Chimeric Antigen Receptor (CAR) T Cell Therapy for CD7-Positive Acute Myeloid Leukemia

Abstract

Expression of CD7 is an adverse prognostic factor in both AML and myelodysplastic syndromes. Because CD7 is not constitutively expressed by mature myeloid cells or early myeloid progenitor cells, we explored the feasibility of targeting these malignancies using T cells expressing a CD7-directed chimeric antigen receptor (CAR7). Due to high expression of CD7 on normal T cells, expression of CAR7 induced substantial fratricide of T cells, preventing their expansion and long term survival. We therefore disrupted the CD7 gene in T cells using CRISPR/Cas9 prior to transduction with the CAR. Electroporation of the Cas9 protein with CD7-specific gRNA produced little toxicity to T cells, and the resulting CD7KOCAR7 T cells (henceforth CAR7 T cells) had no evident fratricide, expanding >300-fold within two weeks.Upon coculture with primary AML blasts, CAR7 T cells eliminated a mean 85% of malignant cells within 48h (Fig. A) and expanded 5-fold. Furthermore, CAR7 T cells effectively targeted primitive AML progenitor cells resulting in a mean >25-fold decrease in the number of leukemic colonies after a 5h coculture (Fig. B). CAR7 T cells demonstrated robust cytotoxicity against CD7+ AML cell lines eliminating >95% of malignant cells after 72h of coculture at a 1:40 effector-to-target ratio but had no activity against CD7-negative tumor cells. To test the protective effect in vivo, we established a mouse xenograft model of disseminated AML by intravenous injection of the CD7+ AML cell line KG-1a. A single injection of 2x10^6 CAR7 T cells produced a >2-log reduction in tumor burden and significantly extended median survival (65 days in control group; median survival not reached at 88 days for the CAR7 T treated animals, P=0.0025). In a second, more aggressive AML model using Kasumi-3 cells, we observed a 2-log reduction in tumor volume following administration of CAR7 T cells.CD7 may also be transiently expressed in normal early myeloid progenitors. To assess the off-tumor hematotoxicity of CAR7 T cells, we cocultured them with cord blood-derived hematopoietic progenitor cells and measured colony formation on a methylcellulose medium. We observed no differences in the numbers of granulocytic, monocytic and erythrocytic colonies after exposure to CAR7 T or control non-transduced T cells indicating a lack of toxicity to normal myeloid and erythroid progenitor cells (Fig. C). Similarly, we observed no reactivity of CAR7 T cells against peripheral monocytes. However, CAR7 T cells were cytotoxic to normal peripheral T- and NK-cells suggesting potential aplasia of these lymphocyte subsets subsequent to in vivo administration of the CAR7 T cells. We may be able to circumvent this toxicity, by infusing pathogen-directed CD7KO T cells from the same donor to repopulate the host, since CD7KO cells appear to retain TCR-mediated activity against a range of pathogens. Hence, CD7KO T cells expressing a CD7-directed CAR can expand without fratricide and have high activity against AML while sparing normal myeloid cells. These data support the feasibility of this approach for the targeted therapy of AML. [Display omitted] DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees.