHeparanase: Regulatory Mechanism and the Development
and Characterization of a Novel Neutralizing mAb
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains, activity that is strongly implicated in cell dissemination associated with tumor metastasis, inflammation and angiogenesis. Heparanase expression is elevated in the vast majority of hematological and solid tumors, and is associated with increased tumor metastasis. Mechanisms responsible for heparanase induction are incompletely understood. Here, we provide evidence that heparanase expression is subjected to post-transcriptional regulation. Employing primers that cover the entire 3'-untranslated region (3'-UTR) of heparanase yielded two distinct PCR products. Sequence analysis revealed that a shorter product results from exclusion of a 185 bp region that contains an adenine (A)/uracil (U)-rich element (ARE). While the full length 3'-UTR conferred reduced heparanase levels, the shorter form lacking the 185 bp region or the ARE motif was associated with a more stable heparanase mRNA, elevated levels of heparanase expression, enzymatic activity and cell invasion capacity, and formation of larger tumor xenografts. These results indicate that loss of the ARE is an important mechanism contributing to heparanase induction in cancer. It also imply that heparanase function is not limited to tumor metastasis but is engaged in progression of the primary lesion, thus critically supporting the intimate involvement of heparanase in tumor progression and encouraging the development of heparanase inhibitors as anti-cancer therapeutics.

Previous study from our laboratory was undertaken to identify functional domains that would serve as targets for drug development. Three potential heparin-binding domains of heparanase were identified based on consensus sequences. Particular attention was given to the Lys158-Asp171 domain since a peptide corresponding to this sequence (termed KKDC) physically interacts with HS with high affinity and inhibits heparanase enzymatic activity. Furthermore, deletion construct lacking this domain exhibits no enzymatic activity and polyclonal antibody (Ab #733) directed to this region inhibit heparanase activity. We have followed this rational and generated a panel of monoclonal antibodies directed against the KKDC peptide, targeting the interaction of heparanase with its HS substrate. We report the generation and characterization of such monoclonal antibody (mAb) 9E8 which neutralizes heparanase activity. The antibody markedly decreases heparanase activity, cellular invasion through reconstituted basement membrane and lung colonization by mouse B16 melanoma and human LM2-4 breast carcinoma cells. Moreover, mAb 9E8 attenuated the progression of tumor xenografts produced by MC38 mouse colon carcinoma cells. These results imply that generation of heparanase-neutralizing mAb is feasible, and enable us to elucidate the role of heparanase in cancer progression and metastasis.