Dual-targeting CD33/CD123 NANOBODY T-cell engager with potent anti-AML activity and good safety profile.

ABSTRACT: Novel therapies are needed for effective treatment of acute myeloid leukemia (AML). Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. CD123 and CD33, 2 clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells in >95% of patients with AML. However, their expression is heterogenous between subclones and between patients, which may affect the efficacy of single-targeting agents in certain patient populations. We present here a dual-targeting CD33/CD123 NANOBODY T-cell engager (CD33/CD123-TCE) that was designed to decrease the risk of relapse from possible single antigen-negative clones and to increase coverage within and across patients. CD33/CD123-TCE killed AML tumor cells expressing 1 or both antigens in vitro. Compared with single-targeting control compounds, CD33/CD123-TCE conferred equal or better ex vivo killing of AML blasts in most primary AML samples tested, suggesting a broader effectiveness across patients. In a disseminated cell-line-derived xenograft mouse model of AML, CD33/CD123-TCE cleared cancer cells in long bones and in soft tissues. As cytokine release syndrome is a well-documented adverse effect of TCE, the compound was tested in a cytokine release assay and shown to induce less cytokines compared to a CD123 single-targeting control. In an exploratory single-dose nonhuman primate study, CD33/CD123-TCE revealed a favorable PK profile. Depletion of CD123 and CD33 expressing cells was observed, but there were neither signs of cytokine release syndrome nor clinical signs of toxicity. Taken together, the CD33/CD123 dual-targeting NANOBODY TCE exhibits potent and safe anti-AML activity and promises a broad patient coverage.

A novel CD123-targeted therapeutic peptide loaded by micellar delivery system combats refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival rate due to the refractory feature and high rate of relapse. CD123 is highly expressed on multiple types of AML cells, especially leukemia stem cells, and closely associated with the poor prognosis of AML. Aiming to meet the urgent demand to targeted therapeutics for the refractory AML patients, herein we synthesize a CD123 antagonistic peptide (PO-6) loaded in nanomicelles (mPO-6), and investigated its therapeutic effect and pharmacokinetics on a lab-established refractory AML mice model (AE & CKITD816V). It is shown that the PO-6 can effectively bind to the CD123+ AML cells and the micellar formulation mPO-6 increases the dissolution stability and the specific binding capacity. When injected intravenously, mPO-6 significantly prolongs the survival of the refractory AML mice by interfering CD123/IL-3 axis, evidenced by the down regulation of phosphorylation of STAT5 and PI3K/AKT and the inhibition of activated NF-κB in the nucleus, as well as by the analysis results of next generation RNA-sequencing (RNA-seq) with the bone marrow of the AML mice. The antagonistic effect leads to the significantly reduction of AML cells infiltration in the bone marrow of the AML mice. In conclusion, mPO-6 could provide a potent antagonistic therapeutic approach for targeted treatment of AML.

A Case of Cutaneous Blastic Plasmacytoid Dendritic Cell Neoplasm Treated With a Bcl-2 Inhibitor.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive primary cutaneous lymphoma characterized by transformed plasmacytoid dendritic cells that overexpress interleukin-3 receptor subunit alpha (IL3RA) also known as CD123. In addition to several therapies currently undergoing clinical trials, Tagraxofusp-erzs (Stemline Therapeutics, Inc., NY) is a single FDA-approved option available for treatment of adults and children over 2 years of age suffering from BPDCN. It was designed to target CD123 overexpression in BPDCN as a CD123-directed cytotoxin consisting of a recombinant human interleukin-3 fused to a truncated diphtheria toxin. We discuss a case of a male patient in his late 70s’ who presented with an asymptomatic rash involving the back and the right knee that initially developed as pink patches, progressed into plaques, and subsequently rapidly evolved into a tumor involving the right knee that was confirmed as BPDCN on skin biopsy and was accompanied by bone marrow involvement. Upon initiation of first line tagraxofusp-erzs therapy, the patient did not achieve improvement. However, off-label use of venetoclax (AbbVie Inc, IL and Genentech-USA, CA), a Bcl2 inhibitor currently in a Phase I clinical trial, resulted in a satisfactory clinical outcome, nearly complete resolution of a right knee tumor lesion, and deferment of bone marrow transplant. We believe that our case exemplifies the complexity of BPDCN, briefly reviews current treatment and management options that are only in their infancy and raises awareness towards success with alternative off-label therapies such as venetoclax when treating BPDCN. J Drugs Dermatol. 20(5):550-551. doi:10.36849/JDD.5373.

Development of Highly Optimized Antibody-Drug Conjugates against CD33 and CD123 for Acute Myeloid Leukemia.

PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

CD123 bi-specific antibodies in development in AML: What do we know so far?

Bispecific antibodies are synthetic molecules designed to simultaneously bind two separate antigens. Given the recent success of blinatumomab in the treatment of acute lymphoblastic leukemia, there is growing interest in the use of bispecific antibodies as T-cell redirecting antibody for the treatment of cancer. In acute myeloid leukemia (AML), CD123 (also known as the interleukin receptor 3 alpha subunit) has emerged as a promising therapeutic target for bispecific antibodies. Prior attempts to target CD123 with unconjugated antibodies and antibody-drug conjugates have been mixed. However, available data from CD123-directed bispecific antibodies currently in clinical trials have been encouraging. In this review, we discuss the biology of CD123 and prior attempts to target this cell surface marker as part of anti-leukemic therapy. We then summarize and discuss the five CD123-directed bispecific antibodies currently in clinical trials for treatment of AML and provide practical insights regarding the use of these agents.

Tagraxofusp and anti-CD123 in blastic plasmacytoid dendritic cell neoplasm: a new hope.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy, characterized by poor prognosis if treated with conventional therapy. Allogenic hematologic stem cell transplant can improve survival and can be curative, but it is available in a small percentage of patients given that the median age at diagnosis is 70 years. In this scenario it is assumed that only the development of precision medicine-driven therapy will change BPDCN patient prognosis. CD123 (the α-subunit of interleukin (IL)-3 receptor) is over-expressed on BPDCN cells surface and seems to be the ideal marker to develop antibody-based therapies. Tagraxofusp (Elzonris®), a recombinant immunotoxin consisting of human interleukin-3 fused to a truncated diphtheria toxin, has been approved by FDA in December 2018 for the treatment of BPDCN in adult and pediatric patients. tagraxofusp has shown promising clinical activity, with a high overall response rate and quite manageable safety profile even in elderly patients. It seems to improve overall survival too, but comparative trials are necessary to confirm this. Adverse events are commonly reported and the most important are transaminitis, thrombocytopenia and capillary leak syndrome (CLS). Therefore, to prevent the onset of severe CLS is recommended to reserve tagraxofusp for patients with preserved hepatic and cardiac functions, and to strictly observe serum albumin level. Further studies are required to resolve many several unanswered questions about tagraxofusp. In this review, we will resume and discuss pharmacological characteristic of tagraxofusp, results of clinical trials leading to its approval by FDA in 2018 and future perspectives about its use in BPDCN and other hematological malignancies.

Blockade of VLA4 sensitizes leukemic and myeloma tumor cells to CD3 redirection in the bone marrow microenvironment.

Redirecting T cells to specifically kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of blinatumomab for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. In hematological malignancies, the bone marrow (BM) niche is protective to leukemic stem cells and has minimized the efficacy of several anti-cancer drugs. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using bispecific antibodies targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia or multiple myeloma cells with BM stromal cells protected tumor cells from bispecific antibody-T cell-mediated lysis in vitro and in vivo. Impaired CD3 redirection cytotoxicity was correlated with reduced T cell effector responses and cell-cell contact with stromal cells was implicated in reducing T cell activation and conferring protection of cancer cells. Finally, blocking the VLA4 adhesion pathway in combination with CD3 redirection reduced the stromal-mediated inhibition of cytotoxicity and T cell activation. Our results lend support to inhibiting VLA4 interactions along with administering CD3 redirection therapeutics as a novel combinatorial regimen for robust anti-cancer responses.

CD123 as a Therapeutic Target Against Malignant Stem Cells.

Hematopoiesis is a tightly regulated process that originates from highly specialized cells, hematopoietic stem cells (HSCs). Many cancers can arise and be maintained by malignant stem cells. In acute myeloid leukemia, leukemic stem cells (LSCs) are identified by their immunophenotype, which is partly shared with normal HSCs (CD34+CD38-). However, LSCs also possess unique immunophenotypic features that can be used to distinguish them from HSCs and therapeutically target them. One such unique immunophenotypic marker is CD123, found to be aberrantly expressed in leukemic stem, progenitor, and blast cells. Thus, CD123 is sought as an attractive target to eliminate LSCs.

Immunotherapies Targeting CD123 for Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, difficult-to-diagnose, highly aggressive myeloid malignancy with poor prognosis and no standard of care. The interleukin-3 receptor α, or CD123, is highly expressed in patients with myeloid malignancies, particularly acute myeloid leukemia and BPDCN. CD123 is overexpressed on leukemic stem cells compared with normal hematopoietic stem cells, suggesting CD123 as an attractive immunotherapeutic target. To date, multiple CD123-targeted therapeutic avenues have been explored to treat BPDCN and other CD123+ hematologic malignancies. This review summarizes immunotherapies targeting CD123 for the treatment of BPDCN and related neoplasms.

Development of a novel fully-human anti-CD123 antibody to target acute myeloid leukemia.

Monoclonal antibodies are being considered as biopharmaceuticals for the in vivo targeting of acute myeloid leukemia. Here we describe the generation and characterization of a fully-human monoclonal antibody specific to CD123, a surface marker which is overexpressed in a variety of hematological disorders, including acute myeloid leukemia. The cloning and expression of the extracellular portion of CD123 as recombinant Fc fusion allowed the selection and affinity maturation of a human antibody, called H9, which specifically recognized the cognate antigen in biochemical assays and on leukemic cells. The H9 antibody and a previously-described anti-CD123 antibody (CSL362) were reformatted into full immunoglobulin human IgG1 formats, including a variant bearing S293D and I332E mutations to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). The two antibodies recognized different epitopes on the surface of the N-terminal domain of CD123, as revealed by crystallography and SPOT analysis. Both H9 and CSL362 in full immunoglobulin format were able to selectively kill leukemic cells in in vitro ADCC assays, performed both with cell lines and with patient-derived AML blasts. Further, the two antibodies, when reformatted as bispecific BiTE™ reagents by fusion with the anti-CD3 scFv(OKT3) antibody fragment, induced selective killing of AML blasts by patient-derived, autologous T-cells in an in vitro setting, but BiTE(CSL362/OKT3) exhibited a 10-fold higher potency compared to BiTE(H9/OKT3). The availability of two classes of CD123-specific biopharmaceuticals, capable of redirecting the cytolytic activity of NK cells and T cells against AML blasts, may enable novel interventional strategies and combination opportunities for the treatment of AML.

CD123: A Novel Biomarker for Diagnosis and Treatment of Leukemia.

Leukemia is a group of progressive hematologic malignancies derived from stem cells in bone marrow which causes a large number of cancer deaths. Even with treatment such as traditional chemotherapy, targeted therapy, and allogeneic stem cell transplantation (allo-HSCT), many patients suffer from relapse/refractory disease, and the overall survival is dismal. Leukemic stem cells (LSCs) are induced by gene mutations and undergo an aberrant and poorly regulated proliferation process which is involved in the evolution, relapse, and drug-resistance of leukemia. Emerging studies demonstrate that CD123, the interleukin 3 receptor alpha (IL-3Rα), is highly expressed in LSCs, while not normal hematopoietic stem cells (HSCs), and associates with treatment response, minimal residual disease (MRD) detection and prognosis. Furthermore, CD123 is an important marker for the identification and targeting of LSCs for refractory or relapsed leukemia. Anti-CD123 target-therapies in pre-clinical studies and clinical trials confirm the utility of anti-CD123 neutralizing antibody-drugs, CD3×CD123 bispecific antibodies, dual-affinity retargeting (DART), and anti-CD123 chimeric antigen receptor-modified T-cell (CAR-T) therapies in progress. This review summarizes the most recent progress on the study of CD123 biology and the development of novel CD123-targeted therapies.

Blastic Plasmacytoid Dendritic Cell Neoplasm.

OPINION STATEMENT: While there is a high initial response rate with standard chemotherapeutic regimens for blastic plasmacytoid dendritic cell neoplasm (BPDCN), the responses are typically not durable and this remains a very aggressive disease with generally poor outcomes. For this reason, the standard approach for eligible patients has been high-dose induction chemotherapy preferably with acute lymphoblastic leukemia (ALL)-based regimens followed by consolidation with allogeneic hematopoietic stem cell transplantation (alloHSCT). Unfortunately, many patients with this disease are elderly and/or frail and cannot tolerate this therapy, and the low-dose regimens being used in this population are generally not as effective. However, this paradigm may be changing with the advent of newer targeted therapies, particularly the exploitation of CD123. SL-401 has shown very promising results with manageable toxicities and durable responses and appears to be a viable option for elderly or frail patients who are not eligible for transplant. The other CD123-directed therapies, especially chimeric antigen receptor-therapy (CAR-T), may also give promising results in trials that are currently underway. CAR-T has shown promise in a number of other hematologic malignancies, and toxicities have become more manageable as its use is becoming more widespread. While SL-401 has shown potential to provide durable responses even without transplant, we do not yet know whether it will be effective as a means to avoid transplant in patients who are otherwise eligible. All transplant-eligible patients should undergo alloHSCT consolidation given the current available data indicating this is the optimal approach to achieve a long-term remission. Once the CD123-directed therapies are established as standard regimens, future studies may be designed to investigate whether these therapies can be utilized without the use of transplant. Furthermore, combination therapy using anti-CD123 agents with high-dose induction chemotherapy or other low-dose regimens for elderly/frail patients should be investigated. Given the promising results in early clinical trials, it appears CD123 is the most viable target for BPDCN, and future studies should continue to exploit its expression on BPDCN cells.

Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1.

CD123 (IL-3Rα) is frequently expressed by malignant Hodgkin lymphoma (HL) cells. Naked monoclonal antibodies (mAb) against HL lack clinical benefit, partially due to absence of natural killer (NK) cells in the tumor microenvironment. Here we show that the combination of a fully humanized anti-CD123 mAb (CSL362) and high-affinity Fcγ-receptor NK-92 cells (haNK) effectively target and kill HL cells in vitro. First, we confirmed high expression of CD123 in 2 of the 3 HL cell lines (KM-H2 and L-428), and its absence in NK cells. Cytotoxicity of haNK cells against CD123-positive HL cells was significantly higher in the presence of CSL362. This was also shown with IL-15-activated primary NK cells, although haNK cells showed a 10.87-fold lower estimated half-maximal stimulatory effective concentration (EC50). CSL362 facilitated a significant increase in the expression of CD107a, intracellular IFN-γ and TNF-α and enhanced expression of c-JUN, PLD-1, and ARF6 by NK cells. Inhibition of the ARF6-PLD-1 axis (NAV2729), but not of the MAPK pathway (U0126), completely abrogated CSL362-facilitated antibody-dependent cell-mediated cytotoxicity (ADCC) in haNK and activated primary NK cells. Our results support CD123 as an immunotherapeutic target for HL and the combination of NK cells and CSL362 as a treatment strategy for HL.

Histone deacetylase inhibitor targets CD123/CD47-positive cells and reverse chemoresistance phenotype in acute myeloid leukemia.

Chemoresistance may be due to the survival of leukemia stem cells (LSCs) that are quiescent and not responsive to chemotherapy or lie on the intrinsic or acquired resistance of the specific pool of AML cells. Here, we found, among well-established LSC markers, only CD123 and CD47 are correlated with AML cell chemosensitivities across cell lines and patient samples. Further study reveals that percentages of CD123+CD47+ cells significantly increased in chemoresistant lines compared to parental cell lines. However, stemness signature genes are not significantly increased in resistant cells. Instead, gene changes are enriched in cell cycle and cell survival pathways. This suggests CD123 may serve as a biomarker for chemoresistance, but not stemness of AML cells. We further investigated the role of epigenetic factors in regulating the survival of chemoresistant leukemia cells. Epigenetic drugs, especially histone deacetylase inhibitors (HDACis), effectively induced apoptosis of chemoresistant cells. Furthermore, HDACi Romidepsin largely reversed gene expression profile of resistant cells and efficiently targeted and removed chemoresistant leukemia blasts in xenograft AML mouse model. More interestingly, Romidepsin preferentially targets CD123+ cells, while chemotherapy drug Ara-C mainly targeted fast-growing, CD123- cells. Therefore, Romidepsin alone or in combination with Ara-C may be a potential treatment strategy for chemoresistant patients.

CD123 expression patterns and selective targeting with a CD123-targeted antibody-drug conjugate (IMGN632) in acute lymphoblastic leukemia.

The potential of CD123-targeted therapies in acute lymphoblastic leukemia/lymphoma remains largely unexplored. We examined CD123 expression levels in a large cohort of patients with acute lymphoblastic leukemia/lymphoma and assessed the in vitro impact of IMGN632, a conjugate of CD123-binding antibody with a novel DNA-alkylating payload. CD123 expression on leukemic blasts was surveyed using multicolor/multiparameter flow cytometry. The in vitro effect of IMGN632 was evaluated on B acute lymphoblastic leukemia/lymphoma cell lines and primary B acute lymphoblastic leukemia/lymphoma blasts. The study cohort (n=213) included 183 patients with B acute lymphoblastic leukemia/lymphoma and 30 with T acute lymphoblastic leukemia/lymphoma. CD123 expression was more prevalent in B acute lymphoblastic leukemia/lymphoma than in T acute lymphoblastic leukemia/lymphoma (164/183, 89.6% versus 13/30, 43.3%; P<0.0001), and within B acute lymphoblastic leukemia/lymphoma CD123 expression was more prevalent in Philadelphia chromosome-positive patients than in Philadelphia chromosome-negative patients (96.6% versus 86.3%; P=0.033). In T acute lymphoblastic leukemia/lymphoma, 12/13 (92.3%) patients with CD123-positive blasts had either early T precursor (ETP) or early non-ETP immunophenotype. IMGN632 was highly cytotoxic to B acute lymphoblastic leukemia/lymphoma cell lines, with half maximal inhibitory concentrations (IC50) between 0.6 and 20 pM. In five of eight patients' samples, low picomolar concentrations of IMGN632 eliminated more than 90% of the B acute lymphoblastic leukemia/lymphoma blast population, sparing normal lymphocytes. In conclusion, CD123 expression is prevalent across acute lymphoblastic leukemia/lymphoma subtypes, and the CD123-targeted antibody-drug conjugate IMGN632 demonstrates promising selective activity in preclinical models of B acute lymphoblastic leukemia/lymphoma.

CD16+NK-92 and anti-CD123 monoclonal antibody prolongs survival in primary human acute myeloid leukemia xenografted mice.

Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

Compound CAR T-cells as a double-pronged approach for treating acute myeloid leukemia.

Acute myeloid leukemia (AML) bears heterogeneous cells that can consequently offset killing by single-CAR-based therapy, which results in disease relapse. Leukemic stem cells (LSCs) associated with CD123 expression comprise a rare population that also plays an important role in disease progression and relapse. Here, we report on the robust anti-tumor activity of a compound CAR (cCAR) T-cell possessing discrete scFv domains targeting two different AML antigens, CD123, and CD33, simultaneously. We determined that the resulting cCAR T-cells possessed consistent, potent, and directed cytotoxicity against each target antigen population. Using four leukemia mouse models, we found superior in vivo survival after cCAR treatment. We also designed an alemtuzumab safety-switch that allowed for rapid cCAR therapy termination in vivo. These findings indicate that targeting both CD123 and CD33 on AML cells may be an effective strategy for eliminating both AML bulk disease and LSCs, and potentially prevent relapse due to antigen escape or LSC persistence.