TCR Gene Editing Achieved In a Single Round Of T Cell Activation Is Sufficient To Redirect T Cell Specificity and Prevent GvHD
Authors: Mastaglio Sara , Genovese Pietro , Magnani Zulma , Provasi Elena , Lombardo Angelo , Reik Andreas , Cieri Nicoletta , Ciceri Fabio , Bordignon Claudio , Holmes Michael C. , Gregory Philip D , Naldini Luigi , Bonini Chiara 
Published: October 2019

Abstract
The genetic transfer of tumor-specific T cell receptors (TCRs) into mature T lymphocytes enables T cell specificity to be redirected towards cancer cells, however the transfer of novel TCRs into polyclonal T cells, while overcoming tolerance barriers, may be limited by factors intrinsic to TCR biology. Specifically, the tumor-specific a and b TCR chains are expressed in lymphocytes that already bear an endogenous TCR on their cell surfaces. Gene-modified cells thus express at least two different TCRs that compete for binding to the CD3 complex, resulting in mutual TCR dilution and reduced avidity. Furthermore, since TCRs are heterodimers, the a and b chains of the endogenous TCR have the potential to mispair with the respective α and β chains of the transgenic TCR to produce a new hybrid TCR, with unpredictable and potentially harmful specificity. This represents a major concern in TCR transfer adoptive immunotherapy, both in autologous and allogeneic settings.
To permanently eliminate the expression of the endogenous TCR and the risk of mispairing, our group recently developed a TCR gene editing approach. This technique is based on the transient transfer of zinc-finger nucleases (ZFN) to induce DNA double strand breaks in the constant regions of the endogenous TCR a and/or b chain genes, leading to permanent gene disruption. Upon lentiviral transfer of a tumor-specific TCR, such fully TCR-edited T cells express only the exogenous tumor-specific TCR transgenes at high levels (Provasi, Genovese et al., Nature Medicine 2012). While the complete editing procedure (both a and b TCR chains) currently requires multiple manipulation steps, ‘single TCR editing’, based on the ZFN-mediated knock-down of a single endogenous TCR chain (a or b) followed by the introduction of the tumor-specific TCR, enables the generation of redirected T cells devoid of their natural TCR repertoire during a single round of T cell activation, improving the feasibility of the clinical translation of this approach. This might be particularly useful to reduce the risk of GvHD after allogeneic hematopoietic stem cell transplantation.
We exploited a HLA-A2 restricted TCR specific for NY-ESO-1, a cancer testis antigen expressed by solid tumors and hematological malignancies, to directly compare the safety and efficacy profile of unedited TCR transferred T cells (TR), single TCR edited (SE) lymphocytes and completely TCR edited (CE) T cells. We observed that gene editing does not detectably affect the phenotype, function or proliferative potential of engineered lymphocytes. Our protocols ensured the maintenance of the early differentiated memory phenotype, with enrichment in central memory and CD45RA+/CD62L+/CD95+ memory stem T (TSCM) cells. Upon lentiviral transfer of the NY-ESO-1-specific TCR, we observed significantly higher levels of the tumor-specific TCR expression, evaluated as NY-ESO-1 specific dextramer binding, in edited versus transferred T cells (relative fluorescence intensity to untransduced cells: CE: 37; SE: 31; TR: 19). Edited T cells were more efficient than unedited-TCR transferred T cells in killing NY-ESO-1-pulsed cell lines (half maximal effective peptide concentration in a 51Cr release assay: 310, 210, 186 nM for TR, SE and CE T cells respectively) and NY-ESO-1+ myeloma cell lines naturally processing the antigen. Importantly our SE and CE T cells displayed no activity against NY-ESO-1- targets. Importantly, in NSG mice, NY-ESO-1 redirected single edited and complete edited T cells completely eliminated an NY-ESO1+ HLA-A2+, WT1- myeloma cell line, that, on the contrary, expanded in bone marrow in the presence of WT1-redirected CE T cells. Our results demonstrate that the TCR single editing approach is effective in redirecting T cell specificity as evidenced by the potent anti-tumor effect observed while potentially eliminating the risk of GvHD associated with the infusion of donor-derived lymphocytes. Moreover, the relative speed and simplicity of the TCR single editing protocol should facilitate its clinical application to patients with hematological malignancies.

Disclosures:
Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.