Upgrading the non-gene-editing, allogeneic ThisCART platform for extending persistence in vivo.

Abstract

e14569 Background: Lack of in vivo persistence is the major challenge in allogeneic CAR-T field. Various HLA-related gene-editing strategies are being explored to prevent rejection by host T and NK cells (Table 1). This study reports a platform upgrade on ThisCART (TCR and HLA-I intracellular sequestered), represented by ThisCART19C cells. Methods: ThisCART19C cells were produced with a single lentivirus vector, encoding a CD19-targeting CAR, a KDEL-tagged anti-CD3 scFv, and the HCMV US11 protein. The surface expression of CAR19, TCRαβ and HLA-A/B/E was measured by flow cytometer. Specific tumor cytotoxicity was evaluated both in vitro and in vivo. Furthermore, the safety of donor derived ThisCART19C cells were tested in a xenogeneic GvHD model. Results: Similar to our ThisCART19A product currently in multi-center trials, ThisCART19C cells were readily produced to a quantity and quality supporting clinical investigation. ThisCART19C cells demonstrated an anticipated and stable surface immunotype: CAR19+, TCRαβ-, HLA-A-, HLA-B-，HLA-E+，with great potential for simultaneous evasion of both host T and NK cells. Similar to autologous CD19-directed CAR-T, ThisCART19C exhibited potent CD19-specific cytotoxicity in vitro and in vivo. No xenogeneic GvHD reaction was observed in murine model over an extended period. Conclusions: To our knowledge, ThisCART19C represents the first application of viral evading strategy in promoting the clinical persistence of allogeneic CAR-T cells. Its efficient processing, potent cytotoxicity and favored preclinical safety profile warrant clinical investigation. [Table: see text]