209. TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD
Authors: Pietro Genovese, Sara Mastaglio, Zulma Magnani, Elisa Landoni, Barbara Camisa, Giulia Schiroli, Elena Provasi, Angelo Lombardo, Andreas Reik, Nicoletta Cieri, Maurilio Ponzoni, Fabio Ciceri, Claudio Bordignon, Michael C. Holmes, Philip D. Gregory, Luigi Naldini, Chiara Bonini
Journal: Molecular Therapy (2015)

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer adoptive immunotherapy. Yet, TCR gene transfer into mature T cells results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. Thus, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes (Provasi et al, Nat. Med. 2012). While successful, the complete editing requires multiple manipulation steps involving repeated cell activation cycles and transductions. To reduce the duration and complexity of cell product generation, we recently developed a 'single TCR editing' (SE) procedure, based on the disruption of the endogenous TCR α chain only followed by the transfer of the tumor specific TCR genes. This SE method generates redirected T cells fully devoid of their natural TCR repertoire in a single round of cell activation. We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by a considerable proportion of high risk multiple myeloma). The SE strategy allowed rapid production of high numbers of tumor specific T cells enriched for an early differentiation phenotype. When fucntionaly tested (co-culture, γ-IFN and 51Cr release) against the U266 myeloma cell line (NY-ESO-1+HLA-A2+), all NY-ESO-1 redirected T cells showed a strikingly high killing activity. However, when we assess the alloreactive potential of the different redirected T cells in mixed lymphocyte reactions, we observed that the allogeneic lysis by SE T cells was markedly lower (p=0.05) than that of conventional TCR transfer cells (TR). These results were validated in NSG mice, where the genetically modified T cells were infused after the engraftment of the U266 myeloma. All animals treated with tumor specific T cells were completely myeloma-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 redirected T cells. However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (p<0,001) corresponding with a significant difference in acute and chronic GvHD occurrence (71% vs 0%, p<0,001). Consistently, histopathological analysis of human T cell infiltration in the organs revealed a significantly higher score in mice treated with TR cells (p<0.001). The relative simplicity of the SE protocol enables rapid generation of highly performing tumor specific T cells, fully devoid of their endogenous TCR repertoire, and thus incapable of participating in GvHD. Such single TCR edited cells thus potentially represent a further advance in adoptive immunotherapy for cancer.