Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells - PubMed (original) (raw)

. 2014 Apr 10;123(15):2343-54.

doi: 10.1182/blood-2013-09-529537. Epub 2014 Mar 4.

Sarah K Tasian, Marco Ruella, Olga Shestova, Yong Li, David L Porter, Martin Carroll, Gwenn Danet-Desnoyers, John Scholler, Stephan A Grupp, Carl H June, Michael Kalos

Affiliations

Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells

Saar Gill et al. Blood. 2014.

Erratum in

Abstract

Many patients with acute myeloid leukemia (AML) are incurable with chemotherapy and may benefit from novel approaches. One such approach involves the transfer of T cells engineered to express chimeric antigen receptors (CARs) for a specific cell-surface antigen. This strategy depends upon preferential expression of the target on tumor cells. To date, the lack of AML-specific surface markers has impeded development of such CAR-based approaches. CD123, the transmembrane α chain of the interleukin-3 receptor, is expressed in the majority of AML cells but is also expressed in many normal hematopoietic cells. Here, we show that CD123 is a good target for AML-directed CAR therapy, because its expression increases over time in vivo even in initially CD123(dim) populations, and that human CD123-redirected T cells (CART123) eradicate primary AML in immunodeficient mice. CART123 also eradicated normal human myelopoiesis, a surprising finding because anti-CD123 antibody-based strategies have been reportedly well tolerated. Because AML is likely preceded by clonal evolution in "preleukemic" hematopoietic stem cells, our observations support CART123 as a viable AML therapy, suggest that CART123-based myeloablation may be used as a novel conditioning regimen for hematopoietic cell transplantation, and raise concerns for the use of CART123 without such a rescue strategy.

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Figures

Figure 1

Figure 1

CD123 is frequently expressed in primary AML. (A) Primary patient AML samples express CD123. AML blasts were gated using standard side scatterlow CD45dim characteristics (n = 35-46, from a diverse range of AML subtypes; see supplemental Table 1). (B) CD123 expression levels vary among leukemia samples, as revealed by gating on blasts and using residual normal lymphocytes or isotype-matched controls (not shown) to establish negative and positive gating for CD123. Two examples of blasts stained with CD123 and CD34 are shown (top), and a panel of 9 representative leukemias is shown (bottom). The median fluorescence intensity (MFI) for CD123 was adjusted for cell size by dividing MFI by the forward scatter (FSC). (C) Sorted CD123dim and CD123bright leukemia cells form methylcellulose colonies with an identical CD123 phenotype. MFI of CD123 is shown adjacent to each peak. PE, phycoerythrin.

Figure 2

Figure 2

CART123 cells manifest multiple effector functions upon in vitro exposure to CD123-expressing targets. (A) Activation of CART123 cells by AML targets. CART123 cells were coincubated with normal bone marrow (NBM) cells, primary AML cells, or with CD123+ MOLM14 cells, and CD107a degranulation was measured via flow cytometry. CAR-expressing cells were identified by staining with goat anti-mouse F(ab′)2 reagent. CART123 cells alone and the CD123− Jurkat cell line were used as negative controls; P < .0001 (Student t test). (B) Antigen-specific cytokine production in response to CD123+ AML. CARs were detected using a goat anti-mouse F(ab′)2 (for CART123) or an anti-CAR19 idiotypic antibody (for CART19). Intracellular cytokines were interferon-γ, MIP1β, and tumor necrosis factor α. (C) Proliferation of CART123 in response to CD123+ primary AML. CART123 or CART19 cells were labeled with CFSE and exposed to primary AML cells for 96 hours. Proliferation was assessed by CFSE dilution; P < .001 (Student unpaired t test). (D) Cytotoxic targeting of primary AML blasts by CART123 after incubation for 16 hours at the indicated effector-to-target (E:T) ratios; CART19 cells were used as controls. Two representative examples are shown. (E) Cytokine profiling of CART123 (black) or CART19 (white) cells in response to 24-hour coincubation with MOLM14 cells. All results are representative of at least 2 experiments with similar results. IFN-γ, interferon-γ; IL-2, interleukin-2; GM-CSF, granulocyte macrophage colony-stimulating factor; TNF-α, tumor necrosis factor α.

Figure 3

Figure 3

CART123 cells eliminate human AML in xenograft models. (A) Schematic of the MOLM14 xenograft model. NSG mice were sublethally irradiated (200 cGy) on day −1 and then injected via tail vein with 1 × 106 green fluorescent protein/luciferase+ MOLM14 on day 0. BLI was performed on day 6 to quantify engraftment and for randomization of treatment groups. Saline vehicle, CART19 cells (1 × 106), or CART123 (1 × 106) cells were injected IV on day 7, and mice were followed with serial BLI. Quantification of BLI radiance was used as a surrogate measurement of AML burden. (B) Elimination of MOLM14 occurred only in xenografted mice treated with CART123 cells, as measured by BLI radiance and displayed colorimetrically. (C) Summary BLI data from 3 MOLM14 xenograft experiments demonstrated rapid leukemic progression in vehicle-treated (black) and CART19-treated (blue) mice, whereas AML rejection was observed in CART123-treated mice (red). Mean radiance (symbols) with standard error of the mean (whiskers) are depicted at each time point. (D) Survival analysis of MOLM14 xenograft mice revealed significant survival for CART123-treated mice in comparison with vehicle- and CART19-treated mice. Attrition of CART123 T-cell–treated mice was primarily due to BLI-detectable AML progression in facial bones and subsequent anorexia and weight loss. Data were summarized from 4 independent experiments.

Figure 4

Figure 4

CD123 is an excellent target in primary AML in vivo. (A) Primary AML xenograft model. NSGS mice were sublethally irradiated on day −1 and injected with 5 to 10 × 106 primary AML blasts via the tail vein on day 0. Engraftment was confirmed by flow cytometric measurement of live mouse CD45neg human CD45dim CD123+ cells in the peripheral blood, usually occurring around day 14. Mice were then injected with CART123 cells or control T cells (CART19 or UTD T cells) and bled weekly to quantify AML burden. (B) Analysis of peripheral blood from mice 8 to 15 days after receiving T cells demonstrated marked reduction of circulating UPN 024 blasts. Note that residual CD45bright T cells are poorly detectable in some CART123 mice at this time point, correlating with the rapid rise and fall of peripheral CART123 cells in response to clearance of AML; P < .0001 (Student t test). (C) Composite survival plot of mice from 3 independent experiments. (D) In vivo upregulation of CD123 on AML blasts. Upon injection into NSGS mice treated with control UTD T cells, the UPN034 sample upregulated CD123 expression in comparison with blood measurements obtained prior to (day 13 [D13], left) and after (day 27 [D27], right) T-cell injection. The CD45bright CD123− population represents adoptively transferred human T cells. MFI of CD123 is shown at top right. All mice receiving control T cells subsequently died of disease, and all CART123-treated mice were long-term survivors (these are a subset of the mice in Figure 4C). Results are representative of 3 independent experiments.

Figure 5

Figure 5

CART123 cells exhibit characteristics of immunologic memory. (A) In a challenge/rechallenge model, NSG mice were sublethally irradiated and then injected via tail vein with saline or with 1 × 106 green fluorescent protein/luciferase+ MOLM14 cells on day 0. After BLI quantification of AML burden and randomization into treatment groups, CART123 cells (1 × 106) were injected IV on day 10, and mice were followed with serial BLI until AML eradication. Approximately 2 weeks after AML clearance, all mice were challenged (right) or rechallenged (left) with 1 × 106 MOLM14 cells and followed with weekly retro-orbital venous bleeding for T-cell quantification and by BLI for AML burden. (B) Summary of BLI radiance in CART123-treated mice following rechallenge or primary challenge with MOLM14. Each symbol represents an individual animal. The dashed line depicts the mean radiance measurement of untreated NSG mice. (C) CART123-treated, MOLM14-treated mice that are rechallenged with MOLM14 demonstrate increased numbers of peripheral CART123 cells in comparison with CART123-treated mice administered MOLM14 cells for the first time. Mice were bled 7 days prior to (day 28) and 7 days following (day 42) injection of MOLM14 cells. CART123 cells were identified as live, singlet, human CD45+ CD3+ CAR+ cells per the gating strategy in supplemental Figure 7 and quantified using Countbright beads. (D) Representative mouse (the highest blue data point from Figure 5B) showing that initial failure to reject MOLM14 is associated with low CART123 cell numbers in peripheral blood and that late rejection is accompanied by emergence of CART123 cells. Results are representative of 2 to 5 independent experiments. NS, nonsignificant.

Figure 6

Figure 6

Eradication of normal hematopoiesis by CART123 in a xenograft model. (A) Healthy bone marrow progenitor populations exhibit moderate to bright expression of CD123. Bone marrow from 4 normal donors was stained for CD123 after gating on live singlet lineage-negative CD45dim cells, and the indicated progenitor subpopulations were identified using CD34, CD38, CD45RA, and CD90 (gating strategy is shown in supplemental Figure 8). CD123 gating was based on normal lymphocytes and confirmed with fluorescence-minus-one controls. MEP, megakaryocyte-erythroid progenitors; CMP, common myeloid progenitors; GMP, granulocyte-monocyte progenitors; MPP, multipotent progenitors; HSC, hematopoietic stem cells. (B) CD123dim/− bone marrow progenitors differentiate to CD123+ in semisolid culture. CD123dim (top panel, red histogram) or CD123intermediate/+ (top panel, blue histogram) CD34+ cells were sorted from normal bone marrow (NBM) and cultured in MethoCult Optimum medium for 14 days. The middle and lower panels show the phenotype of colonies that developed from sorted CD123dim/− and CD123intermediate/+ populations, respectively. The sorted cultured populations exhibited similar CD123 expression and an indistinguishable ability to form myeloid or erythroid colonies. MFI of CD123 is shown at top right. (C) CART123 cells markedly impair hematopoietic function. CD34+ cells selected from normal human cord blood were incubated at a 1:10 target-to-effector ratio with CART123 or control UTD T cells for 4 hours, followed by a 14-day culture in Methocult Optimum. Coculture with UTD T cells was used to control for the allogeneic effect. Hematopoietic function was assessed by manual colony counts (not shown) or quantified by flow cytometry for the indicated cell populations using Countbright beads. (D) Cycling bone marrow cells upregulate CD123. Mice previously engrafted with human CD34+ cells were treated with 5-fluorouracil (5-FU) or vehicle. Fourteen days later, bone marrow was harvested from these mice and analyzed for the intracellular proliferation marker Ki67 and for CD123 after gating on live lineage-negative human cells; P < .01 (Student t test). (E) Schematic of xenograft model to evaluate potential CART123-mediated myeloablation. NSG mice were engrafted with human fetal liver CD34+ cells (HIS mice) and bled for confirmation of engraftment after 6 to 8 weeks. On day 0, mice received CART123, control UTD T cells, or saline vehicle. Flow cytometric quantification of human hematopoietic cells in peripheral blood (days 7, 14, 21) and in bone marrow (day 28) was performed. (F) Specific decline in circulating human B cells, myeloid cells, monocytes, and platelets is seen after treatment with CART123. Representative plots are shown after control (top) or CART123 (bottom) infusion and quantified in the lower panel (control T cells, open column; CART123, solid column). (G) Specific myeloablation of human bone marrow in CART123 mice. On day 28 after T-cell injection, bone marrow was harvested and analyzed for human progenitor cell populations after gating on live singlet human lineage-negative cells. (H) Sections of femur taken from HIS mice 1 month after treatment with control (top) or CART123 (bottom). Hematoxylin and eosin staining; Zeiss microscope original magnification ×1 and ×10 shown. Results are representative of at least 2 independent experiments. NS, nonsignificant.

Figure 6

Figure 6

Eradication of normal hematopoiesis by CART123 in a xenograft model. (A) Healthy bone marrow progenitor populations exhibit moderate to bright expression of CD123. Bone marrow from 4 normal donors was stained for CD123 after gating on live singlet lineage-negative CD45dim cells, and the indicated progenitor subpopulations were identified using CD34, CD38, CD45RA, and CD90 (gating strategy is shown in supplemental Figure 8). CD123 gating was based on normal lymphocytes and confirmed with fluorescence-minus-one controls. MEP, megakaryocyte-erythroid progenitors; CMP, common myeloid progenitors; GMP, granulocyte-monocyte progenitors; MPP, multipotent progenitors; HSC, hematopoietic stem cells. (B) CD123dim/− bone marrow progenitors differentiate to CD123+ in semisolid culture. CD123dim (top panel, red histogram) or CD123intermediate/+ (top panel, blue histogram) CD34+ cells were sorted from normal bone marrow (NBM) and cultured in MethoCult Optimum medium for 14 days. The middle and lower panels show the phenotype of colonies that developed from sorted CD123dim/− and CD123intermediate/+ populations, respectively. The sorted cultured populations exhibited similar CD123 expression and an indistinguishable ability to form myeloid or erythroid colonies. MFI of CD123 is shown at top right. (C) CART123 cells markedly impair hematopoietic function. CD34+ cells selected from normal human cord blood were incubated at a 1:10 target-to-effector ratio with CART123 or control UTD T cells for 4 hours, followed by a 14-day culture in Methocult Optimum. Coculture with UTD T cells was used to control for the allogeneic effect. Hematopoietic function was assessed by manual colony counts (not shown) or quantified by flow cytometry for the indicated cell populations using Countbright beads. (D) Cycling bone marrow cells upregulate CD123. Mice previously engrafted with human CD34+ cells were treated with 5-fluorouracil (5-FU) or vehicle. Fourteen days later, bone marrow was harvested from these mice and analyzed for the intracellular proliferation marker Ki67 and for CD123 after gating on live lineage-negative human cells; P < .01 (Student t test). (E) Schematic of xenograft model to evaluate potential CART123-mediated myeloablation. NSG mice were engrafted with human fetal liver CD34+ cells (HIS mice) and bled for confirmation of engraftment after 6 to 8 weeks. On day 0, mice received CART123, control UTD T cells, or saline vehicle. Flow cytometric quantification of human hematopoietic cells in peripheral blood (days 7, 14, 21) and in bone marrow (day 28) was performed. (F) Specific decline in circulating human B cells, myeloid cells, monocytes, and platelets is seen after treatment with CART123. Representative plots are shown after control (top) or CART123 (bottom) infusion and quantified in the lower panel (control T cells, open column; CART123, solid column). (G) Specific myeloablation of human bone marrow in CART123 mice. On day 28 after T-cell injection, bone marrow was harvested and analyzed for human progenitor cell populations after gating on live singlet human lineage-negative cells. (H) Sections of femur taken from HIS mice 1 month after treatment with control (top) or CART123 (bottom). Hematoxylin and eosin staining; Zeiss microscope original magnification ×1 and ×10 shown. Results are representative of at least 2 independent experiments. NS, nonsignificant.

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