Complex interactions of tumor cells with different stromal cell types and extracellular signals are critically involved in cancer progression. In particular, mesenchymal stromal cells (MSC) have been shown to imprint key cancer traits, including chemoresistance and stem-cell behavior. We have recently found the co-culturing leukemia and multiple myeloma cells with bone-marrow derived MSC selectively induces the up-regulation of CD44v6 and, therefore, developed a T-cell gene therapy strategy based on the redirection of T cells by means of a specific chimeric antigen receptor (CAR).
Unfortunately, MSC also display a strong immunosuppressive activity that hampers the antitumor efficacy of CAR-redirected T cells. In this project, we aim at identifying the molecular factors involved in the immunosuppressive activity of MSC on CD44v6-specific CAR-redirected T cells, with the final goal overcoming them for better antitumor efficacy.
Preliminary results produced in our laboratory suggest VEGF as a good candidate. However, to obtain a more comprehensive picture, CAR-redirected T cells exposed to MSC will be studied by gene expression profiling and analysis of the kinome by phosphoproteomics (in collaboration with ETH, Zurich). After identification, we will screen all potential candidates by assaying the proliferation and the cytotoxicity of CAR-redirected T cells in the presence of recombinant molecules. Moreover, we will attempt pharmacological inhibition of MSC-induced immunosuppression with drugs already in the clinic, including the mAb trastuzumab (anti-VEGF) and the VEGF-R inhibitor sunitinib. Alternatively, CAR-redirected T cells will be rendered insensitive to the immunosuppressive activity of MSC by RNA interference for the receptors of candidate molecules (i.e. VEGF-R2). As a final readout, the efficiency of the different strategies will be tested in vivo in humanized models based on the co-grafting of MSC and tumor cells in NSG mice.

This project is intended for the Basic and Applied Immunology Section of the PhD Program

Specific signals within the bone-marrow niche determine the neoplastic phenotype of acute myeloid leukemia (AML) and multiple myeloma (MM) cells. We have recently found that a specific variant (v6) of the hyaluronic acid receptor CD44 is selectively up-regulated upon co-culture of AML and MM cells with bone marrow-derived mesenchymal stromal cells (MSC). We have also shown that targeting CD44v6 with a chimeric antigen receptor (CAR)-redirected T cells has major antileukemia and antimyeloma activity both in vivo and in vitro, in xenograft mouse models.
In the absence of CD44, CD44 functions can be compensated for by the receptor for hyaluronic acid-mediated motility (RHAMM). In order to circumvent potential immune escape mechanism due to CD44 loss, we aim at developing a novel T-cell gene therapy strategy for the treatment of hematological tumors based on the dual redirection of T cells towards CD44v6 and RHAMM.
Dual redirection will be achieved by genetic modification of T cells with a bi-directional lentiviral vector encoding for the already available CD44v6-specific CAR and a newly generated RHAMM-specific CAR. The RHAMM-specific CAR will be generated by inserting multiple sequences of RHAMM antagonistic peptides within the CAR back-bone. The efficacy of dual CAR-redirected T cells will be evaluated both in vitro and in vivo. During the project, RNA interference will be employed to generate CD44v6 and RHAMM knock-downs. The neoplastic phenotype of the knock-downs as well as their relative sensitivity to single or dual redirected T cells will be studied in artificial bone-marrow niches produced in vitro by nanotechnology (in collaboration with Università del Salento, Bio-engineering Dep) and in vivo, in humanized models based on the grafting of primary leukemia and myeloma cells in NSG mice.

This project is intended for the Cancer Biology and Biotherapy Section of the PhD Program