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Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results

Nature Medicine volume 29, pages 1379–1388 (2023)Cite this article

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An Author Correction to this article was published on 09 January 2024

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Abstract

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954.

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Fig. 1: GD2-CAR.15 NKTs expand in peripheral blood, traffic to metastatic tumor sites and induce complete remission.
Fig. 2: GD2-CAR.15 NKT in vivo expansion and CD62L+ expression in infusion products correlate with anti-tumor activity in patients.
Fig. 3: BTG1 knockdown enhances GD2-CAR.15 NKT anti-tumor activity.

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Data availability

All requests for raw and analyzed data and materials should be directed to L.S.M. and will be promptly reviewed by the Baylor College of Medicine Licensing Group to verify if 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 may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. All raw data for single-cell sequencing are deposited in the Gene Expression Omnibus under accession number GSE223071. Source data are provided with this paper.

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Acknowledgements

The authors are grateful to M. Brenner, H. Heslop and personnel of the cGMP facility at the Center for Cell and Gene Therapy for manufacturing CAR-NKTs, including A. Jacques, J. Sritabal-Ramirez, H. Hu, K. Matzar and R. Al Hussien. We also thank K. Kukreja of the Interventional Radiology section and P. Srivaths, director of the Pheresis Service, of Texas Children’s Hospital, as well as staff of the Flow Cytometry Core Laboratory of the Texas Children’s Cancer and Hematology Center and the Single Cell Genomics Core at Baylor College of Medicine, for excellent technical assistance. This work was supported by grants or contracts from Alex’s Lemonade Stand Foundation for Childhood Cancer, St. Baldrick’s Foundation, the American Cancer Society (to L.S.M. and A.H.) and Athenex, Inc. We thank the Cancer Prevention and Research Institute of Texas (CPRIT) for support under grant no. RP180785.

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Authors and Affiliations

  1. Department of Pediatrics, Center for Advanced Innate Cell Therapy, Baylor College of Medicine, Houston, TX, USA

    Andras Heczey, Xin Xu, Amy N. Courtney, Gengwen Tian, Gabriel A. Barragan, Linjie Guo, Claudia Martinez Amador, Nisha Ghatwai, Purva Rathi, Michael S. Wood, Yanchuan Li, Chunchao Zhang, Thorsten Demberg, Erica J. Di Pierro & Leonid S. Metelitsa

  2. Department of Medicine, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA

    Andras Heczey, Huimin Zhang, Birju Mehta, Sachin G. Thakkar, Bambi Grilley & Leonid S. Metelitsa

  3. Department of Radiology, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA

    Andrew C. Sher

  4. Biostatistics and Data Management Resource, Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Tao Wang

  5. Department of Pediatrics, Cincinnati Children’s Hospital, Cincinnati, OH, USA

    Brian D. Weiss

  6. Immunai, Inc., New York, NY, USA

    Antonino Montalbano, Meena Subramaniam, Chenling Xu, Chirag Sachar & Daniel K. Wells

  7. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Gianpietro Dotti

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  1. Andras Heczey
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Contributions

A.H. and L.S.M. designed the clinical trial. A.H. wrote the clinical protocol and was the principal investigator of the clinical trial. G.D., L.S.M., A.H., X.X. and G.T. developed the CAR construct selected for the trial and tested it in NKT cells. A.H., A.C.S. and B.D.W. performed pre-therapy and post-therapy patient clinical evaluation. H.Z. and B.M. generated CAR-NKT cell products in the cGMP facility. A.N.C., C.M.A., N.G., S.G.T. and P.R. processed clinical samples and performed flow cytometry, qPCR and Luminex. X.X. performed serial tumor challenge assay. A.M., M.S., C.X., C.S. and D.K.W. performed and analyzed scRNA-seq. C.Z. analyzed bulk RNA-seq. X.X., G.A.B., M.S.W., T.D. and Y.L. tested in vitro BTG1 function in NKT and T cells. X.X. and L.G. performed therapeutic experiments in mice. B.G. and T.W. provided regulatory and statistical support for the clinical trial. The manuscript was written by A.H. and L.S.M. and edited by E.J.D.P. All authors discussed and interpreted results.

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Correspondence to Andras Heczey or Leonid S. Metelitsa.

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Competing interests

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

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Extended data

Extended Data Fig. 1 Consort flow diagram summarizing enrollment on the GINAKIT2 study.

Enrollment is conducted in two phases: procurement (manufacturing of the cell product) and treatment (infusion of the cell product). The study is a single arm, Phase 1 clinical trial and patients were enrolled on a first come/first served basis. Received allocated intervention on DL1: 3, DL2: 3, DL3: 3 and DL4 4 patients.

Extended Data Fig. 2 Serum cytokine and chemokine levels in patients infused with GD2-CAR.15 NKTs.

A. Mean fold change (FC) compared to baseline (day −4) of 38 evaluated cytokines and chemokines in patient peripheral blood samples collected at indicated timepoints quantified by Luminex. B. Absolute values (pg/mL) of IL1, IL6 and TNFbeta over four weeks in all patients.

Extended Data Fig. 3 Frequency and severity of adverse events following first and second GD2-CAR.15 NKT infusions.

Toxicity parameters were assessed and graded according to the Common Terminology Criteria for Adverse Events version 4 from the start of lymphodepletion (day −4) until four weeks after each infusion (day 28). n = 4 patients. Data presented as mean +/− SD.

Source data

Extended Data Fig. 4 Antitumor response in patient 12.

Pre- and post-GD2-CAR NKT infusion images from MIBG scans. Planar images (top row) and fused SPECT-CT images (bottom row) show pelvic bone metastasis (red circle) visible prior to infusion (left column) and complete resolution after infusion (right column). Note: MIBG-avid regions in the middle of the image correspond to bladder with urine containing MIBG.

Extended Data Fig. 5 Long-term expansion and persistence of GD2-CAR.15 NKTs in patients with relapsed or refractory neuroblastoma.

A. Peripheral blood (PB) samples were collected at indicated timepoints and the CAR transgene was detected by quantitative PCR. Patients 10 and 12 were reinfused at approximately week 8 (W8); patients 2 and 11 were reinfused at approximately three months (M3). B. AUC of peripheral blood CAR-NKT absolute numbers in four patients receiving repeat infusions. Two-tailed paired T test.

Extended Data Fig. 6 BTG1 expression is elevated in exhausted CAR-NKTs.

A. Cytotoxicity of CAR-NKTs from patient pre-infusion products against CHLA255 NB cells over the course of five repeat co-culture cycles. B. UMAP projections of gene expression profiles measured by scRNAseq from pre-infusion products (IP) and CAR-NKTs following five-cycle repeat co-culture with tumor cells (5RcC). C. UMAP projection of gene expression in CAR-NKTs from pre-infusion products (IP), post-5RcC (5RcC), and isolated from peripheral blood (PB) post-infusion. D, E. Volcano plots showing differentially expressed genes in pre-infusion products versus peripheral blood CAR-NKTs (D) and versus post-5RcC CAR-NKTs (E).

Extended Data Fig. 7 Overexpression (OE) of BTG1 reduces global RNA expression and proliferative capacity in NKTs.

A. Total RNAseq reads from BTG1-OE and control NKTs. Red and blue lines indicate fold change from nuclear or mitochondrial transcripts, respectively. B. Volcano plot showing differentially expressed genes in BTG1-OE NKTs versus control NKTs. Blue vertical lines correspond to two-fold change on log2 scale; green dashed and solid lines represent p < 0.05 and <0.01, respectively. C. Pathway enrichment analysis showing gene expression programs enriched in BTG1-OE NKTs. Fisher’s exact test was used to calculate the probability of observing overlap between the input gene set and the pathway or gene set database by chance. P-values were adjusted for multiple testing using the Benjamini-Hochberg method to control for false discovery rate. D. Fold expansion of BTG1-OE vs control NKTs, n = 6 donors, two-tailed paired T test. E, F. Survival of NKTs stimulated with anti-CD3/CD28 monoclonal antibodies and evaluated with annexin V and fixable viability dye e780 on day 3, n = 4 (E) and CellTrace Violet dilution measured on day 5, n = 3 (F), (E-F data presented as mean +/− SD: two-tailed paired T test).

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Heczey, A., Xu, X., Courtney, A.N. et al. Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results. Nat Med 29, 1379–1388 (2023). https://doi.org/10.1038/s41591-023-02363-y

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