T cells expressing chimeric antigen receptors (CAR-T cells) are very effective in killing tumor cells in patients with acute lymphocytic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL), evidenced by the FDA approval of two CD19 CAR-T products (Yescarta and Kymriah) in 2017. While the clinical results with CAR-T cells in blood-based cancers have been impressive, similar results have not been forthcoming in the treatment of solid tumors. This has been attributed to not only the lack of persistence and "exhaustion" of the administered CAR-T cells but also to the physical and immunosuppressive barrier that the tumour stroma presents. This is comprised of multiple cellular and molecular components that can interact with cytokine receptors and checkpoint proteins to activate the complex inhibitory signaling pathways in T cells. To overcome this immunosuppression of CAR-T cells, the most common approach is to use checkpoint blockade monoclonal antibodies (mAbs), which rely on both the surface expression of the target molecule and appropriate level of mAb saturation. To overcome these hurdles, we undertook deletion of a group of genes implicated in inhibition of T cell function in CAR-T cells using CRISPR/CAS9 editing. Our CAR-T cells are second generation and directed against the pan-adenocarcinoma glycoslation mutant TAG-72. We evaluated the effect of the gene deletions on enhancement anti-tumor activity and persistence of CAR-T cells in the Ovcar3 ovarian tumor xenograft model in NSG mice. The TAG-72 CAR-T cells alone were able to reduce the size of pre-established tumors initially but recurrence was observed from around 30 days post CAR-T cell administration. After gene editing, we found multiple gene knockouts could delay the recurrence of the tumor. Most importantly, a double gene knockout TAG-72 CAR-T cell group showed the best anti-tumor activity and persistence of CAR-T cells in these groups, as was evidenced by the complete prevention of tumor recurrence for up to 100days. Genomic DNA sequencing-based quantification results showed that CRISPR/CAS9 efficiently introduced the indels into the target genes without significant off-target effects. Furthermore, the double gene knockout TAG-72 CAR-T cells could be expanded in vitro as well as the non-edited TAG-72 CAR-T cells. Therefore, we have selected the double gene knockout TAG-72 CAR-T cells as a development candidate (CTH-004), and are aiming for a phase I/IIa clinical study in ovarian cancer patients to commence in 2021. Current pipeline studies are examining whether multiple deletions of different immune suppression genes in different signalling pathways provide even greater function and longevity in the CAR-T cells.