Gene Editing of TRAC Locus Utilizing Megatal Nucleases Increases Expression of Transgenic TCRs Delivered Via Lentiviral Vector-Mediated Gene Transfer

T-cell receptor (TCR) transgenic T cell therapies have demonstrated efficacy in late stage and refractory oncologic malignancies, including melanoma and synovial cell sarcoma. However, these responses are generally either short in duration or occur in a minority of patients. One of the limitations of transgenic TCR therapies may be related to the mispairing of the introduced transgenic TCR alpha and beta chains with the endogenous TCR chains, resulting in non-productive TCR transgenic T cells. Using genome editing, endogenous TCR genes can be eliminated, potentially reducing mispairing with a subsequent increase in the activity of the transgenic TCR. It is essential that therapeutic genome editing be achieved with sufficient specificity to minimize off-target editing events. MegaTALs, derived by fusing a DNA binding array to an engineered meganuclease, can be iteratively refined in response to off-target detection, achieving a genome-wide increase in on-target versus off-target discrimination. To this end, we developed a megaTAL targeting the T-cell receptor alpha chain (TRAC) locus and identified a prominent off-target site using unbiased double strand break detection methods coupled with targeted amplicon sequencing. Optimization of this megaTAL using a combination of protein engineering approaches resulted in a reagent achieving >90% editing of the TRAC locus in primary human T cells and the off-target site was eliminated. The TRAC specific megaTAL resulted in a 92% reduction in CD3 surface expression a surrogate marker of TCR expression (5.6±0.4% vs 97.6±0.2%, p<0.0001). To further evaluate this nuclease we transduced primary human T cells with a lentiviral vector encoding an anti-NY-ESO-1 transgenic TCR. Expression of the transgenic TCR was followed by staining for the TCR Vbeta13.1 protein and we observed 74% transduction efficiency accounting for Vbeta13.1 usage in the endogenous TCR repertoire (79.4±2.6% vs 5.1±0.7%, p<0.0001). Of the transduced Vbeta13.1 positive cells, 64.3% were positive for the transgenic TCR as determined by tetramer staining, suggesting 35.7% of transduced cells exhibit TCR mispairing with components of the endogenous TCR. In parallel, T cells were treated with a combination of megaTAL and TCR vector transduction. Transgenic TCR expression in TRAC megaTAL edited T cells restores CD3 surface expression in all Vbeta13.1-positive transduced cells (74.0±2.4% vs 5.6±0.4%, p < 0.0001). In addition, 96% of the TRAC edited Vbeta13.1 positive cells were now positive for the transgenic TCR as shown by the tetramer staining (95.5±1.0% vs 64.3±4.8%, p<0.01). Treatment of transgenic TCR expressing human T cells with the TRAC specific megaTAL resulted in a 31% reduction in mispairing of the transgenic TCR (4.4±1.0% vs 35.7±4.8%, p<0.01). Moreover, TRAC megaTAL treatment produced a 1.7-fold increase in the expression of the transgenic TCR in tetramer positive cells (gMFI 6292±662 vs 3800±200, p <0.05). TRAC edited TCR-transgenic T cells exhibit a similar phenotypic profile as un-edited TCR transgenic T cells and TRAC megaTAL editing does not result in a significant impact on T cell expansion, CD4 to CD8 T cell ratios, T cell memory subsets, or lentiviral integration during manufacturing. These results demonstrate that the megaTAL architecture facilitates the iterative refinement of nuclease specificity and activity, generating exquisitely selective, single-component reagents, and highlight the potential of megaTAL driven TRAC gene editing to further improve expression of transgenic TCRs.