Garofalo, Sonia (2008) Antitumor activity of a novel TLR9 agonist and cooperativity with targeted agents. [Tesi di dottorato] (Unpublished)

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Novel modified Toll-like receptor 9 (TLR9) agonists, termed immune modulatory oligonucleotides (IMOs), exhibit antitumor activity and are currently investigated in cancer patients. Intriguingly, their mechanisms of action on tumor growth and angiogenesis are still incompletely understood. Using an IMO recognizing human and murine TLR9, we recently discovered that IMO acts by impairing Epidermal Growth Factor Receptor (EGFR) signalling, cooperates with anti-EGFR drugs and boosts the antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-EGFR antibodies. IMO in fact cooperates with anti-EGFR antibody cetuximab in GEO human colon cancer xenografts, through the potentiation of cetuximab ADCC and the inhibition of EGFR signalling. IMO is instead ineffective in VEGF overexpressing cetuximab-resistant GEO-CR tumors. Therefore, we used IMO and the anti-VEGF antibody bevacizumab as tools to study IMO’s role on EGFR and angiogenesis and to explore its therapeutic potential in GEO, LS174T and GEO-CR cancer xenografts. We found that bevacizumab has no ADCC and IMO is unable to enhance it. Nevertheless, IMO plus bevacizumab combination cooperatively inhibits the growth of GEO and LS174T preceded by inhibition of signalling proteins expression, microvessels formation and human, but not murine, VEGF secretion. Interestingly also the growth of GEO-CR tumors is cooperatively inhibited by this drug combination. Moreover, IMO shows a direct antiangiogenic activity, inhibiting survival, adhesion, migration and capillary formation of VEGF-stimulated endothelial cells. The antitumor activity was irrespective of the TLR9 expression on tumor cells. Therefore, considering that IMO creates the opportunity to take advantage of multiple chance for cooperativity, involving EGFR- and ADCC-dependent and -independent mechanisms and neoangiogenesis, we have evaluated IMO ability to revert mechanisms of resistance in different tumor models from colon cancer. Since intrinsic and acquired resistance to anti-ErbB2 antibody trastuzumab is becoming an increasingly relevant issue in breast cancer, we investigated whether the antitumor, antiangiogenic and immunostimulatory activity of IMO could enhance trastuzumab effect in trastuzumab-resistant breast cancers. We found that IMO potentiates trastuzumab-mediated ADCC on KPL-4 and JIMT-1 cells, naturally resistant to trastuzumab in vitro. IMO inhibits the growth of KPL-4 and JIMT-1 xenografts, and potentiates the inhibitory effect of trastuzumab, with an almost complete suppression of tumor growth and expression of several ErbB-related signal transducers by the combined treatment. The combination produces also a cooperative direct antiangiogenic effect. Surprisingly, TLR9 is expressed in KPL-4 cells and seems to functionally interact with EGFR. These studies demonstrate that TLR9 agonists interfere with tumor and its microenvironment by multiple mechanisms providing a strong rationale to combine IMO with several targeted agents in cancer patients.

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