Dual-armoring of CLL-1 CAR-T cells with IL-18 and an RQR8 suicide switch enhances efficacy and safety in Acute Myeloid Leukemia

Abstract Introduction: Relapsed/refractory acute myeloid leukemia (R/R AML) remains a therapeutic challenge with limited options. While CLL-1-targeted CAR-T therapy shows promise, poor response rates persist. Our prior proteomics identified elevated serum IL-18 in responders, suggesting its role in efficacy. To amplify potency while addressing toxicity risks, we engineered CLL-1 CAR-T cells with IL-18 co-expression and an RQR8 safety switch. Methods: We engineered a doubly armored lentiviral vector for co-expression of a CLL-1-specific CAR (anti-CLL-1 scFv/CD8α hinge/4-1BB/CD3ζ), IL-18, and an RQR8 safety switch using T2A and P2A peptide linkers. CAR-T cells were tested in vitro against CLL-1-high (U937, HL-60) and CLL-1-low (MOLM13) AML cell lines and primary AML blasts, assessing proliferation, cytotoxicity, cytokine secretion, and impact on normal hematopoietic stem/progenitor cells (HSPCs) via colony-forming unit (CFU) assays. In vivo efficacy and survival were evaluated in HL-60 and MOLM13 cell line-derived xenograft (CDX) models. Results: Dual-armored CLL-1/IL-18 CAR-T cells demonstrated significantly elevated IL-18 mRNA and protein secretion compared to conventional CLL-1 CAR-T or untransduced T cells. These cells exhibited potent, antigen-specific cytotoxicity against CLL-1-expressing AML cell lines (HL-60, MOLM13, THP-1) and primary AML blasts, accompanied by significantly increased secretion of IFN-γ and IL-6 upon target engagement. RNA-seq revealed upregulation of IL-18, IL-23R, IL-21, IFNG, CXCL10, and IL-7R in CLL-1/IL-18 CAR-T cells, potentially contributing to enhanced effector function. Critically, CFU assays showed no significant impairment of normal HSPC clonogenic potential versus controls, indicating no increased on-target/off-tumor toxicity. In CDX models, CLL-1/IL-18 CAR-T cells mediated superior tumor control and significantly prolonged median survival compared to conventional CAR-T cells. The integrated RQR8 safety switch enabled significant elimination of CAR-T cells by rituximab within 48h in vitro, completely abrogating cytotoxicity and cytokine secretion, thereby providing a critical safeguard against potential IL-18-driven cytokine release syndrome or neurotoxicity. Conclusion: IL-18 co-expression significantly enhances the anti-leukemic efficacy of CLL-1 CAR-T cells against AML in vitro and in vivo, without exacerbating on-target/off-tumor toxicity to HSPCs. The incorporation of the RQR8 safety switch provides a critical safeguard against potential toxicity. This dual-armoring strategy represents a promising novel therapeutic approach for R/R AML.