In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

Abstract

Background: Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia.

Methods and findings: We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85-90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination.

Conclusions: This strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a 'bridge" therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Generation and genetic modification of human activated T-cells.

(a) Diagram of CAR.CD33, and iC9-ΔCD19-CAR.CD33 plasmid constructs used for the genetic modification of human activated T-cells (ATCs). (b) Schematic representation of the protocol for ATCs generation and transduction. (c) Representative experiment displaying the CAR expression on the surface of ATCs generated from a healthy donor. (d) CAR expression on non-transduced (NT), CAR.CD33, or iC9-ΔCD19-CAR.CD33 transduced ATCs assessed on day 11 after transduction, and on ΔCD19 selected (sel.) iC9-CAR.CD33 assessed on day 28 after transduction. (e) Lymphocyte subset marker’s expression on NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs assessed by flow cytometry on day 11 after transduction. (f) Memory marker’s expression on NT, CAR.CD33, and ΔCD19 sel. iC9-CAR.CD33 ATCs (assessed on day 11, 11, or 28 after transduction, respectively); (mean± standard error of the mean (SEM); N: 4–7 experiments from 5 healthy donors for all the experiments). LTR: long terminal repeat; Ψ: packaging signal; scFv: single chain variable fragment; ζ: Zeta chain of the T-cell receptor; PBMC: peripheral blood mononuclear cells; CM: central memory; EM: effector memory; TEMRA: terminal differentiated effector memory.

Fig 2. CAR.CD33, and ΔCD19 sel. iC9-CAR.CD33 ATCs displayed effectors function after stimulation with the CD33+ acute myeloid leukemia cell line, MV4-11.

(a) Overnight co-culture experiments using non transduced (NT), CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 activated T-cells (ATCs) against the MV 4-11eGFP+ CD33+ cell line, followed by evaluation of enhanced green fluorescent protein (eGFP)/CD33+ cell killing by flow cytometry, as compared with co-culture employing NT ATCs as effectors; (mean±SEM of 3 experiments using ATCs from 3 healthy donors) (left). Annexin V and 7-AAD expression from one representative co-culture experiment (right). (b) Luciferase-based cytotoxicity assay using NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs after overnight incubation with the CD33+ MV4-11 cell line engineered to express the eGFP-firefly-luciferase (eGFP-ffLuc) construct. Effector:target (E:T) ratios ranged from 10:1 to 0.6:1; (N: 3–7 experiments with ATCs generated from 5 healthy donors). (c) Evaluation of CD107a degranulation marker expression on CAR.CD33 or ΔCD19 sel. iC9-CAR.CD33 ATCs (as compared with NT ATCs) after 4 hours of stimulation with MV4-11 CD33+ targets (E:T = 4:1); (N: mean±SEM of 4–6 experiments with ATCs from 4 healthy donors). (d) CFSE-based proliferation assay stimulating NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs with irradiated (40 Gy) MV4-11 CD33+ cell line (gray), or no target cells (blank), and cultured for additional 5 days (E:T = 1:1). Data from one representative experiment is shown. (e) Cytokines production on supernatant collected after overnight incubation of NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs with the MV4-11 CD33+ cell line (E:T = 4:1); (mean±SEM of 3–9 independent experiments from 3–4 different healthy donors). SEM: standard error of the mean; NS: not statistically significant.

Fig 3. CAR.CD33, and ΔCD19 sel. iC9-CAR.CD33 ATCs exhibit anti-tumor activity against samples from patients with active AML.

(a) CD33 expression on freshly isolated samples from patients with active acute myeloid leukemia (AML) evaluated by flow cytometry after gating on side scatterlow/CD45dim cells; (N:9 peripheral blood; N:5 bone marrow samples from different patients). (b) Overnight co-culture assay of non transduced (NT), CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs, generated from 3 healthy donors, with samples collected from patients with active AML labeled with the PKH-26 dye (effector:target (E:T) = 4:1), (patients#C, P, Q, R, and S). Results from a representative co-culture experiment (patient#C) gated on viable residual cells displaying the expression of (i) PKH vs.CD33, or (ii) PKH vs.CD3, or (iii) CD3 vs. CD33, (left). Summary results from 3–9 independent experiments displaying average (±SEM) percentage of cell killing is shown, (right). (c) Correlation (coefficient R2) between the reduction of PKH+CD33+ cells and CD33 expression level of the ΔCD19 sel. iC9-CAR.CD33 ATCs co-cultured with patient samples as represented in Fig 2B; (N: 9 independent experiments using ATCs from 3 healthy donors). (d) Evaluation of CD107a expression on CAR.CD33 or ΔCD19 sel. iC9-CAR.CD33 ATCs (as compared with NT ATCs) assessed 4 hours after stimulating them with samples collected from patients with AML (effector:target (E:T) = 4:1); (N:8–12 experiments using ATCs from 4 healthy donors). (e) Cytokines production (picograms per mL: pg/mL) on supernatant collected after overnight incubation of NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs generated from healthy donor with samples isolated from 8 patients with AML (#C, G, L, N, O, P, Q, R, S, and U) (E:T = 4:1); (mean±SEM from 5–18 experiments using ATCs generated from 4 different healthy donors). ATCs: activated T-cells; Pt(s): patient(s); lymph: lymphocyte; SEM: standard error of the mean; NS: not statistically significant.

Fig 4. ΔCD19 sel. iC9-CAR.CD33 ATCs generated from a patient with AML.

(a) Peripheral blood mononuclear cells isolated from a patient with acute myeloid leukemia (AML) (patient#3) were used to generate activated T-cells (ATCs). Non transduced (NT), CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs were stained with the appropriate monoclonal antibody, and analyzed by flow cytometry after gating on the CD3+ population to analyze CD8 and CAR expression, (left) or CD45RA and CD62L expression, (right). (b) Dot plots from a representative co-culture experiment employing NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs from patient#3 challenged with the MV4-11 CD33+ AML cell line genetically modified to express the enhanced green fluorescent protein (eGFP) marker (left), or with an autologous CD33+ AML sample (right); the co-cultures were performed in the presence of medium (top panels) or in the presence of autologous patient’s plasma (bottom panels).

Fig 5. Co-culture of CAR T-cells with PD-L1+ AML blasts.

(a) Non Transduced (NT), CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 activated T-cells (ATCs) generated from a healthy donor were co-cultured overnight with PD-L1+(top panels) or PD-L1neg(bottom panels) blasts collected from a patient with acute myeloid leukemia (AML), (patient#T). AML blasts were sorted by flow cytometry for PD-L1 expression, and labeled with the PKH-26 dye prior to co-culture. After overnight incubation 20,000 events were acquired by flow cytometry after gating on the PKH+ population and the reduction of CD33+ AML cells, as compared with the retained CD3+ cells is shown in the dot-plots. (b) ATCs co-cultured as per 5a were stained with a PD-1 monoclonal antibody, and PD-1 expression on the PKHneg/CD3+ population was analyzed by flow cytometry.

Fig 6. ΔCD19 sel. iC9-CAR.CD33 ATCs demonstrate limited toxicity against normal hematopoietic stem cells.

(a) Human CD34+-selected hematopoietic stem cells (CD34+ HSC) from healthy volunteer donors were co-cultured overnight with or without non transduced (NT) or ΔCD19 sel. iC9-CAR.CD33 activated T-cells (ATCs), generated from healthy donors at an effector:target (E:T) ratio of 4:1. Afterwards, CD34+ HSC were isolated with hematopoietic lineage depletion kit, and enumerated by flow cytometry; (mean±SEM, N:4, from duplicate experiments using ATCs from 2 different healthy donors). (b) 500 CD34+lyneage- HSC, from each respective condition outlined above, were plated on cytokine conditioned semisolid medium, (MethoCult H4434). After 14 days, hematopoietic colony formation was scored using standard morphologic criteria; (mean±SEM, N:4, from duplicate experiments using ATCs from 2 different healthy donors). CFU: Colony-forming unit: BFU-E: burst-forming unit-erythroid: CFU-M/G/GM: CFU-macrophage/granulocyte/granulocyte, macrophage; CFU-GEMM: CFU-granulocyte, erythroid, macrophage, megakaryocyte; SEM: standard error of the mean; NS: not statistically significant.

Fig 7. Pharmacologic elimination of ΔCD19 sel. iC9-CAR.CD33 ATCs.

(a) NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs generated from healthy donors were treated overnight with ABT-199 or ABT-737 [both at 2 or 10μM], or mafosfamide [0.5 or 2 μg/mL] with or without the CID [10 nM], and thereafter washed by centrifugation. On day 1, cells were harvested, stained with Annexin V/7-AAD and analyzed by flow cytometry. Ten to twenty thousand total events were acquired for each sample (the same number of events was acquired within each experiment). Average percentage (±SEM) of cell killing is represented in the histogram graph. Conditions without application of the CID are shown as shaded bars, while conditions involving the application of the CID are shown as black bars; (N:3–15 experiments using ATCs from 3–8 different healthy donors). (b) Cells treated overnight with ABT-199 or ABT-737 [both at 2 μM], or mafosfamide [0.5 μg/mL] (with or without CID [10 nM]) were harvested, washed by centrifugation and cultured for additional 4–7 days in the presence of culture medium supplemented with rhIL2 50 I.U./mL. Outline of the experimental plan, (left), and histogram data from a representative experiment displaying residual viable cells is shown, (right). UnTx: untreated; SEM: standard error of the mean; CID: chemical inducer of dimerization to activate the iC9 suicide gene; rhIL2: recombinant human interleukin-2; ABT-199: BCL-2 inhibitor; ABT-737: Pan-BCL inhibitor; MAF: Mafosfamide; NS: not statistically significant.