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Cytokine-mediated CAR T therapy resistance in AML

Nature Medicine volume 30, pages 3697–3708 (2024)Cite this article

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Abstract

Acute myeloid leukemia (AML) is a rapidly progressive malignancy without effective therapies for refractory disease. So far, chimeric antigen receptor (CAR) T cell therapy in AML has not recapitulated the efficacy seen in B cell malignancies. Here we report a pilot study of autologous anti-CD123 CAR T cells in 12 adults with relapsed or refractory AML. CAR T cells targeting CD123+ cells were successfully manufactured in 90.4% of runs. Cytokine release syndrome was observed in 10 of 12 infused individuals (83.3%, 90% confidence interval 0.5–0.97). Three individuals achieved clinical response (25%, 90% confidence interval 0.07–0.53). We found that myeloid-supporting cytokines are secreted during cell therapy and support AML blast survival via kinase signaling, leading to CAR T cell exhaustion. The prosurvival effect of therapy-induced cytokines presents a unique resistance mechanism in AML that is distinct from any observed in B cell malignancies. Our findings suggest that autologous CART manufacturing is feasible in AML, but treatment is associated with high rates of cytokine release syndrome and relatively poor clinical efficacy. Combining CAR T cell therapies with cytokine signaling inhibitors could enhance immunotherapy efficacy in AML and achieve improved outcomes (ClinicalTrials.gov identifier: NCT03766126).

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Fig. 1: Pilot clinical study of CART-123 results in brief responses in patients with relapsed or refractory AML.
Fig. 2: Cytokines promote AML resistance to CAR T cells.
Fig. 3: AML exposure to cytokines results in CART-123 exhaustion.
Fig. 4: Targeting cytokine-induced STAT5 signaling restores AML sensitivity to CART-123.

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Data availability

All requests for raw and analyzed data and materials are promptly reviewed by the University of Pennsylvania Center for Innovation to see whether the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical trials and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a Material Transfer Agreement. Raw sequencing data are deposited in the National Center for Biotechnology Information Database of Genotypes and Phenotypes, with accession number phs001707.v4. Source data are provided with this paper.

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Acknowledgements

We thank the patients who participated in this study and their families. This work was supported by NIH P01CA214278-05 (to W.-T.H., D.L.P., S.I.G. and C.H.J.), a Commonwealth of Pennsylvania Research Formula Fund no. 585499 (to S.I.G.), Leukemia and Lymphoma Society Blood Cancer Discovery grant (to S.I.G.); NIH 5T32HD043021 (supporting A.S.B.), as well as grant 2023-0221 from the Doris Duke Foundation (to A.S.B.) and a Fellowship Grant Award with generous support by JJ’s Angels from the St. Baldrick’s Foundation (to A.S.B.). The clinical trial was funded by Novartis Pharmaceuticals. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Anand S. Bhagwat, Han R. Fisher, Saamia N. Farooki & Richard Aplenc

  2. Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Anand S. Bhagwat, Leonel Torres, Olga Shestova, Maksim Shestov, Patrick W. Mellors, Benjamin F. Frost, Avery L. Gaymon, Diane Frazee, Walter Rogal, Carl H. June & Saar I. Gill

  3. Immunology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Leonel Torres

  4. Hematologics Inc., Seattle, WA, USA

    Michael R. Loken

  5. Division of Hematology–Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Noelle Frey, Elizabeth O. Hexner, Selina M. Luger, Alison W. Loren, Mary Ellen Martin, Shannon R. McCurdy, Alexander E. Perl, Edward A. Stadtmauer, David L. Porter & Saar I. Gill

  6. Center for Cell Therapy and Transplant, University of Pennsylvania, Philadelphia, PA, USA

    Noelle Frey, Elizabeth O. Hexner, Alison W. Loren, Mary Ellen Martin, Shannon R. McCurdy, Alexander E. Perl, Edward A. Stadtmauer, David L. Porter & Saar I. Gill

  7. Novartis Biomedical Research, Cambridge, MA, USA

    Jennifer L. Brogdon

  8. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Joseph A. Fraietta

  9. Department of Biostatistics, Epidemiology, and Informatics, University of Pennsylvania, Philadelphia, PA, USA

    Wei-Ting Hwang

  10. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Don L. Siegel, Gabriela Plesa & Carl H. June

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  1. Anand S. Bhagwat
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  29. Saar I. Gill
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Contributions

A.S.B. and S.I.G. conceptualized the project, analyzed the data and wrote the paper. A.S.B., L.T., O.S., M.S., H.R.F., S.N.F., M.R.L. and B.F.F. carried out the translational experiments. M.R.L. performed antigen quantification on patient marrow samples. A.S.B., L.T., M.S., P.W.M. and B.F.F. visualized the data. S.I.G. supervised the project. W.-T.H. provided statistical guidance and analysis. C.H.J., J.A.F., J.L.B., D.L.S., W.-T.H. and D.L.P. edited the paper. D.L.S., A.L.G., D.F., W.R., N.F., E.O.H., S.M.L., A.W.L., M.E.M., S.R.M., A.E.P., E.A.S., J.L.B., J.A.F., W.-T.H., G.P. and S.I.G. participated in and executed the clinical trial, including CAR T cell manufacturing. R.A. supervised serum proteomic analysis.

Corresponding author

Correspondence to Saar I. Gill.

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Competing interests

D.L.P. declares funding from the National Marrow Donor Program; membership on an entity’s Board of Directors or advisory committees of Kite/Gilead, Janssen, Genentech, DeCart, Sana Biotechnology, Verismo and Novartis; is a current equity holder of the American Society for Transplantation and Cellular Therapy and Verismo; declares honoraria for Incyte; and has patents and royalties in Tmunity and Wiley and Sons Publishing. J.A.F. is a member of the scientific advisory boards of Cartography Bio and Shennon Biotechnologies and has patents, royalties and other intellectual property. M.R.L. is an employee of Hematoloics, Inc. J.L.B. is an employee of Novartis. C.H.J. is an inventor of patents related to CAR therapy products and may be eligible to receive a select portion of royalties paid from Kite to the University of Pennsylvania. C.H.J. is a scientific cofounder and holds equity in Capstan Therapeutics, Dispatch Biotherapeutics and Bluewhale Bio. C.H.J. serves on the board of AC Immune and is a scientific advisor to BlueSphere Bio, Cabaletta, Carisma, Cartography, Cellares, Cellcarta, Celldex, Danaher, Decheng, ImmuneSensor, Kite, Poseida, Verismo, Viracta and WIRB-Copernicus group. S.I.G. has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. S.I.G. is a scientific cofounder and holds equity in Interius Biotherapeutics and Carisma Therapeutics. S.I.G. is a scientific advisor to Carisma, Cartography, Currus, Interius, Kite, NKILT and Mission Bio. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Patient responses and correlative data.

a, Flow cytometric measurable residual disease (MRD) in bone marrow aspirates using the different-from-normal (DfN) method. b, Copy number of CAR-123 per microgram of genomic DNA as measured by qPCR at indicated time points for each patient. c, Flow cytometry quantification of surface CD123 molecules on AML blasts of each patient (using “difference from normal” approach) at the indicated time points. d, Serial serum cytokine levels as measured serially in subjects on study via clinical Luminex panel. Data presented in log scale, normalized to the first time point ( ~ 1 week prior to initiation of LD chemo). Light red lines represent values from individual subjects, dark red line represents the mean of all patients at each time point for which >1 patient had a detectable value. e, IL-3 ELISA to complement Olink data, comparing clinical subjects’ baseline serum IL-3 level to “peak” IL-3 level. Statistical analysis performed using Welch’s two-tailed t-test.

Extended Data Fig. 2 Responder and non-responder CART-123 infusion products are similar.

a, Percentage of CD4 + CAR+ (left) or CD8 + CAR+ (right) populations expressing the indicated activation markers. b, Dot plot of t-distributed stochastic neighbor embedding (t-SNE) of CAR T products from all available patients (left). Distributions of cells from individual patients are labeled with patient ID (right). Red plots indicate initial partial (patient 10) or complete (patients 5 and 13) responses defined by day 14 bone marrow aspirate/biopsy. Blue plots indicate stable progressive disease on day 14 marrows. All analyzed patients are represented in two main populations. c, Percentage of CD4 + CAR+ (left) or CD8 + CAR+ (right) populations designated as naïve or stem cell memory (TN/TSCM), central memory (TCM), effector memory (TEM), or terminally differentiated effector memory (TEMRA) by CD45RA and CCR7 expression. d, Normal donor CART-123 (two independent replicates, black bars) and left-over infusion product from the indicated clinical trial subjects were incubated with an AML cell line (MOLM14) at a 1:5 effector:target ratio for 5 days. T cells were counted, and proliferation is represented as the fold-change in the MOLM14-stimulated CAR T cells compared with no stimulation control. Normal donor replicates are shown in black. Two of three subjects with early marrow blast reduction (UPN10, UPN13) were available and are shown in green. Four subjects with no change in marrow blasts (UPN 1, 12, 16, 20) are shown in yellow (note that UPN 1 blasts were 0% prior to CART-123 infusion and 0% at day 28 after CART-123 infusion). Five subjects with increase in marrow or blood blasts (UPN 2, 8, 9, 21 and 22) are shown in red. e, Correlation of fold expansion in vitro of subjects’ infusion products with their peak VCN when measured after infusion (expressed as copies/ug gDNA). Each circle represents an individual subject. Pearson r = -0.19, P = 0.57.

Extended Data Fig. 3 Cytokines selectively undermine myeloid-directed cell therapy by supporting AML cell survival.

a, Number of live primary AML or primary ALL cells in culture after 7 days in serum free medium with the addition of G-CSF. Data shown as live, non-apoptotic leukemia cells relative to number of similar cells cultured in serum-free medium for each independent experiment. Data presented as mean +/- SEM. n = 8 independent experiments involving 6 independent AML samples, and n = 5 independent ALL samples. b, Flow cytometry analysis of CD45 and CD34 expression on primary AML sample cultured in serum-free medium supplemented with PBS control or GM-CSF (1 ng/mL) for one week. Data are representative of at least two independent AML samples. CD45-dim / CD34+ cells are gated as “blasts”. c-e, Quantification of proportion of cells expressing the indicated cell surface markers by flow cytometry analysis of primary AML samples cultured in serum-free medium supplemented with PBS control or GM-CSF (1 ng/mL) for one week. Data presented as mean +/- SEM of two independent AML samples. f, Expression of cell-surface CD-123 on primary AML sample. Data representative of multiple independent AML samples. g, Count of live AML cells after 96 hours co-culture with CART-123 at an E:T ratio of 1:1. n = 8 independent experiments involving 4 distinct primary AML samples and 4 CART-123 donors. h, Count of live AML cells after 96 hours co-culture with CART-123 at an E:T ratio of 1:16. n = 13 independent experiments involving 7 primary AML samples and 5 CART-123 donors, presented as mean +/- SEM. i, Count of live AML cells after 96 hours co-culture with CART-123 at an E:T ratio of 1:4. GM-CSF was washed out of media prior to addition of CART-123 cells, and remainder of experiment conducted in serum-free and supplement-free media. Data normalized to AML cell count in PBS control condition for each experiment, and presented as mean +/- SEM. n = 7 independent experiments using 3 distinct primary AML samples. j, Count of live AML cells after 96 hours co-culture with CART-33 at E:T ratio of 1:4 with indicated cytokine supplement. n = 3 independent experiments involving a primary AML sample. Statistical analysis for g-j performed on independent experiments by two-tailed Wilcoxon signed rank test for g-j. k, Number of live CART-123 cells in serum-free medium supplemented with PBS control or GM-CSF for four days. Data presented as mean +/- SEM, n = 9 including 5 individual T cell donors, and statistical analysis performed by two-tailed student’s t-test. l-n, As in a, but depicting number of live CART-123 cells in indicated culture conditions after four days’ co-culture with AML at indicated E:T ratios. Data presented as mean +/- SEM, n = 8 for l, n = 9 for m, n = 9 for n, including 5 individual T cell donors, and statistical analysis performed by student’s t-test. o, CellTrace Violet measurement of proliferation of CART-123 cells upon exposure to AML cells at the indicated E:T ratio in the presence of PBS control or GM-CSF. Results representative of at 5 individual T cell donors. p-q, Count of indicated live primary ALL sample after 96 hours of co-culture with CART-123 at indicated E:T ratios. ALL cells were primed with 48 hours of cytokine (or control PBS) addition, as in Fig. 2h-j. Two independent ALL patient samples are presented, with individual dots indicating technical replicates, presented as mean +/- SEM.

Source data

Extended Data Fig. 4 CART-123 dysfunction caused by inability to clear AML antigen.

a, Flow cytometry analysis of expression of additional inhibitory receptors CD38 and CD39 (top) and CTLA4 and CD94 (bottom). Representative of two experiments with different primary AML and CART-123 donors, complements distinct donors shown in Fig. 3. b, Intracellular expression of TCF-1 in the indicated CART-123 + T cell subsets, measured by flow cytometry. MFI displayed to the right. Representative of two experiments with different primary AML and CART-123 donors complements distinct donors shown in Fig. 3. See also, Supplementary Fig. 3. c, Flow cytometry analysis of live single cells depicting residual CD3- (presumed AML cells) after 2 weeks of co-culture. d, Intracellular cytokine analysis of IFN\(\gamma\) and TNF\(a\) in two different CART-123 donors after 3 weeks of UPA or CPA co-culture. e, Surface expression of inhibitory receptor ligands on two distinct primary AML samples after one week in co-culture with CART-123 cells. f, UMAP plots of SCFV-expressing, CAR + T cells recovered from patients at time points at least 10 days after infusion. On the left coloration is determined by patient response, and on the subsequent 3 the indicated “gene module score” is overlaid.

Extended Data Fig. 5 Cytokines activate STAT5-mediated survival signaling in AML blasts.

a, PathfindR analysis of selected KEGG pathways in primary AML transcriptomic analysis. Primary AML was exposed to PBS or GM-CSF (1 ng/mL) for 48 hours before RNA was processed from single cells. Blue bars highlight signaling pathways, while red bars highlight pathways relevant to observed phenotypes. b, Phospho-flow cytometry analysis of pSTAT3 in primary AML treated for 30 minutes with the indicated supplement to serum-free medium. “Baseline” serum refers to pre-LD chemotherapy serum sample, “peak” serum refers to serum drawn during peak T cell expansion, as in Fig. 2a. Statistical analysis performed with a two-sided unpaired t test. c, Phospho-flow cytometry analysis of pSTAT5 and pSTAT3 in primary AML samples treated with 30 minutes of PBS, 1 ng/mL GM-CSF, 10 ng/mL FLT3L, or 50 ng/mL IL-6. d, Western blot of total STAT5 and pSTAT5 in primary AML cells treated for one hour with PBS control or 1 ng/mL GMCSF. Shown is a representative blot consistent with sample across two independent AML donors. e, Number of live primary AML in culture after 7 days in serum free medium with the addition of PBS or GM-CSF and DMSO or 300 nM Ruxolitinib. Data shown relative to number of live cells serum free medium for each experiment. Lines represent mean. Statistical analysis performed by two-tailed Wilcoxon signed-rank test, n = 6 for PBS Rux condition, n = 9 for all other conditions. f, Number of live primary AML cells after 96 hours’ co-culture with CART-123 at an E:T ratio of 1:4, normalized to PBS + DMSO condition for each experiment. Data presented as mean +/- SEM of n = 3 replicates of 2 independent primary AML samples and 2 distinct CART-123 donors.

Source data

Extended Data Table 1 Treatment and Response
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Extended Data Table 2 Adverse Events
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Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–6.

Reporting Summary

Supplementary Data 1

Study protocol.

Source data

Source Data Figs. 1–4 and Extended Data Figs. 3 and 5

Statistical source data for Figs. 1–4 and Extended Data Figs. 3 and 5.

Source Data Fig. 4 and Extended Data Fig. 5

Unprocessed western blots for Fig. 4 and Extended Data Fig. 5.

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Bhagwat, A.S., Torres, L., Shestova, O. et al. Cytokine-mediated CAR T therapy resistance in AML. Nat Med 30, 3697–3708 (2024). https://doi.org/10.1038/s41591-024-03271-5

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