Effective Dual Targeting of PRAME and WT1 with CD33-Gated ARTEMIS® Antibody-TCR Platform in Acute Myeloid Leukemia

PRAME (Preferentially Expressed Antigen in Melanoma) is a cancer-testis antigen known to be aberrantly expressed in high-risk subtypes of AML. We have previously demonstrated the preclinical efficacy of targeting the PRAME peptide/HLA-A2 complex with a TCR mimic CAR T in AML (Kirkey et al, Blood Adv. 2023). Wilms' Tumor 1 (WT1) is another therapeutic target in hematologic malignancies that is highly expressed in AML. Both PRAME and WT1 are tumor-specific antigens with no expression in normal hematopoietic cells, providing ideal therapeutic targets without risk for hematopoietic toxicity. In order to circumvent AML tumor heterogeneity and limit on-target, off-tumor toxicity, we developed a novel approach to target cancer antigens through an ARTEMIS®* cell receptor platform that comprises an antibody-TCR (AbTCR) and a chimeric signaling receptor (CSR). This approach utilizes T cells co-expressing a dual-targeting AbTCR (PRAME- and WT1- peptide/MHC binding antibody moieties in tandem fused to the gamma/delta TCR transmembrane and intracellular domains) and a CD33-targeting CSR with a CD28 costimulatory signaling domain. This novel approach allows for dual-targeting of PRAME- or WT1-positive leukemia cells with a logic gating mechanism to restrict T-cell directed killing of CD33+ cells, thus limiting on-target/off-tumor toxicity. To evaluate the preclinical efficacy of T cells expressing the PRAME/WT1 dual-targeting AbTCR and CD33-targeting CSR combination, we tested engineered cancer cell lines in vitro, complemented with in vivo assays using an aggressive AML patient-derived xenograft (PDX) model.

PRAME/WT1 AbTCR-CD33CSR T cells demonstrated potent cytolytic activity across multiple effector:target cell ratios to OCI-AML2 (PRAME+/WTI+/CD33+/ HLA-A*02+) cells following a 24-hour co-incubation, but not to NOMO-1(PRAME-/WT1-/CD33+/HLA-A*02-) cells, highlighting target-specific cytolytic activity of the dual-targeting AbTCR T cells. To evaluate whether the CD33 co-stimulatory domain may be utilized as an “AND”-gated mechanism, we further tested the efficacy of this construct in CD33 knock-out (PRAME+/WT1+/CD33-/HLA-A*02+) OCI-AML2 cells. Potent cytolytic activity of the PRAME/WT1 AbTCR-CD33CSR T cells seen in the parental cells was abrogated in the CD33-knock-out counterpart, demonstrating successful implementation of the “AND”-gating strategy with the ARTEMIS AbTCR-CSR platform.

As IFN-γ has been shown to increase the expression of MHC and peptide:MHC complexes, which can enhance efficacy of peptide/MHC complex-recognizing T cells , we co-incubated OCI-AML2 cells with IFN-γ prior to treatment with the PRAME/WT1 AbTCR-CD33CSR T cells. Exposure to IFN-γ enhanced cytolytic activity of the AbTCR-CSR co-expressing cells at all effector:target cell ratios at both 6 and 24 hours compared to DMSO-treated controls.

We further evaluated the preclinical efficacy of the PRAME/WT1 AbTCR-CD33CSR T in an aggressive KMT2A-r PDX model. PDX cells were transduced with ffluciferase for noninvasive bioluminescent imaging to monitor leukemic progression. Mice were transplanted with 1x10⁶ PDX leukemia cells, and one week later, the PDX leukemia-bearing mice were treated with unmodified T cells or PRAME/WT1 AbTCR-CD33CSR T cells at 5 x10⁶ cells (1:1 CD4:CD8 T cells) per mouse. Leukemia burden was measured by IVIS imaging and flow cytometric peripheral blood analysis. Mice treated with unmodified T cells developed significant leukemia by day 50 post T cell infusion, and all mice required euthanasia by day 100. In contrast, mice that received the PRAME/WT1 AbTCR-CD33CSR T Cells remained leukemia-free until day 80 and had significantly prolonged survival (average survival 145 days) in comparison to the unmodified T cell treated controls.

Here we describe the superior preclinical efficacy of a novel, PRAME- and WT1-dual-targeting AbTCR T cell therapy candidate that significantly expands accessibility and counteracts inherent AML heterogeneity. Furthermore, we have complemented the dual-targeting AbTCR with a CSR to include logic gating for CD33, which limits on-target/off-tumor toxicity. These results demonstrate a robust and expanded immunotherapeutic approach that embodies both the effectivity of TCR mimic antibody-derived AbTCR and the ability to minimize off-target toxicity.

* ARTEMIS is a registered trademark owned by Eureka Therapeutics, Inc.

Xiong:Eureka Therapeutics: Current Employment. Zhang:Eureka Therapeutics: Current Employment. Liu:Eureka Therapeutics: Current Employment.