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Background CAR-T cells exhibit limited efficacy against solid tumors, due to factors including extensive antigen heterogeneity. The NKG2D ligands MICA and MICB are broadly expressed in human cancers upon DNA damage, and as such represent an ideal target antigen for CAR-T cell therapy. In this study, we generated MICA/B-targeting CAR-T cells that significantly delayed tumor growth in an immunodeficient mouse model. We also identified CARM1 knockout (CARM1 KO) as a genetic perturbation which significantly enhanced CAR-T cell efficacy in mice bearing large tumors. We have also designed and executed a focused in vivo CRISPR screen to identify additional regulators of CAR-T cell function.Methods Human primary T cells were isolated from healthy blood donors and activated using anti-CD3/CD28 Dynabeads. Activated T cells were electroporated with a Cas9 ribonucleoprotein targeting the CARM1 gene, followed by transduction with a lentivirus encoding a CAR targeting the MICA/B antigens. Immunodeficient NSG mice were subcutaneously injected with 1x10 6 PC3 human prostate cancer. Once the tumors reached ~250 mm3 in size, mice were treated with 1x106 CARM1 knockout or wildtype CAR-T cells. Tumor growth was monitored using calipers, and mice were euthanized upon reaching humane endpoints. T cells were transduced with lentivirus encoding a sgRNA library in addition to the CAR, and injected into PC3 tumor-bearing mice. After 14 days, CAR-T cells were harvested from tumor tissue and genomic DNA was submitted for sequencing.Results Both wildtype and CARM1 KO anti-MICA/B CAR-T cells were efficiently generated from human primary T cells and exhibited potent in vitro killing. CARM1KO CAR-T cells exhibited complete loss of CARM1 protein and no differences were observed between wildtype and CARM1KO CAR-T cells during CAR-T production. When injected into NSG mice, wildtype anti-MICA/B CAR-T cells significantly delayed tumor growth. However, in mice with large tumors (~250mm3), CARM1KO CAR-T cells showed potent tumor growth inhibition that was significantly better compared to the wildtype CAR-T cells (figure 1A). Consequently, the mice treated with CARM1KO CAR-T cells had prolonged survival (figure 1B). The in vivo CRISPR screen resulted in efficient CAR-T cell recovery, and sequencing is currently being performed to identify enriched genes.Conclusions Primary anti-MICA/B CAR-T cells can be readily generated and exhibit antitumor activity in a human prostate solid tumor model. CARM1 KO CAR-T cells significantly improved tumor control and survival of mice with large tumors. The in vivo CRISPR screen is ongoing, and we hope to identify additional genetic regulators of CAR-T cell activity in solid tumors.Ethics Approval The animal work described in this abstract was performed in accordance with protocol 08-049 as was approved by the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (IACUC). Human PBMCs were isolated from blood sourced from anonymous donors at the Brigham and Women’s Hospital Crimson Core under study T0214.Abstract 244 Figure 1CARM1 knockout CAR-T cells enhance tumor control and survival in a solid tumor model. NSG mice were implanted with PC3 prostate cancer tumor cells via subcutaneous injection, followed by CAR-T cell treatment once the tumors were larger (~200-300 mm3) on day 19. Mice were euthanized once tumor growth exceeded 2.0 cm in either dimension