Allogeneic CAR T-cells resistant to both T- and NK-cell cytotoxicity

Abstract

Background & Aim The last years have seen the adoptive transfer of engineered autologous T-cells making great strides in the development of new treatments against cancer. The use of third-party donor-derived T-cells represents an attractive alternative to generate CAR T-cells readily accessible to patients. These off-the-shelf cells derived from a possibly non-HLA-matched donor nevertheless carry the risk of graft-versus-host-disease (GvHD) through the expression of their endogenous T-cell receptors (TCRs). The recent advances in precise genome editing using designer nucleases allowed to mitigate this issue as demonstrated by the molecular remissions observed in patients after infusion of universal TALEN® multiplex gene-edited CAR T-cells. In particular, these universal CAR T-cells were engineered to reduce the risk of graft versus host disease (GvHD) by TALEN® inactivation of TCRαβ. The clinical outcome of CAR T-cell therapies is intimately linked to the ability of effector cells to engraft and proliferate in order to eradicate tumor cells within patients. Although the transient activity of off-the-shelf CAR T-cells represents an important safety feature, the possibility to extend their therapeutic window may be desirable in particular disease indications and after lymphodepleting regimens where the patient's immune system has been restored. Thus, we are developing novel approaches to render these cells less visible to both host T- and NK-cells. Methods, Results & Conclusion The single genetic disruption of the beta-2 microglobulin (B2M) gene, a required component of all MHC class I molecules, promotes resistance to host CD8+ T-cell attack but may trigger NK-cell activation, leading to the rejection of the edited T-cells lacking MHC-I. We therefore further engineered these B2M negative cells via a TALEN®-mediated and B2M gene-specific targeted integration matrix to express inhibitors of NK cytotoxicity. We have identified an NK-cell inhibitor that successfully blocks the so-called NK-cell “missing self-response” in vitro and in vivo. Altogether, the precise TALEN®-mediated inactivation of B2M coupled with insertion of an NK-cell inhibitor enabled the generation of allogeneic CAR T-cells, resistant to host T- and NK-cells, with improved persistence and long-term anti-tumor activity.