T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia

Abstract

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

Figure 1

CD123-specific CARs can be expressed in healthy donor human T cells. (A) Schematic diagram of the CAR containing a modified immunoglobulin G4 hinge, a modified transmembrane and intracellular signaling domain of CD28, and the CD3ζ signaling domain. The T2A ribosomal skip sequence and the EGFRt transduction marker are also indicated. (B) Representative phenotype of mock- and lenti-transduced T cells derived from a single healthy donor. After immunomagnetic selection and 1 cycle of expansion, CAR-modified T cells were stained with biotinylated anti-Fc or biotinylated anti-cetuximab followed by phycoerythrin-conjugated streptavidin and anti-TCRα/β, anti-CD4, or anti-CD8 and analyzed by flow cytometry. Quadrant placement is based on staining with isotype controls, and the percentage of cells falling in each quadrant is indicated. (C) Expression of indicated cell surface markers from 3 different healthy donor T-cell lines following immunomagnetic selection and 1 cycle of expansion. Data represent mean values ± standard error of the mean (SEM).

Figure 2

CD123-specific CAR-expressing T cells lyse CD123-expressing tumor cell lines. (A) Flow cytometric analysis of 293T cells transiently transfected to express CD123 (top, black line) or CD19 (bottom, black line). Parental mock-transduced 293T cells were stained with either anti-CD123 or anti-CD19 antibodies (gray filled, top and bottom) to determine background expression levels. (B) Specific cytotoxicity of CD123-CAR–expressing T cells (26292 and 32716) against 293T cells expressing either CD123 (293T-CD123) or CD19 (293T-CD19) by chromium release assay. Data represent mean values of triplicate wells ± standard deviation (SD). (C) Flow cytometric analysis of CD123 on the AML cell line KG1a, the Epstein-Barr virus–transformed LCL cell line, and the CML cell line K562. Percentage of cells positive for CD123 staining (black line) over isotype controls (gray filled) are indicated in each histogram. (D) Specific cytotoxicity of CD123-CAR T cells (26292 and 32716) against the CD19⁺CD123⁺ LCL cell line and the CD19⁻CD123⁺ cell line KG1a by chromium release assay. OKT3-expressing LCL (LCL-OKT3) and the CD19⁻ CD123⁻ K562 cell lines were used as positive and negative control cell lines, respectively. Data represent mean values of triplicate wells ± SD. (E) CD123 CAR T cells, or control pair-matched T cells, from 3 healthy donors were cocultured with the indicated cell lines for 24 hours at an E:T of 10:1 and the release of IFN-γ and TNF-α were quantified by Luminex multiplex bead technology. Fold elevation of IFN-γ production against KG1a compared with K562 for 26292 and 32716 were 2.3 and 19.1, respectively. Fold elevation of TNF-α production against KG1a compared with K562 for 26292 and 32716 was 5.5 and 16.5, respectively. (F) Pair-matched carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD19- or CD123-specific T cells were cocultured with the indicated stimulator cell lines for 96 hours at an E:T of 2:1 and analyzed by flow cytometry for CFSE dilution. Unstimulated T cells (filled histograms) were used as baseline T-cell proliferation controls. (G) CFSE-labeled target cells were cocultured with CAR T cells for 5 days in the absence of exogenous cytokines at an E:T ratio of 0.5:1. At the end of the culture, cells were stained using anti-CD3 to distinguish between T cells and CSFE-labeled tumor cells.

Figure 3

Activation of multiple CD4 and CD8 effector functions by CD123-specific CARs following coculture with primary AML samples. Pair-matched CAR-engineered T cells were cocultured for 6 hours with 3 different primary AML patient samples (AML 179, 373, and 605) and analyzed for surface CD107a expression and intracellular IFN-γ or TNF-α production. (A, bar graphs) Percentage of DAPI⁻CD3⁺CD8⁺ EGFRt⁺ cells expressing CD107a. Data represent mean values + SD. (A, pie charts) The fractions of CD3⁺CD8⁺EGFRt⁺ cells undergoing degranulation and producing IFN-γ and/or TNF-α are plotted in the pie charts. Percentages in each subset are indicated. (B) DAPI⁻CD3⁺CD4⁺EGFRt⁺ population data from the same experiment as described in panel A. (C) Pair-matched CFSE-labeled CD19- or CD123-specific T cells were cocultured with the indicated stimulator cells for 72 hours at an E:T of 2:1 and analyzed by flow cytometry for CFSE dilution in the DAPI⁻CD3⁺EGFRt⁺ population. LCL and K562 cell lines served as positive and negative controls, respectively. Pre-B ALL 802 is a primary patient sample double positive for CD19 and CD123. Quadrant placement is based on unstimulated T cells.

Figure 4

Primary AML cells are specifically targeted by CD123 specific T cells. (A) Pair-matched CD19 or CD123-specific T cells were cocultured for 4 hours with ⁵¹Cr labeled CD34⁺ primary AML samples at an E:T of 25:1. Pre-B ALL 802 is a primary patient sample double positive for CD19 and CD123. Data represents mean values of triplicate wells + SD (B) Specific lysis of AML blasts from the 3 primary AML patient samples in panel A. Data represent mean values ± SEM. *P < .05 and **P < .0005 using the unpaired Student t test comparing 26292 and 32716 to CD19R. (C-D) CD34-enriched primary AML or CB cells were cocultured with CAR T cells or left untreated for 4 hours prior to flow cytometric analysis. Percentages in each quadrant are indicated. For CD123 histograms (second and third rows), the y-ordinate scale was adjusted according to the number of events captured and the relative fluorescence (RFI) index is indicated. The RFI is the ratio of the median of the anti-CD123 antibody (clone 9F5)-stained signal to isotype-matched control stain. Gates were placed according to fluorescence-minus-one controls.

Figure 5

The effect of CD123-CAR–expressing T cells on normal and leukemic progenitor cells in vitro. (A-B) CD34⁺ CB cells (n = 3) were CD34-immunomagnetically selected and cocultured with either CD19- or CD123-specific pair-matched T cells from a healthy donor or media alone (untreated) for 4 hours at an E:T of 25:1. The cells were then plated in semisolid methylcellulose progenitor culture for 14 to 18 days and scored for the presence of BFU-E (A), CFU-GM (B) and colonies. Colony numbers (left) and normalized colony formation percentages (right) are presented. Percentages are normalized to CD19-specific T cell controls. Data represent mean values + SEM for 3 different CB samples. (C) CD34⁺ primary AML patient samples (AML 493, 519, or 545) were immunomagnetically selected and cocultured with either CD19 or CD123-specific CAR T cells from a healthy donor or media alone (untreated) for 4 hours at an E:T of 25:1. The cells were then plated in semisolid methylcellulose progenitor culture for 14 to 18 days and scored for the presence of leukemia colony-forming units (CFU-L). Colony numbers (left) and normalized colony formation percentages (right) are presented. Percentages are normalized to CD19-specific T cell controls. Data represent mean values + SEM for 3 different primary AML patient samples. *P < .05 using the unpaired Student t test comparing 26292 and 32716 to CD19R.

Figure 6

CD123-CAR–redirected T cells derived from AML patients specifically lyse autologous blasts in vitro. (A) T cells from 3 AML patients were lentivirally transduced to express either CD19R, 26292, or 32716 CARs. Shown are T cell lines from AML 722 19 days posttransduction. (B) CD123 expression on target cells used in ⁵¹Cr release assay. The percentage of CD123⁺ cells and the RFI of each sample is indicated. (C) Results of 4-hour autologous killing assays using T cells engineered from 3 AML patient samples as effectors and ⁵¹Cr-labeled autologous CD34-enriched blasts as target cells. Data represent mean values of triplicate wells ± SD.

Figure 7

CD123 CAR T cells exhibit antileukemic effects in vivo. Mice were sublethally irradiated with 300 cGy from a ¹³⁷Cs γ-irradiator 24 hours prior to intravenous transplantation of 0.5 × 10⁶ KG1a-GFP-firefly luciferase cells. Five days later, mice received a single intravenous injection of 5.0 × 10⁶ CAR⁺ T cells. (A) Flow cytometric analysis of CAR-expressing T cells prior to use in vivo. Percentage of cells in each quadrant is indicated. (B) Bioluminescent imaging prior to T-cell treatment (day 4), on day 12, and on day 33 following KG1a-GFP-firefly luciferase transplantation. (C) Bioluminescent signal for each treatment group over time. Dotted line represents day of T-cell treatment. Data represent mean values of each group ± SD. Results represent pooled data from 2 separate experiments. PBS n = 3; CD19R, 26292, 32716 n = 6. (D) Representative flow cytometric analysis of peripheral blood 32 days after leukemia transplant. Percentage of viable human CD45⁺GFP⁺ KG1a cells is indicated. (E) Summary of leukemic cell engraftment in mouse peripheral blood 32 days after leukemia transplant. The percentage of viable human CD45⁺GFP⁺ KG1a cells is indicated. Each symbol indicates 1 mouse; bars represent mean values, and mean values for each group are indicated. (F) Kaplan-Meier analysis of survival for each group (PBS n = 3, CD19R n = 4, CD123-specific n = 10). Log-rank (Mantel-Cox) tests were used to perform statistical analyses of survival between groups. *P < .05.