Targeting Of Leukemia Stem Cells By Interfering With Niches Using CD44 Variant 6 Chimeric Antigen Receptor Redirected Central Memory T Cells

Leukemic stem cells (LSCs), are resistant to radio/chemotherapy and responsible for disease relapse. Therefore, prevention of leukemia recurrence relies on the eradication of LSCs, which remains challenging for the treatment of hematopoietic malignancies. CD44 and its ligand are critical components of LSC niches that contain crucial signals for LSCs self-renewal. A CD44-specific mAb (clone H90) has been shown to specifically eradicate LSCs in immunodeficient mouse models. Further, a splice variant of CD44, CD44 variant 6 (CD44v6) is over expressed on malignant hematopoietic cells including ALL, AML, CML, NHL and multiple myeloma and plays an important role for proliferation, homing, and metastasis of LSCs.

With the aim of targeting LSCs, we constructed a second generation Chimeric Antigen Receptor (CAR) specific for CD44v6 containing truncated EGFR as a selection and ablation molecule (CD44v6CAR/EGFRt). Central memory CD8⁺ T cells (CD62L⁺CD45RO⁺CD8⁺; CD8⁺T_CM) were isolated from a healthy donor and transduced with lentivirus encoding CD44v6CAR/EGFRt after CD3/CD28 beads activation. Gene modified cells were immunomagnetically purified after labeling with biotinylated Erbitux followed by anti-biotin microbeads and expanded in rapid expansion medium (REM) containing OKT3 and feeder cells in the presence of IL-2 (50U/ml) and IL-15 (1ng/ml). The purified CD44v6CAR/EGFRt CD8⁺ T cells were expanded 300 fold following each cycle of 2 stimulations. 40-50% of the expanded CD44v6CAR/EGFRt CD8⁺T cells preserved central memory T-cell phenotype markers such as CD62L, CD28 and CD27. In a 4 hour chromium release assay, CD44v6CAR/EGFRt CD8⁺ T cells specifically recognized and lysed AML cells (THP-1) and lymphoblastoid cell lines (LCL) cells expressing CD44v6, while sparing CD44v6-negative KG1-a target cells (Fig. 1).

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To study in vivo efficacy of the CD44v6CAR/EGFRt CD8⁺ T cells, a xenogeneic AML model was established by intravenous injection of 2x10^6 firefly luciferase expressing THP-1 cells (THP-1-ffluc) into NSG mice. Adoptive transfer of 5x10^6 CD44v6CAR/EGFRt CD8⁺ T cells dramatically reduced THP-1-ffluc tumors as evidenced by bioluminescent imaging 14 days post T cell infusion. To prove the specificity of targeting, CD19⁺LCL-ffluc cells that are 50% CD44v6 positive were systemically infused into NSG mice. As expected, CD44v6CAR/EGFRt CD8⁺ T exhibited less antitumor activity against LCL tumor than THP-1 tumor. Accordingly, irrelevant CE-7CAR⁺T cells (L1CAM specific) that were derived from the CD8⁺ T_CM of the same donor had no antitumor activity. These results indicate that targeting CD44v6 with CAR-transduced CD8⁺ T_CM cells has the potential to treat hematological malignancies. Moreover, CD44v6 redirected T cell therapy would apply to various hematopoietic malignancies that aberrantly over-express CD44v6.