CD7 CAR T Cells for the Therapy of Acute Myeloid Leukemia

Abstract

Chimeric antigen receptor (CAR) T cell therapy for the treatment of acute myeloid leukemia (AML) has the risk of toxicity to normal myeloid cells. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients but is absent on normal myeloid and erythroid cells. Since CD7 expression by malignant blasts is also linked with chemoresistance and poor outcomes, targeting this antigen may be beneficial for this subset of AML patients. Here, we show that expression of a CD7-directed CAR in CD7 gene-edited (CD7^KO) T cells effectively eliminates CD7⁺ AML cell lines, primary CD7⁺ AML, and colony-forming cells but spares myeloid and erythroid progenitor cells and their progeny. In a xenograft model, CD7 CAR T cells protect mice against systemic leukemia, prolonging survival. Our results support the feasibility of using CD7^KO CD7 CAR T cells for the non-myeloablative treatment of CD7⁺ AML.

Keywords: CAR T cells; CD7; acute myeloid leukemia; chimeric antigen receptors; mouse model.

Figure 1

CD7 Expression in Normal and Malignant Cells (A) Representative histograms of CD7 expression in immune subsets from peripheral blood of healthy donors. (B) Surface expression of CD7 measured by flow cytometry in primary AML samples collected from pediatric and adult patients. (C) CD7 expression in AML cell lines. Iso Ctrl, Isotype control.

Figure 2

CD7-Edited CD7 CAR T Cells Are Cytotoxic against AML Cell Lines (A) Expression of CD7 in non-treated (NTR) and CD7 and CAR in CD7-edited T cells. (B) Frequency of residual live GFP⁺ KG-1a AML cells upon coculture with control or CD7 CAR T cells for 3 days at a 1:40 effector-to-target (E:T) ratio. (C) Absolute counts of live AML cells at the end of the coculture are plotted on the bar graphs. (D) Absolute counts of residual live KG-1a cells after coculture with CD7 CAR T cells at the indicated E:T ratios. (E) IFNγ production by CD8⁺ and CD4⁺ CD7 CAR T cells upon 4-hr coculture with indicated AML cell lines by intracellular cytokine staining and flow cytometry. Individual data points for each donor are shown. (F) Repeated cytotoxicity against KG-1a AML cells in a sequential killing assay. CD7 CAR T cells were plated with KG-1a AML cells at a 1:4 E:T ratio, and the ratio was restored every 3–4 days after quantification of residual live tumor cells (left) and CAR T cells (right). Data are shown as mean ± SD. Data represent 2–3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by unpaired Student’s t test.

Figure 3

Elimination of Primary AML Blasts and Colony-Forming Cells by CD7 CAR T Cells (A) Normalized MFI of CD7 in primary AML blasts compared with KG-1a (positive control) and Raji (negative control). (B) Representative dot plots showing frequencies of live primary AML blasts after 48 hr coculture with control or CD7 CAR T cells at a 1:1 E:T ratio. (C) Absolute counts of residual CD33⁺ myeloid cells from individual patients at the end of coculture with control or CD7 CAR T cells. (D) Control or CD7 CAR T cells were cocultured with AML blasts for 5 hr at a 5:1 E:T ratio and plated on MethoCult media. The bar graphs show relative numbers of colonies (pooled duplicate measurements) formed after 14 days of culture.

Figure 4

CD7 CAR T Cells Are Protective in a Mouse Xenograft Model of AML (A) General outline of the experiment. NSG mice received FFluc-expressing KG-1a cells 24 hr after sublethal irradiation with 116 cGy. Eight days later, mice received a single injection of control or CD7 CAR T cells intravenously and were monitored for tumor progression. (B) Representative images showing leukemia progression in individual mice. (C) Kinetics of leukemia progression in individual mice that received either control or CD7 CAR T cells by IVIS imaging. (D) Kaplan-Meier curves showing survival of mice in each experimental group. p < 0.0001 by Mantel-Cox log rank test. (E) Expression of CD7 in residual unmodified and CRISPR/Cas9-edited CD7^KO KG-1a AML. (F) Kinetics of leukemia progression in CD7 CAR T-treated mice receiving unmodified (CD7⁺) or CD7KO KG-1a leukemia. **p < 0.01 by unpaired Student’s t test.

Figure 5

Lack of Reactivity of CD7 CAR T Cells against Mature Myeloid Cells and Cord Blood Precursors (A) CD14⁺ monocytes were purified from PBMC using magnetic beads and labeled with eFluor 670 prior to coculture with control or CD7 CAR T cells at a 1:1 ratio. Representative dot plots show the numbers of residual live monocytes after 24 hr of coculture. (B) Data from four donors are summarized in a bar graph. (C) Total blood cells after RBC lysis were cocultured with autologous CD7 CAR T cells for 24 hr. Live granulocytes were quantified at the end of coculture by flow cytometry. (D) Cord blood cells were cocultured with control or CD7 CAR T cells at a 10:1 E:T ratio for 5 hr and plated on the MethoCult media. Numbers of erythrocytic, granulocytic, and monocytic colonies were quantified 14 days later. Data from individual donors are shown. Data represent two independent experiments. ns, Not significant by unpaired Student’s t test.