CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia - PubMed (original) (raw)

. 2015 Aug;29(8):1637-47.

doi: 10.1038/leu.2015.52. Epub 2015 Feb 27.

M Ruella 2, O Shestova 2, M Klichinsky 2, V Aikawa 3, J J D Morrissette 3, J Scholler 2, D Song 2, D L Porter 4, M Carroll 5, C H June 2, S Gill 4

Affiliations

CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia

S S Kenderian et al. Leukemia. 2015 Aug.

Abstract

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

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Conflict of interest statement

Conflict of Interest: CHJ and DLP have filed patent applications related to CAR technology and could potentially receive licensing royalties from Novartis corporation. The other authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Expression of CD33 on malignant and normal myeloid cells. (a) CD33 is expressed in AML. (1) CD33 is expressed on blasts in most patient samples with AML (AML blasts were gated using standard side scatterlow CD45dim characteristics, n = 36) (2) Histogram mean fluorescence intensity (MFI) from an AML patient (gated on AML blasts). (b) CD33 is expressed in bone marrow from MDS patients. (1) CD33 is expressed on the CD34+CD38+ compartment in MDS patients. (2) CD33 is expressed on the CD34+CD38– hematopoietic stem cell compartment in MDS patients. (3) Histogram MFI from a MDS patient (gated on CD34+CD38- cells). (c) Tissue expression of CD33. CD33 is expressed on normal bone marrow myeloid progenitors (1), liver Kupffer cells (2), no expression on neurological tissue (3), occasional lung macrophage expression (4), occasional myeloid cells in the kidneys (5) and non-specific cardiac staining (6). (d) CAR constructs used in this study. All are second-generation CARs composed of: an extracellular domain (the light-to-heavy orientation of the scFV clone MY96 of GO), a hinge derived from either human CD8 or IgG4 as indicated, a transmembrane domain (TM) derived from CD8, 41BB (CD137) costimulatory domain and CD3zeta intracellular signaling domain.

Figure 2

Figure 2

CART33 cells exhibit robust in vitro effector functions in response to the CD33+ cell line MOLM14 (plots are representative of four independent experiments). (a) CART33 and CART123 cells undergo specific degranulation to the CD33+/CD123+ MOLM14. CART33, CART123 and untransduced T cells (UTD) were incubated with the CD33+/CD123+ cell line MOLM14, PMA/ionomycin as a positive non-specific T-cell stimulant (not shown) and the control T-cell ALL cell line Jurkat, in the presence of CD49d, CD28 costimulatory molecules and monensin. CD107a degranulation was measured by flow cytometry after 4 h of incubation. (b) CART33 cells result in specific killing of MOLM14 cells that is comparable to CART123. CART33, CART123 and UTD were incubated with MOLM14-luc or Jurkat-luc for 24 h at different E:T ratios as indicated, and bioluminescence imaging was then performed as a measure of residual living cells. (c) CART33 cells undergo specific proliferation to MOLM14 and not to Jurkat. T cells were labeled with CFSE and incubated with MOLM14, PMA/ionomycin as a positive nonspecific T-cell stimulant or with Jurkat as a negative control, for 120 h. The number of proliferating T cells was significantly higher in response to MOLM14 as compared with Jurkat and was comparable to CART123. (d) CART33 cells produce robust levels of cytokines in response to MOLM14, comparable to CART123 cells. CART33, CART123 and UTD cells were incubated with MOLM14, Jurkat and PMA/ionomycin for 24 h. Supernatant was then harvested, and a 30-plex Luminex assay was performed. Levels of the rest of cytokines are presented in Supplementary Figure S4. There was some variability in cytokine production between the donors, but when averaged out, CART123 and CART33 produced similar levels of cytokines. (e) CART33 cells produce more than one cytokine per cell in response to MOLM14. CART33, CART123 and UTD cells were incubated with MOLM14, or PMA/ionomycin. The cells were then fixed and permeabilized, stained for five different cytokines (tumor necrosis factor alpha, interferon gamma, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein 1b and interleukin-2), and flow cytometric analyses were performed. The majority of CART33 cells produce more than one cytokine in response to MOLM14, similar to their response to PMA/Ionomycin.

Figure 3

Figure 3

CART33 and CART123 cells exhibit antitumor activity in MDS. (a) CART33 and CART123 cells undergo specific degranulation in response to bone marrow cells from MDS patients. Bone marrow samples from MDS patients were CD34 enriched (≥85% purity) and then incubated with CART33, CART123 or UTD cells at E:T ratio of 1:5 for 4 h, in the presence of CD49d, CD28 co-stimulation and monensin. CD107a degranulation was then measured by flow cytometry. (b) CART33 and CART123 cells kill CD34-enriched bone marrow cells from MDS patients. CD34-enriched bone marrow from patients with MDS were incubated with either UTD, CART33 or CART123 for 24 h, and then live leukemic cells were measured by flow cytometry. There was a significant reduction in live CD45dimCD34+ cells in samples treated with CART33 or CART123. (c) Treatment with CART33 results in specific killing of the MDS clone. CD34-enriched bone marrow sample from a patient with MDS and 5q deletion was incubated with CART33, UTD cells or with no treatment at 1:1 E:T ratio for 4 h. Sample was then harvested, and fluorescence in situ hybridization for 5q- was performed. There was significant reduction in the 5q- clone percentage in the group treated with CART33 when compared with UTD and No treatment groups (as T cells comprised 50% of the sample, interphase nuclei were corrected by a factor of two). Results are representative of three experiments.

Figure 4

Figure 4

CART33 treatment results in reduction in disease burden and prolonged survival in MOLM14-engrafted xenografts (three independent experiments). (a) Experiment schema: NOD-SCID-gamma chain knockout (NSG) mice were injected with the AML cell line MOLM14 1×106 intravenously and imaged for engraftment after 4–6 days. Between day 5 and 7, mice were treated with CART33 (5×106), or control vehicle (UTD cells 5 × 106). The mice were followed with serial weekly imaging to assess the burden of AML. (b) Representative Images of tumor burden by bioluminescent imaging (BLI) from one experiment. (c) CART33 treatment results in reduction in leukemia in MOLM14-engrafted xenografts. Tumor burden over time by BLI; data from one experiment (_n_= 5 per group), each mouse is represented by a line. (d) Composite survival of three independent experiments. Treatment with CART33 resulted in significant survival advantages when compared with treatment with UTD.

Figure 5

Figure 5

CART33 treatment results in leukemia eradication and long-term disease-free survival in primary AML engrafted xenografts. (a) Experiment schema: NSG mice transgenic for the human cytokines stem cell factor, interleukin-3, granulocyte macrophages colony-stimulating factor (NSG-S mice) were injected with a primary AML sample (5 × 106 intravenously (IV)). Engraftment was confirmed by retro-orbital bleeding after 2–4 weeks, and then mice were treated with CART33, CART123 or control vehicle (UTD cells). Total number of T cells injected was 1 ×105 IV. CART33 and CART123 cells had comparable transduction efficacy. The mice were followed with serial retro-orbital bleedings to assess the burden of leukemia. (b) Analysis of peripheral blood from mice treated with UTD, CART33 or CART123 at baseline, day 14 and day 70 after T cells injection. AML was not detected in mice treated with CART33 or CART123 starting 4 weeks after T-cell treatment. (c) Summary of disease burden measured by blasts/ul from retro-orbital bleedings at different time points as indicated. (d) Survival of mice treated with CART33, CART123 or UTD (_n_=8 per group) (P < 0.001 when treatment with CART33 or CART123 is compared with UTD).

Figure 6

Figure 6

CART33 and CART123 treatment results in similar hematopoietic toxicity in two different humanized xenografts models (representative of six independent experiments). (a) Schema of Model no. 1: humanized immune system (HIS) mice were bled retro-orbitally 6–8 weeks after injection of human CD34+ cells derived from the fetal liver to confirm engraftment of human cells. Mice were then treated with either CART33, CART123, UTD cells (1 ×106 cells each) or with no treatment and followed by serial weekly retro-orbital bleedings. Mice were then euthanized on day 28, and organs were harvested and analyzed. (b) CART33 or CART123 treatment results in significant reduction in myeloid progenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38 –). Representative FACS plots and summary statistics from bone marrow analysis by flow cytometry on day 28 at the conclusion of the experiment, gated on singlets, huCD45dim, Lineage negative. (c) CART33 treatment results in reduction of the CD34+ compartment in the bone marrow by immunohistochemistry. Sections of the femur were taken from the mice on day 28 after treatment with UTD cells or CART33 cells. huCD45 and CD34 staining by immunohistochemistry was performed. No difference in huCD45 between control T cells and CART33, although both these groups show less huCD45 staining likely consistent with an allogeneic human-anti-human effect. There was specific reduction of CD34+ cells in mice treated with CART33. (d) Schema of Model no. 2: NSGS mice received busulfan intraperitoneally followed by 2 × 106 T-cell depleted bone marrow cells from a normal donor the following day. Engraftment was confirmed by flow cytometric analysis of peripheral blood after 4 weeks, and mice were then treated with 1 × 106 autologous CART33, autologous CART123 or UTD cells. Mice were then followed with retro-orbital bleeding on days 7 and 14 and were euthanized for necropsy on day 21. (e) CART33 or CART123 treatment results in significant reduction in myeloid progenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38 –). Representative plot of bone marrow analysis by flow cytometry on day 21. CART33 and CART123 treatment resulted in significant reduction in myeloid progenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38 –), Gated on huCD45dim, Lin – . (f) Summary statistics from peripheral blood analysis by flow cytometry on day 14.

Figure 7

Figure 7

RNA-modified CART33 cells have transient CAR expression and result in significant in vitro and in vivo activity. (a) Transient expression of CAR following RNA modification. RNA-modified CAR33 expression as correlated with time postelectroporation. CAR expression reaches the peak at 24 h postelectroporation and gradually decreases after that. (b) The mean fluorescence intensity (MFI) of CAR expression in mRNA CART33 compared with lentivirally transduced CART33. The MFI of CAR expression decreases with time postelectroporation, while there is stable expression of CAR in lentivirally transduced T cells. (c) RNA-CART33 treatment results in specific killing against MOLM14 that is comparable to LV-transduced CART33. RNA-modified CART33 and LV-transduced CART33 cells were incubated with the CD33-positive cell line MOLM14-luc and a control mantle cell lymphoma cell line JEKO-luc at different E:T ratios as indicated. Bioluminescence imaging was performed after 24 h as a measure of living cells. This experiment was repeated at different time points postelectroporation of T cells. Twenty-four hours postelectroporation, RNA CART33 resulted in the most specific killing of MOLM14 cell line (comparable to LV-transduced CART33) that decreased with time post electroporation. (d) RNA-CART33 therapy combined with lympho-depleting chemotherapy results in further reduction of leukemic burden in MOLM14-engrafted xenografts. NSG mice were injected with MOLM14-luc (1 × 106 intravenously (IV)) and imaged to confirm engraftment 4 days later. Mice were then randomized to receive either RNA-CART33 with cyclophosphamide or UTD cells with cyclophosphamide (60 mg/kg intraperitoneally). T cells were given at a dose of 10 ×106 IV on days 5, 9 and 16. Lympho-depleting doses of cyclophosphamide were given on days 8 and 14, prior to T-cell infusion. (e) RNA-CART33 treatment combined with lympho-depleting chemotherapy result in prolonged survival compared with control T cells with chemotherapy.

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