Figure 1. The researchers used the Luciferase Reporter Cell Line - NALM6 to measure cell death via luciferase activity in untreated and digitonin-treated cells, using a Matador-Glo assay in a 384-well plate. (Choi S, et al., 2021)
HIP CAR T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice.
Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice
Paper
2023
Nature Communications |
DOI
Background
This study develops hypoimmune (HIP) allogeneic anti-CD19 CAR T cells by CRISPR-Cas9 disruption of B2M, CIITA, and TRAC, plus lentiviral expression of CD47 and anti-CD19 CAR. HIP edits did not impair cytotoxicity or exhaustion. In immunodeficient mice, tumor clearance was comparable to conventional allogeneic CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells persisted, expanded, and provided lasting CD19+ tumor control, whereas allo CAR T cells were rejected. CD47-targeting fusion proteins safely eliminated HIP CAR T cells, supporting an off-the-shelf universal therapeutic strategy.
Key Findings
HIP CAR T cells were engineered by CRISPR-Cas9 disruption of B2M, CIITA, and TRAC and lentiviral expression of CD47 and anti-CD19 CAR. In vitro, HIP and conventional allogeneic CAR T cells showed similar CD19+ Nalm6 killing and no increased exhaustion marker expression or cytokine changes. In immunodeficient NSG mice, HIP CAR T cells cleared CD19+ tumors comparably to allo CAR T cells. In fully immunocompetent allogeneic humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, with improved persistence and expansion, durable tumor suppression, and rapid clearance of re-injected Nalm6 cells. HLA-negative HIP subpopulations avoided host T cell responses, while HLA-expressing cells primed immunity. SIRPα-Fc or poly-His CD47-targeting proteins selectively eliminated HIP CAR T cells in vitro and in vivo without affecting benign cells. These findings show HIP editing enables allogeneic CAR T persistence and lasting antitumor efficacy without host immune suppression.
Contribution
Creative Biogene provided the Nalm6 fluc+ cell line (CSC-RR0361) used as the CD19+ tumor target in cytotoxicity and in vivo bioluminescence efficacy studies. This reagent supported key comparisons of HIP and conventional allogeneic CAR T cells, enabling assessment of tumor killing and persistence.
This study develops hypoimmune (HIP) allogeneic anti-CD19 CAR T cells by CRISPR-Cas9 disruption of B2M, CIITA, and TRAC, plus lentiviral expression of CD47 and anti-CD19 CAR. HIP edits did not impair cytotoxicity or exhaustion. In immunodeficient mice, tumor clearance was comparable to conventional allogeneic CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells persisted, expanded, and provided lasting CD19+ tumor control, whereas allo CAR T cells were rejected. CD47-targeting fusion proteins safely eliminated HIP CAR T cells, supporting an off-the-shelf universal therapeutic strategy.
Key Findings
HIP CAR T cells were engineered by CRISPR-Cas9 disruption of B2M, CIITA, and TRAC and lentiviral expression of CD47 and anti-CD19 CAR. In vitro, HIP and conventional allogeneic CAR T cells showed similar CD19+ Nalm6 killing and no increased exhaustion marker expression or cytokine changes. In immunodeficient NSG mice, HIP CAR T cells cleared CD19+ tumors comparably to allo CAR T cells. In fully immunocompetent allogeneic humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, with improved persistence and expansion, durable tumor suppression, and rapid clearance of re-injected Nalm6 cells. HLA-negative HIP subpopulations avoided host T cell responses, while HLA-expressing cells primed immunity. SIRPα-Fc or poly-His CD47-targeting proteins selectively eliminated HIP CAR T cells in vitro and in vivo without affecting benign cells. These findings show HIP editing enables allogeneic CAR T persistence and lasting antitumor efficacy without host immune suppression.
Contribution
Creative Biogene provided the Nalm6 fluc+ cell line (CSC-RR0361) used as the CD19+ tumor target in cytotoxicity and in vivo bioluminescence efficacy studies. This reagent supported key comparisons of HIP and conventional allogeneic CAR T cells, enabling assessment of tumor killing and persistence.