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

Successful 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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All studies must disclose on these points even when the disclosure is negative. Antibodies Antibodies used dataview.ncbi.nlm.nih.gov/object/PRJNA766490?reviewer=jvclq763e4cg7bptp553ikjbks), respectively. All other processed data generated in this study as depicted in the main and supplementary figures are provided as a source data file.
For the sample size throughout vivo experiments a sample size of at least n=5 per group was used to allow for accurate statistical analyses and comparisons across groups . For In Vitro experiments, a sample size of at least n=3 was used to allow for proper statistcal analyses of the data.
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All the experimental findings were reliably reproduced. All experiments were performed independently at least 3 times with each condition set up with at least technical duplicates.
For In vivo experiments, mice were first imaged using BLI to confirm engraftment. Thereafter, mice with similar engraftment levels were randomized into the experimental treatment groups. All samples or mice were included in our analysis.
The experiments were performed in a non-blinded manner. Only tissue damage histological scoring was done in a blinded setting. In order to obtain unbiased data, the histopathological scoring and analysis was performed by a pathologist blinded to the treatment groups. Only after finalization of the quantitative scores, the samples were allocated to their designated groups. These information are also provided in the supplementary methods. All antibodies were used at a dilution of 1:100 except for Protein-L which was used at a conc. of 1ug/mL and streptavidin-PE was used at 1:20 dilution.
Antibodies were validated by titration experiments where the base line was the recommended working concentration provided by the manufacturer. In most cases, the manufacturer always recommended a dilution of 1:20. Validation was performed per experimental setup, and cell type that was used.
Human The cell lines used in this study were obtained from a batch that had been prevoiusly authenticated at DSMZ, Germany using PCR assays with species-specific primers.
The cell lines used in this study were obtained from a batch that had been prevoiusly tested for Mycoplasma contamination and were found to be negative. Cell lines were subsequently routinely tested for mycoplasma contamination.
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October 2018
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Sample preparation Rag2-/-Il2r"-/-immunocompromised mice were bred in-house at the University clinic (University of Freiburg) animal facility under standard room temperatures (~26oC) with a 12 h light/dark cycle and humidity ranging 40-60%. Mice were housed in individually ventilated cages and received acidified and autoclaved water. Male and female mice were used between 8-10 weeks of age. Mice were bred and housed under specific pathogen-free (SPF) conditions in the animal facility of University Medical centre Freiburg (ZKF).
NSG (NOD-scid IL2r"null) mice and humanized cytokine knock-in mice (CSF1h/hIL-3/CSF2h/hhSIRPAtgTPOh/hRag2-/-IL2r"-/-), also known as MISTRG-SKI, were bred and maintained at the University Hospital Zürich animal facility according to the Swiss Federal Veterinary Office guidelines and the Cantonal Veterinary Office Zürich. Mice were housed in similar conditions as stated above.
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The study did not involve wild animals.
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All mouse experiments were approved by the Federal Ministry for Nature, Environment and Consumers' Protection of the state of Baden-Württemberg, Germany (Protocol numbers: G-18/019) or the Cantonal Veterinary Office Zürich (194/2018).
Human samples used in the study included patients with acute myeloid leukemia (AML) that were collected at diagnosis or had relapsed/refractory disease following transplantation (Australia and Germany). Samples were obtained following written consent. Healthy Donor BM (Australia) samples were obtained following written consent from the donors. Samples were obtained from either female or males ranging who were at least of legal age (>18 years old).
Further details of the AML patients are supplied in the supplementary materials.
Samples from patients of interest, who acquired written informed consent at University Medical Center Freiburg and SAHMRI, Adelaide, Australia were only used. for the AML patients, written consent was obtained to take extra material during standard diagnostics testing. No other specific criteria was used for the selection of the AML samples. For the healthy donors, specimens were taken on a complete voluntary basis following the written consent. No selection criteria other than legal age was used.
Human sample collection and analysis were approved by the Institutional Ethics Review Board of the Medical center, University of Freiburg, Germany (protocol number: 509/16) and the Australian Institutional Human Research Ethics Committee (R20150526, HREC/15/RAH/221) and and the Cantonal Ethics Board Zürich, Switzerland (Ethics approval no's: 2009-0062). Written informed consent was obtained from each patient. All analysis of human data was carried out in compliance with relevant ethical regulations Cells analysed by flow cytometry were either fresh mouse bone marrow, spleen, or peripheral blood or PBMCs or BMMCs derived from healthy donor or AML patient samples. blood was first processed using density gradient centrifugation using Lymphoprep or Panoll. Then, based on the experimental set up, cells were harvested according to the cell type, counted using a haemocytometer to examine cell count. Cell viability was analysed using the LIVE/DEAD fixable dead cell stain kit (molecular Probes) or 7-AAD (BD Biosciences). Following 20min incubation on ice, the cells were washed twice in FACs buffer. For cells