NG2/CSPG4 transmembrane proteoglycan (PG) is a multivalent cell surface macromolecule whose impact on tumour growth and dissemination has been extensively documented in a variety of tumours and animal models. The PG is abundantly expressed in numerous soft-tissue sarcoma subtypes where it tightly links to disease progression and evolvement of metastases. Several of our previous studies have highlighted primary mechanisms through which NG2/CSPG4 may enhance malignancy and promote metastasis formation in sarcomas and have established a clear value of the PG a theranostic factor.
With the intent to generate highly unique, proprietary anti-NG2/CSPG4 monoclonal antibodies (mAbs) that could be proposed as potential immunotherapeutic compounds, we have recently created the largest anti-NG2/CSPG4 hybridoma selection worldwide. Following appropriate cellular screens, we discovered that, upon binding to the antigen, a subset of 8 of these mAbs – currently under patenting (PCT/IB2015/059921/Europe/USA) – was capable of inducing apoptosis/autophagy in sarcoma cells, and effectively interfered with sarcoma growth in vivo. Collectively, this series of observations led to the identification of a lead anti-neoplastic antibody (out of the 8 evaluated) for which we are proposing the denomination: “engituzumab” (according to the WHO International Nonproprietary Names guidelines). We aim here to establish the pre-clinical rationale for a direct transfer of the mAbs to a more advanced pre-clinical stage. To this end, we will focus on the following objectives:
1) investigate the molecular mechanisms through the antibody induced apoptosis and autophagy;
2) determine the precise biodistribution and pharmacodynamics of the antibody in rodent “non-metastatic” and metastatic soft-tissue sarcoma models.
To better resolve the detailed molecular mechanisms underlying the pro-apoptotic/pro-autophagic activity of the mAb, immunosorted NG2/CSPG4+ sarcoma cells and their counterpart NG2/CSPG4- ones, stably transfected with a signaling-death NG2/CSPG4 construct, will be exposed to engituzumab and processed for a global phospho-proteomic mapping by combined antibody array and mass spectometry. For detailed pharmacodynamic and biodistributional analyses we will use xenographic/orthotopic implantations and systemic intravenous infusions into athymic nude and NOD-SCID mice. Animals will receive variants of the above described immunosorted NG2/CSPG4+ sarcoma model cell lines that have been engineered to display a dual-tracer (luminescence-fluorescence) IRES-based construct for non-invasive, high-resolution whole-body monitoring of tumour growth and spreading. We envision that the outcome of the study will consolidate the anti-neoplastic nature of a novel immunotherapeutic agent and favor its progression through the necessary preclinical development for reaching clinical experimentation on sarcoma patients.
Roberto Perris, PhD,
Universita degli Studi di Parma (Italy)
Recipient of the: $50,000 Christopher Langbein Research Award