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4821 Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Biological therapies, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Diseases, Therapies, Myeloid Malignancies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Danielle C Kirkey, MD1,2, Leila Robinson1*, Tiffany Hylkema, BS3*, Anisha Loeb, BA1*, Rhonda E Ries, MA3*, Cyd McKay1*, Christina Root1*, Laura Pardo, MS1,4*, Keith R. Loeb, MD, PhD5, Quy Le, PhD5*, David A. Scheinberg, MD, PhD6 and Soheil Meshinchi, MD, PhD7

1Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA
2Division of Pediatric Hematology/Oncology, Seattle Children's Hospital, Seattle, WA
3Translational Sciences and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA
4Hematologics, Inc., Seattle, WA
5Fred Hutchinson Cancer Center, Seattle, WA
6Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY
7Translational Sciences and Therapeutics, Fred Hutchinson Cancer Research Center, Seattle, WA

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME+/HLA-A2+ pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x106 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x106 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis.

Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free >180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group.

We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis.

We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Disclosures: Pardo: Hematologics Inc: Current Employment. Scheinberg: Actinium Pharma: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties.

*signifies non-member of ASH