e14532 Background: The prognosis of patients with R/R AML is poor and treatment remains challenging. Human C-type lectin-like molecule 1(CLL1) is a type II transmembrane glycoprotein that is highly expressed on AML but is absent on normal HSCs. Clinical studies have shown that autologous CLL-1 is a novel and promising target for the treatment of AML. But autologous CAR-T therapy has been complicated by long production time, high-cost and risks of manufacturing failure, and allogeneic CAR-T cells can overcome these hurdles. Methods: A novel non-gene-editing allogeneic CAR-T platform was developed on the base of intracellular retention of membrane proteins, and named ThisCART (This = TCR and/or HLA-I intracellular sequestered). CLL1-directed ThisCART cells are readily produced with a single lentiviral vector, encoding both a CLL1-targeting CAR and an anti-CD3 single chain antibody with the KDEL peptide fused to its C-termini. The surface expression of CAR and TCRαβ/CD3 was measured by flow cytometer. Specific tumor cytotoxicity was evaluated both in vitro and in vivo. Furthermore, the safety of donor derived ThisCART cells were tested in a xenogeneic GvHD model. Results: The manufacturing platform of CLL1 ThisCART was able to achieve over 150-folds of ex vivo CAR-T expansion in all batches, with the purity of products (CAR-positive/TCRαβ-negative) above 99%. In preclinical models, CLL1 ThisCART did not induce GvHD, and exhibited superior antitumor function compared to benchmark (WO2017091615A) and conventional CLL1 CAR-T cells. CLL1 ThisCART cells were readily produced to a quantity and quality supporting clinical investigation. Conclusions: CLL1 ThisCART cells based on intracellular retention of TCRαβ/CD3 complex demonstrated superior CLL1-dependent cytotoxicity and no xenogeneic GvHD in murine models. With streamlined single-vector-based production, enhanced CAR signaling and potent cytotoxicity and favored preclinical safety profile, CLL1 ThisCART warrant clinical investigation.