Driving CAR T Stem Cell Targeting in Acute Myeloid Leukemia: The Roads to Success

Current treatment outcome for acute myeloid leukemia (AML) patients is unsatisfactory and characterized by high rates of relapse and poor overall survival. Increasing evidence points to a crucial role of leukemic stem cells (LSC) and the bone marrow (BM) leukemic niche, in which they reside, in AML evolution and chemoresistance. Thus, future strategies aiming at improving AML therapeutic protocols are likely to be directed against LSC and their niche. Chimeric antigen receptor (CAR) T-cells have been extremely successful in the treatment of relapsed/refractory acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma and comparable results in AML are highly desirable. At present, we are at the dawn of CAR T-cell application in AML, with several preclinical studies and few early phase clinical trials. However, the lack of leukemia-specific targets and the genetic and phenotypic heterogeneity of the disease combined with the leukemia-induced remodeling of the BM microenvironment are limiting CAR T-cell exploitation in AML. Here, we reviewed AML-LSC and AML-BM niche features in the context of their therapeutic targeting using CAR T-cells. We summarized recent progress in CAR T-cell application to the treatment of AML, and we discussed the remaining therapeutic challenges and promising novel strategies to overcome them.

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Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia

Nature Communications ◽

10.1038/s41467-021-26683-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nadia El Khawanky ◽

Amy Hughes ◽

Wenbo Yu ◽

Renier Myburgh ◽

Tony Matschulla ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Intracellular Signaling ◽

Myeloid Leukemia ◽

Epithelial Tissue ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Acute Myeloid

AbstractSuccessful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5′-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

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CD33-Directed Chimeric Antigen Receptor (CAR) T Cells for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v128.22.2825.2825 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2825-2825 ◽

Cited By ~ 4

Author(s):

Sarwish Rafiq ◽

Terence J. Purdon ◽

Liora M. Schultz ◽

Renier J. Brentjens

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Myeloid Leukemia ◽

Term Survival ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Acute Myeloid

Abstract Therapies for acute myeloid leukemia (AML) have not improved patient long-term survival for decades. Novel treatment options, such as chimeric antigen receptor (CAR) T cells, are needed for patients with this disease. However, AML cells lack ideal targeting antigens that are safe to target with CAR T cells. CD33, a commonly targeted antigen, is expressed in about 85-90% of AML cases but is also present on normal myeloid progenitors and myelocytes. Our aim was to engineer and validate CD33-directed CAR T cells, with the intention to open a phase I clinical trial in patients with relapsed AML. Given the potential toxicity associated with targeting CD33 in patients, an elimination gene was included in the construct design to allow CAR T cell clearance after disease eradication. We constructed a CAR specific for CD33 by utilizing the variable heavy and light chains of the humanized M-195 antibody (HuM-195), CD28 and zeta signaling domains, and the IL-12 gene. To facilitate CAR T cell elimination if necessary, a truncated epidermal growth factor receptor (EGFRt) gene was also inserted into the retroviral vector to create the EGFRt/HuM195-28z/IL-12 CAR construct. Retroviral transduction with this tri-cistronic construct resulted in high transduction efficiency of human T cells, as determined by detection of EGFRt with fluorescently-labeled cetuximab or CAR with fluorescently-labeled CD33 molecule by flow cytometry. T cells transduced with the EGFRt/HuM195-28z/IL-12 CAR secreted functional IL-12, as assessed by culturing CAR cell supernatant with peripheral blood mononuclear cells and detecting IFN-g produced in response to IL-12. When stimulated through the CAR by co-culturing with the CD33+ AML cell line Molm-13, EGFRt/HuM195-28z/IL-12 CAR T cells proliferated and produced significant levels of the pro-inflammatory cytokines IFN-g and IL-2. In addition, EGFRt/HuM195-28z/IL-12 CAR T cells mediated significantly cytotoxicity against Molm-13 at a range of effector-to-target ratios in standard 51Cr-release assays, as compared to control CAR T cells. The in vivo anti-tumor efficacy of EGFRt/HuM195-28z/IL-12 CAR T cells was tested in two preclinical mouse models of AML. First, SCID/Beige mice were xenografted with Molm-13 cells. Mice subsequently treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited significant long-term survival, as compared to untreated or control CAR T cell-treated mice (p = <0.0001). In addition, NSG mice were engrafted with patient-derived CD33+ AML (PDX) cells. PDX mice treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited reduction of peripheral CD33+ disease, as compared to untreated or control CAR T cell-treated mice. These studies demonstrate the capacity of the EGFRt/HuM195-28z/IL-12 CAR to redirect the specific anti-tumor function of T cells to CD33+ AML tumor cells. Ongoing studies aim to validate elimination of CAR T cells via EGFRt in vitro in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, as well as in mice are currently underway. These data support the utilization of CD33-specific CAR T cells in the clinic for patients with relapsed AML as a means to decrease disease burden prior to consolidative therapies such as allogeneic transplantation. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Acute myeloid leukemia chimeric antigen receptor T-cell immunotherapy: how far up the road have we traveled?

Therapeutic Advances in Hematology ◽

10.1177/2040620718774268 ◽

2018 ◽

Vol 9 (6) ◽

pp. 135-148 ◽

Cited By ~ 29

Author(s):

Sarah K Tasian

Keyword(s):

Acute Myeloid Leukemia ◽

T Cell ◽

Chimeric Antigen Receptor ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Antigen Receptor ◽

Successful Implementation ◽

Car T Cell ◽

Car T ◽

Acute Myeloid

Chemotherapy resistance and relapse remain significant sources of mortality for children and adults with acute myeloid leukemia (AML). Further intensification of conventional cytotoxic chemotherapy is likely not feasible due to the severity of acute and long-term side effects upon normal tissues commonly induced by these drugs. Successful development and implementation of new precision medicine treatment approaches for patients with AML, which may improve leukemia remission and diminish toxicity, is thus a major priority. Tumor antigen-redirected chimeric antigen receptor (CAR) T-cell immunotherapies have induced remarkable responses in patients with relapsed or chemorefractory B-lymphoblastic leukemia, and similar strategies are now under early clinical study in adults with relapsed/refractory AML. However, potential on target/off tumor toxicity of AML CAR T-cell immunotherapies, notably aplasia of normal myeloid cells, may limit broader implementation of such approaches. Careful selection of optimal target antigens, consideration of toxicity mitigation strategies, and development of methodologies to circumvent potential CAR T-cell resistance are essential for successful implementation of cellular immunotherapies for patients with high-risk AML.

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Harnessing T Cells to Target Pediatric Acute Myeloid Leukemia: CARs, BiTEs, and Beyond

Children ◽

10.3390/children7020014 ◽

2020 ◽

Vol 7 (2) ◽

pp. 14

Author(s):

Rebecca Epperly ◽

Stephen Gottschalk ◽

Mireya Paulina Velasquez

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Targeted Therapies ◽

Myeloid Leukemia ◽

Immune Therapy ◽

Bispecific Antibodies ◽

Car T Cells ◽

Car T ◽

Acute Myeloid

Outcomes for pediatric patients with acute myeloid leukemia (AML) remain poor, highlighting the need for improved targeted therapies. Building on the success of CD19-directed immune therapy for acute lymphocytic leukemia (ALL), efforts are ongoing to develop similar strategies for AML. Identifying target antigens for AML is challenging because of the high expression overlap in hematopoietic cells and normal tissues. Despite this, CD123 and CD33 antigen targeted therapies, among others, have emerged as promising candidates. In this review we focus on AML-specific T cell engaging bispecific antibodies and chimeric antigen receptor (CAR) T cells. We review antigens being explored for T cell-based immunotherapy in AML, describe the landscape of clinical trials upcoming for bispecific antibodies and CAR T cells, and highlight strategies to overcome additional challenges facing translation of T cell-based immunotherapy for AML.

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Modulating TNFα activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001229 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001229

Author(s):

Pinar Ataca Atilla ◽

Mary K McKenna ◽

Haruko Tashiro ◽

Madhuwanti Srinivasan ◽

Feiyan Mo ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Acute Toxicity ◽

Myeloid Leukemia ◽

Tnf Alpha ◽

Car T Cells ◽

Car T ◽

Anti Tumor Activity ◽

Acute Myeloid

BackgroundC-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor (CAR) T-cell therapy. Here, we optimized our CLL-1 CAR for anti-leukemic activity in mouse xenograft models of aggressive AML.MethodsFirst, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vector to coexpress transgenic IL15. We measured the effects of each construct on T cell phenotype and sequential (recursive) co culture assays with tumor cell targets to determine the durability of the anti tumor activity by flow cytometry. We administered CAR T cells to mice engrafted with patient derived xenografts (PDX) and AML cell line and determined anti tumor activity by bioluminescence imaging and weekly bleeding, measured serum cytokines by multiplex analysis. After euthanasia, we examined formalin-fixed/paraffin embedded sections. Unpaired two-tailed Student’s t-tests were used and values of p<0.05 were considered significant. Survival was calculated using Mantel-Cox log-rank test.ResultsIn vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (p<0.0001) and had superior expansion compared with CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CLL-1 CAR T coexpressing transgenic IL15 initially expanded better than CD28z-CD8 CAR T without IL15 (p<0.0001), but produced severe acute toxicity associated with high level production of human tumor necrosis factor α (TNFα), IL15 and IL2. Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by two strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody, while excessive T cell expansion could be arrested by activation of an inducible caspase nine safety switch by administration of dimerizing drug. Both strategies successfully prolonged tumor-free survival.ConclusionCombinatorial treatment with a TNFα blocking antibody and subsequent activation of the caspase-9 control switch increased the expansion, survival and antileukemic potency of CLL-1 CAR T-cells expressing transgenic IL15 while avoiding the toxicities associated with excessive cytokine production and long-term accumulation of activated T-cells.

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CD70-specific CAR T-cells have potent activity against Acute Myeloid Leukemia (AML) without HSC toxicity

Blood ◽

10.1182/blood.2020008221 ◽

2021 ◽

Author(s):

Tim Sauer ◽

Kathan Parikh ◽

Sandhya Sharma ◽

Bilal Omer ◽

David Nikolov Sedloev ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Myeloid Leukemia ◽

Target Antigen ◽

Single Chain ◽

Hematopoietic Stem ◽

Car T Cells ◽

Car T ◽

Acute Myeloid

The prognosis of patients with acute myeloid leukemia (AML) remains dismal highlighting the need for novel innovative treatment strategies. The application of chimeric antigen receptor (CAR) T-cell therapy to AML patients has been limited in particular by the lack of a tumor-specific target antigen. CD70 is a promising antigen to target AML as it is expressed on the majority of leukemic blasts, whereas little or no expression is detectable in normal bone marrow samples. To target CD70 on AML cells, we generated a panel of CD70-CARs that contained a common single chain variable fragment (scFv) for antigen detection but differed in size and flexibility of the extracellular spacer, and in the transmembrane and the co-stimulatory domains. These CD70scFv CARs were compared with a CAR construct that contained the human CD27, the ligand of CD70 fused to the CD3z chain (CD27z). The structural composition of the CAR strongly influenced expression levels, viability, expansion and cytotoxic capacities of CD70scFv CAR T-cells, but the CD27z-CAR T-cells demonstrated superior proliferation and anti-tumor activity in vitro and in vivo, compared to all CD70scFv-CARs. While CD70-CAR T-cells recognized activated virus-specific T-cells (VSTs) that expressed CD70, they did not prevent colony formation by normal hematopoietic stem cells (HSCs). Thus, CD70-targeted immunotherapy is a promising new treatment strategy for patients with CD70-positive AML that does not affect normal hematopoiesis but will require monitoring of virus-specific T-cell responses.

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