L1 cell adhesion molecule (L1CAM) is involved in cancer development and aberrantly over-expressed in various tumors, including neuroblastoma, ovarian cancer, and gastric cancers. High L1CAM expression is associated with aggressive progression, metastasis, and poor prognosis in these patients. We developed CX804, a novel and optimized 3rd generation chimeric antigen receptor (CAR) targeted to L1CAM. CX804 incorporates a novel scFv (Clone H8) obtained from bio-panning of a phage displaying. H8 scFv was linked to IgD-IgG1 hinge, CD28 and ICOS co-stimulation domains. CX804 CAR-T cells were generated by transduction of T cells expressing CX804 CAR. CX804 CAR-T cells were mainly composed of cells expressing markers related to early memory cells such as CD62L and CCR7. When reacted with L1CAM-expressing target cells, but not with L1CAM-negative cells, CX804 demonstrated robust proliferation and potent cytotoxicity against target cells with concomitant secretion of effector cytokines including IFN-γ and GM-CSF. Combined CD28 and ICOS co-stimulation was especially efficient in promoting IFN-γ secretion. In xenograft models of metastatic ovarian cancer, IP injection of CX804 showed strong antitumor efficacy with long-term T cell persistence consistent with early memory phenotype of CX804. Increase of GM-CSF was also observed in peripheral blood, confirming actual T-cell activation. Preliminary biodistribution and toxicity studies showed the presence of CX804 in various organs without toxicity except for a potential graft-versus-host reaction from the human T cells. In conclusion, CX804 is a novel and optimized 3rd generation L1CAM-targeted CAR-T that exerts potent and specific antitumor effect against L1CAM-positive cells in vitro and in vivo with long-term persistence and safety.