Effective Dual Targeting of PRAME and WT1 with CD33-Gated ARTEMIS® Antibody-TCR Platform in Acute Myeloid Leukemia
Authors: Blankenfeld Melia , Kirkey Danielle C. , Xiong Guangyan , Zhang Hongbing , Hawkins Grace , Lira Isabel , McKay Cyd , Root Christina , Quintarelli Concetta , Locatelli Franco , Liu Cheng , Meshinchi Soheil 
Published: December 2024

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.