752. Single Chain TCR Gene Editing in Adoptive Cell Therapy for Multiple Myeloma
Authors: Sara Mastaglio, Pietro Genovese, Zulma Magnani, Elisa Landoni, Barbara Camisa, Giulia Schiroli, Elena Provasi, Angelo Lombardo, Andreas Reik, Nicoletta Cieri, Martina Rocchi, Giacomoq Oliveira, Giulia Escobar, Monica Casucci, Bernhard Gentner, Antonello Spinelli, Anna Mondino, Attilio Bondanza, Luca Vago, Maurilio Ponzoni, Fabio Ciceri, Micheal Holmes, Luigi Naldini, Chiara Bonini
Journal: Molecular Therapy (2016)

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. The TCR gene editing technology, based on the knockout of the endogenous TCR alpha and beta genes, followed by the introduction of exogenous tumor-specific TCR genes, proved safer and more effective than conventional TCR gene transfer in vitro and in animal studies. While successful, complete editing requires multiple manipulation steps and four transduction procedures. To reduce the duration and complexity of the procedure, we developed and tested the 'single TCR editing' (SE) approach, based on the disruption of the endogenous TCR alpha chain only, followed by the transfer of the tumor specific TCR genes. We validated SE using an HLA-A2 restricted TCR specific for NY-ESO-1157-165, a cancer testis antigen expressed by numerous solid tumors and hematological malignancies, including high-risk multiple myeloma. Conventional TCR gene transfer (TR) and SE cells were compared in terms of efficacy and safety in vitro (phenotypical analysis, gamma-IFN EliSPOT, 51Cr release and co-culture assays) and in vivo (adoptive T cell transfer in an experimental humanized-mouse model of multiple myeloma). SE rapidly produced high numbers of tumor specific T cells, with an early differentiation phenotype. When tested in vitro, SE T cells showed a high killing activity against the U266 multiple myeloma cell line, similar to that of T cells redirected with conventional TCR gene transfer; however, while TR cells proved highly alloreactive, SE cells showed a favorable safety profile. The SE and TR cells were then compared in vivo by infusing the cells into NSG mice previously engrafted with myeloma. In contrast to the TR cells, SE cells mediated tumor rejection without inducing xenogeneic graft versus host disease, thus promoting a significantly higher survival than that observed in mice treated with TR cells. The detrimental alloreactive effect mediated by TR cells led to a xenogeneic GvHD rate comparable to that of untransduced allogenic T cells. This finding was confirmed by histopathological examination which revealed TR cell infiltrations and GvHD-like lesions in several murine organs and tissues. Conversely, the abrogation of the endogenous TCR in the SE tumor-redirected lymphocytes prevented off-target reactivity that normally leads to GvHD in mice, potentially providing a safer T cell therapy against cancer. Overall, the single TCR gene editing procedure provides a rapid and efficient method for generating primary T cells that highly express a tumor specific TCR and are devoid of their endogenous TCR, and thus represents a protean platform for the effective and safe adoptive transfer of allogeneic and autologous T lymphocytes redirected towards any desired tumor associated antigen.