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A combination of innovative geosteering techniques, aided by state-of-the-art logging-while-drilling (LWD) technologies, allowed Mobil E&P Australia to remain within a narrow 2-m band while avoiding existing wellbores. The 1997 project [...]

Background:Acute myeloid leukemia (AML) chimeric antigen receptor (CAR) T cell therapies are at early stages of testing in human clinical trials. We previously described the design of a CD33-specific dimerizing agent regulated immunoreceptor complex (DARIC33) that, in the presence of rapamycin (RAPA), switches from an “OFF” to “ON” state that activates T cells in response to tumor antigen. Here, we describe RAPA controlled activation and anti-AML activity of DARIC33 using human AML xenograft NSG mouse models and GMP-compliant T cell manufacturing methodologies. We find that low nanomolar whole blood concentrations of RAPA, below levels used for immunosuppression, are needed for DARIC33 to become active in vivoand exhibit potent CD33-specific anti-AML activity.

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 TRAClocus in primary human T cells and the off-target site was eliminated. The TRACspecific 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 TRACmegaTAL 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 TRACedited 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 TRACspecific 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, TRACmegaTAL 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). TRACedited TCR-transgenic T cells exhibit a similar phenotypic profile as un-edited TCR transgenic T cells and TRACmegaTAL 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 TRACgene editing to further improve expression of transgenic TCRs.