Trop-2 is an epithelial transmembrane glycoprotein that transduces a calcium signal and activates a growth-signaling network that converges on AKT. Trop-2 is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation, and in its mature, glycosylated/functionally-competent form associates with worse prognosis. Trop-2 extracellular domain contains an N-terminal cysteine-rich globular region followed by a cysteine-less region as a connecting “stem” to the transmembrane domain. Trop-2 molecules engage in homophylic interactions between adjacent cells and establish multimeric complexes with tight junction proteins, which may hinder accessibility by therapeutic antibodies. Up to now, Trop-2-targeted approaches have employed anti-Trop-2 monoclonal antibodies (mAb) which essentially recognize a single immunodominant epitope poised between the globular and stem regions. Such mAb have limited or no therapeutic effıcacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specifıcity towards the globular versus stem regions. Hybridoma diversity was maximized by immunization with soluble human Trop-2 extracellular region produced in different transformed mammalian cell lines (human 293 and murine L) and in insect cells/baculovirus expressing system. These were expected to provide native folding of Trop-2 together with a broad spectrum of differential glycosylation. Trop-2-binding hybridomas were further selected by multiple rounds of flow cytometry analysis using live 293 cells expressing different Trop-2 extracellular portions. Two classes of mAb were identifıed, that bound the stem versus the globular region. These mAb effıciently bound Trop-2 expressing cancer cells and were able to inhibit cell growth in vitro. In vivo the naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of distinct tumors, including colon, ovary and prostate cancers. Notably, they showed differential effıcacy for established tumors versus isolated-cell models of metastatic dissemination, consistent with our strategy of maximizing differential accessibility of Trop-2 according to growth mode. Most remarkably, we demonstrated in vivo synergy of these anti-Trop-2 mAb, paving the way for game-changing anti-cancer mAb therapy. The differential effıcacy of the OXG64 and OX-S55 anti-Trop2 mAb against different tumor histotypes and growth stages further allows to exploit their cancer-killing potential in pathological stage-tailored therapeutic approaches.

Novel anti-Trop-2 monoclonal antibodies with unique binding specifıcities show therapeutic synergy against most human cancers

TREROTOLA, MARCO;PEDICONE, CHIARA;D'AMORE, ANTONELLA;ALBERTI, SAVERIO
2017

Abstract

Trop-2 is an epithelial transmembrane glycoprotein that transduces a calcium signal and activates a growth-signaling network that converges on AKT. Trop-2 is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation, and in its mature, glycosylated/functionally-competent form associates with worse prognosis. Trop-2 extracellular domain contains an N-terminal cysteine-rich globular region followed by a cysteine-less region as a connecting “stem” to the transmembrane domain. Trop-2 molecules engage in homophylic interactions between adjacent cells and establish multimeric complexes with tight junction proteins, which may hinder accessibility by therapeutic antibodies. Up to now, Trop-2-targeted approaches have employed anti-Trop-2 monoclonal antibodies (mAb) which essentially recognize a single immunodominant epitope poised between the globular and stem regions. Such mAb have limited or no therapeutic effıcacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specifıcity towards the globular versus stem regions. Hybridoma diversity was maximized by immunization with soluble human Trop-2 extracellular region produced in different transformed mammalian cell lines (human 293 and murine L) and in insect cells/baculovirus expressing system. These were expected to provide native folding of Trop-2 together with a broad spectrum of differential glycosylation. Trop-2-binding hybridomas were further selected by multiple rounds of flow cytometry analysis using live 293 cells expressing different Trop-2 extracellular portions. Two classes of mAb were identifıed, that bound the stem versus the globular region. These mAb effıciently bound Trop-2 expressing cancer cells and were able to inhibit cell growth in vitro. In vivo the naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of distinct tumors, including colon, ovary and prostate cancers. Notably, they showed differential effıcacy for established tumors versus isolated-cell models of metastatic dissemination, consistent with our strategy of maximizing differential accessibility of Trop-2 according to growth mode. Most remarkably, we demonstrated in vivo synergy of these anti-Trop-2 mAb, paving the way for game-changing anti-cancer mAb therapy. The differential effıcacy of the OXG64 and OX-S55 anti-Trop2 mAb against different tumor histotypes and growth stages further allows to exploit their cancer-killing potential in pathological stage-tailored therapeutic approaches.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11564/686367
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