Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis


Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 106 CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3–4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.

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Fig. 1: CAR-NKT cells expand and persist in patients with relapsed or refractory NB.
Fig. 2: CAR-NKT cells infiltrate tumor sites and mediate tumor regression.

Data availability

All requests for raw and analyzed data and materials will be promptly reviewed by the Baylor College of Medicine Licensing Group to verify 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 the clinical trial and might be subject to patient confidentiality restrictions. Any data and materials that can be shared will be released via a material transfer agreement. All raw data for single-cell sequencing have been deposited in the Gene Expression Omnibus with the accession code GSE154037. Source data are provided with this paper.


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The authors are grateful to M. Brenner, H. Heslop and personnel from the cGMP facility at the Center for Cell and Gene Therapy for manufacturing CAR-NKT cells; A. Sher, K. Kukreja and P. Srivaths; staff of the Flow Cytometry Core Laboratory of the Texas Children’s Cancer and Hematology Center; P. Saha for excellent technical assistance; and E. Chomsky, E. Kiner and H. Sharim for help with scRNA-seq. This work was supported by grants and contracts from Alex’s Lemonade Stand Foundation for Childhood Cancer, Kuur Therapeutics (to L.S.M. and A.H.), the American Cancer Society (to A.H.), Cookies for Kids’ Cancer Foundation (to L.S.M.) and the Cancer Prevention and Research Institute of Texas (Baylor College of Medicine Comprehensive Cancer Center Training Program, RP160283, to H.N.).

Author information




A.H. and L.S.M. designed the clinical trial. A.H. wrote the clinical protocol and was the principal investigator of the study. G.D., L.S.M., A.H. and B.S. developed and tested the CAR construct selected for the trial. S.R. wrote CAR-NKT cell manufacturing SOPs. S.R., K.L. and M.L. performed CAR-NKT cell manufacturing. A.N.C., H.N. and N.G. performed flow cytometry, O.D. performed qPCR, A.N.C. performed Luminex assay and B.L. performed Taqman RT–PCR. C.N.C.-F. performed bioenergetics analysis. X.X. performed the serial tumor challenge assay. T.R.-S. and A.M. performed scRNA-seq and analyzed data. The manuscript was written by A.H., A.N.C. and L.S.M. and edited by E.J.D.P. All authors discussed and interpreted the results.

Corresponding authors

Correspondence to Andras Heczey or Leonid S. Metelitsa.

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Competing interests

A.H., A.N.C., G.D. and L.S.M are co-inventors on pending patent applications that relate to the use of NKT cells in cancer immunotherapy and have been licensed by Baylor College of Medicine to Kuur Therapeutics for commercial development. Kuur Therapeutics provided research support for this project (to L.S.M.) via a sponsored research agreement with Baylor College of Medicine. A.M., S.R., K.L., M.L., N.G., O.D., B.L., T.R.-S., C.N.C.-F., X.X., H.N., E.J.D.P. and B.S. declare no competing financial interests.

Additional information

Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Clinical trial design and enrollment.

a, Trial design: Cyclophosphamide/fludarabine (Cy/Flu) chemotherapy is given on days -4, -3, and -2 (purple arrows). CAR-NKTs are infused on day 0 (green arrow). Tumor biopsy is performed at week 2 (blue arrow) and response evaluation with imaging at week 4 (range 4-6; orange arrow). Black arrows indicate clinic visits and peripheral blood analysis. b, Enrollment CONSORT flow diagram.

Extended Data Fig. 2 Patient characteristics.

Indicated variables shown for the three patients enrolled on dose level 1. INSS: International Neuroblastoma Staging System; **Cy: cyclophosphamide, Flu: fludarabine. $: Dose: CAR-NKT cells / m2. Response criteria determined by revised International Neuroblastoma Response Criteria; partial response = elimination of at least 50% of bone metastatic lesions. DOD: Died of disease. AWD: Alive with disease.

Extended Data Fig. 3 Characteristics of GD2-CAR-NKT cell products.

Patient cells were manufactured in the cGMP facility of the Center for Cell and Gene Therapy at Texas Children’s Hospital and evaluated using multi-color flow cytometry. a, NKT frequency (CD3+iNKT+) in initial apheresis product for the three patients. b, NKT purity and CAR expression at the end of manufacturing prior to cryopreservation. Shown are results from one of two independent experiments that produced similar results.

Extended Data Fig. 4 Adverse events.

Patients were monitored for adverse events from the beginning of lymphodepletion (day -4) until day 28 (four weeks post-infusion). Adverse events were described using Common Terminology Criteria for Adverse Events version 4.

Extended Data Fig. 5 Peripheral blood analysis pre- and post-infusion.

Peripheral blood samples were collected at indicated timepoints and a) the frequency of total white blood cells (WBCs), absolute lymphocyte counts (ALC), and absolute monocyte counts (AMC) were quantified in a standard clinical laboratory at the Texas Children’s Cancer Center. b, Frequency of myeloid and lymphoid cell subsets were quantified by flow cytometry staining. Absolute numbers of these subsets in peripheral blood were determined using flow cytometry data and AMC or ALC, respectively. Live PBMCs were gated and cell subsets were defined as shown in Supplementary Fig. 5. For patient 3, analyses of Treg and monocyte subsets were not performed at the week 4 timepoint.

Extended Data Fig. 6 Detection of CAR-NKT cells in treated patients pre- and post-infusion.

Flow cytometry staining showing iNKT+Vβ11+ cells after gating on CD3+ cells and corresponding GD2-CAR expression in patients at indicated timepoints. T-cells served as a negative control for CAR staining. N.D.: not defined; indicates that assay was not performed at given timepoint.

Extended Data Fig. 7 Expression of central memory and WNT pathway genes in pre-infusion patient products.

a, Gene expression in pre-infusion products from patients 1-3 for TNFRSF4 (encodes OX40), SELL (encodes CD62L), and genes related to Wnt-mediated signal transduction ( overlaid on the UMAP 2D projection. b, Cryopreserved patient products were thawed and rested overnight prior to evaluating surface expression of OX40 (CD134) and CD62L (bottom) by flow cytometry using the corresponding fluorochrome-conjugated isotype controls (top). c, LEF1 expression in relation to CD62L was analyzed using intracellular flow cytometry (bottom) with the corresponding PE-conjugated isotype controls (top). Results are from one of two independent experiments with similar results.

Extended Data Fig. 8 Expression of metabolic genes and bioenergetic analysis of pre-infusion patient products.

a, Gene expression in pre-infusion products from patients 1-3 related to glycolysis ( and oxidative phosphorylation ( overlaid on the UMAP 2D projection. b, Extracellular acidification rates (ECAR) and c) oxygen consumption rates (OCR) measured at baseline and in response to glucose (Glc), oligomycin (Oligo), 2-deoxy-D-glucose (2-DG), FCCP, or rotenone/antimycin A (R/A); data shown as mean ± SEM. d, Glycolytic and e) mitochondrial parameters derived from assay in b) and c) respectively; data shown as mean ± SD; P values by two-way ANOVA with Tukey’s correction for multiple comparisons (d,e). Shown are results from two independent experiments with 3 - 6 replicates per experiment. Source data

Extended Data Fig. 9 Serial tumor challenge assay.

a, Patient GD2-CAR NKTs and NB cell line CHLA-255 were co-cultured in a 24-well plate. Five days later, cells were harvested for quantification by trypan-blue exclusion and FACS analysis. NKT cells were then re-plated at a 1:1 effector-to-target ratio with fresh tumor cells in fresh cell culture medium to start the next round with five rounds total. b, Representative flow cytometry analysis after round 4. Summary of c) tumor cell cytotoxicity and d) GD2-CAR NKT proliferation at the end of each five-day cycle. Data shown as mean ± SD from three technical replicates; P values by two-way ANOVA with Tukey’s correction for multiple comparisons (c and d). Source data

Extended Data Fig. 10 Peripheral blood cytokine levels.

Peripheral blood was collected at indicated timepoints from the three patients and cytokine levels were quantified in triplicate by Luminex® assay. a, Cytokine expression is shown as log10 of pg/mL. b, IL7 and c) IL15 levels for the three patients at indicated timepoints. d, Peak IL15 concentration. Data shown as mean ± SD from three technical replicates (b-d); P values by unpaired two-tailed t-test (d). Source data

Supplementary information

Supplementary Information

Supplementary Tables 1, 2 and 4; Supplementary Figs. 1–5; and Clinical Protocol.

Reporting Summary

Supplementary Table 3

Supplementary Table 3

Source data

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

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Heczey, A., Courtney, A.N., Montalbano, A. et al. Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis. Nat Med (2020).

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