Developing an Optimal Fully-Human CD371-Targeting IL-18 Secreting CAR T Cell for the Treatment of Acute Myeloid Leukemia
Authors: Shahid Sanam , Mathew Serena , Souness Sydney , Burns Erin R. , Um Jasmine S. , Yoo Sarah , Cai Winson , Pohl Mary Ann , Fujino Takeshi , Zhang Pu , Lewis Jennifer , Devlin Sean M. , Lorenz Ivo , Brentjens Renier , Daniyan Anthony F. 
Published: December 2024

Introduction: Relapsed/refractory acute myeloid leukemia (AML) has a poor prognosis, highlighting the critical need for more targeted and less toxic therapeutic options. CD371 (CLEC12A, CLL-1), enriched on leukemic stem cells (LSCs) and chemo-resistant AML cells but absent on hematopoietic stem cells (HSCs), has emerged as an attractive target for AML chimeric antigen receptor (CAR) T cell therapy.

Methods: Fully human CD371-specific antibodies were generated via enrichment from a naïve human scFv phage display library. The lead candidate was reconfigured into VH-VL (HL) and VL-VH (LH) orientations, and then integrated into CD28/CD3Z-based CAR T cells for functional in vitro and in vivo evaluation. CD371-targeting CAR T cells were produced by gammaretroviral transduction of peripheral blood-derived primary T cells. The superior orientation was then optimized by assessing different HnL linker lengths (H3L-H5L, where n=G4S) in vitro and in vivo, and the best performing product was further augmented with constitutive IL18 secretion and evaluated in vitro and in vivo. For in vitro assays, U937 (11,380 surface molecules/cell) and HL60 (14,487 surface molecules/cell) were used as CD371-high expressing cell lines, and MOLM14 (4,304 surface molecules/cell) was used as a CD371-low expressing cell line. In vitro, CAR T cell proliferation, 24- and 72-hour cytotoxicity, cytokine secretion, and colony forming unit (CFU) assays were evaluated. In vivo, tumor control and overall survival in antigen-high (U937, HL60) and antigen-low (MOLM14) AML cell-line derived xenograft (CDX) murine models were assessed.

Results: CD371-directed CD28/CD3Z-based CAR T cells with single-chain variable fragments (scFvs) in the HL orientation performed better than those in the LH orientation both in vitro against U937 and HL60 and in vivo in a U937 AML CDX model. Although CAR constructs with five linkers (H5L) between the VH and VL chains of the scFv were relatively comparable to those with three (H3L) or four (H4L) linkers in vitro, they exhibited superior tumor control in vivo in a U937 AML CDX model. The addition of IL18, a potent immunomodulatory molecule, to CD371-directed CAR T cells further augmented efficacy against antigen-high and antigen-low tumors in vitro and in vivo. Importantly, IL18-secreting CD371-directed CAR T cells were more effective than non-armored CD371-directed CAR T cells at killing CD371-low MOLM14, likely due to bystander killing mediated by increased secretion of interferon-gamma and upregulation of FASL (CD95L). Further, IL18-secreting CD371-directed CAR T cells showed superiority in vivo compared to non-armored CAR T cells in a MOLM14 CDX model, with mice exhibiting significantly prolonged survival with treatment of 50,000 CAR T cells per mouse. In vitro CFU assays with isolated CD34+ HSCs from umbilical cord blood revealed that CD371-directed CD28/CD3Z-based CAR T cells did not exert significant toxicity against healthy HSCs.

Conclusions: Our results demonstrate that CD371-targeted CD28/CD3Z-based IL18-secreting CAR T cells lead to eradication of both antigen-high and antigen-low AML without toxicity against HSCs. Based on this rigorous preclinical evaluation, our validated clinical grade CD371-directed scFv is now under clinical evaluation with and without an IL18 transgene in first-in-human phase 1 trials for patients with relapsed/refractory AML (NCT06017258 and NCT06128044).