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Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial

Nature Cancer volume 5, pages 517–531 (2024)Cite this article

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Abstract

We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII+ glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9–6.0 months) and median overall survival (11.8 months; 90% CI, 9.2–14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies.

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Fig. 1: Study overview and outcomes.
Fig. 2: Peripheral blood CAR T-cell levels, target expression and infusion product characterization.
Fig. 3: CAR T cells were detectable in one brain tumor after infusion.
Fig. 4: No major changes in the immune composition of the tumors overall.
Fig. 5: Increased T-cell exhaustion and IFN-stimulated T cells after treatment.

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Data availability

scRNAseq and TCRseq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE242790. Source data for Figs. 1–5 and Extended Data Fig. 1 have been provided as source data files. All other data supporting the findings of this study including de-identified individual participant clinical data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The scRNAseq and TCRseq analyses presented in the paper were performed with open-source algorithms as described in Methods. Further details will be made available by the authors on request. No custom code was generated in the course of this study.

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Acknowledgements

We thank the patients who participated in this study and their families for their dedication to furthering GBM treatment. We also thank the Neurosurgery Clinical Research Division, J. Fraietta and the Translational and Correlative Sciences Laboratory and A. Brennan and the Clinical Cell and Vaccine Production Facility at the University of Pennsylvania Perelman School of Medicine for all of their clinical trial contributions and support. This work was funded by Novartis, the Abramson Cancer Center Glioblastoma Translational Center of Excellence, The Templeton Family Initiative in Neuro-Oncology, the Maria and Gabriele Troiano Brain Cancer Immunotherapy Fund, the Parker Institute for Cancer Immunotherapy (PICI Award Number C-01919), a public grant overseen by the French National Research Agency (Agence Nationale de la Recherche (ANR)) as part of the Investment Programme France 2030 under grant agreement ANR-21-RHUS-0016, and the French National Cancer Institute, the Ministry of Health and Prevention and Interm. High-throughput sequencing was performed by the ICGex NGS platform of the Institut Curie supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the ANR (“Investissements d’Avenir” program), by the ITMO-Cancer Aviesan (Plan Cancer III) and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS-465 and INCa-DGOS- Inserm_12554). We also acknowledge ANR-10-IDEX-0001-02 PSL, CIC IGR-Curie 1428, INCa-DGOS-Inserm-ITMO cancer_18000 and LabEx DCBIOL. We thank the Parker Institute for Cancer Immunotherapy members for all of their constructive feedback, as well as Clinical Immunology laboratory and U932 at the Institut Curie (Paris, France) for insightful discussions, O. Lantz for critical feedback on the ancillary work, N. Amzallag for her support and the genomic and cytometry platforms of Institut Curie for their help.

Author information

Author notes

  1. These authors contributed equally: S. J. Bagley, Z. A. Binder, L. Lamrani.

Authors and Affiliations

  1. Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Stephen J. Bagley, Arati S. Desai, Kelly Markowitz, Devora Delman, Gregory L. Beatty & Elizabeth Hexner

  2. Department of Neurosurgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Zev A. Binder, Eileen Maloney, Steven Brem, Meghan Logun & Donald M. O’Rourke

  3. Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Zev A. Binder, Meghan Logun, Amy Marshall & Donald M. O’Rourke

  4. GBM Translational Center of Excellence, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Zev A. Binder, MacLean P. Nasrallah, Steven Brem, Oliver Y. Tang, Meghan Logun, Meghna Bhattacharyya, Gregory L. Beatty & Donald M. O’Rourke

  5. Clinical Immunology Laboratory, Institut Curie, Paris, France

    Lamia Lamrani & Cecile Alanio

  6. INSERM U932, PSL University, Immunity and Cancer, Institut Curie Research Center, Paris, France

    Lamia Lamrani, Pierre-Emmanuel Bonté, Christel Goudot, Wilfrid Richer, Eliane Piaggio, Sebastian Amigorena & Cecile Alanio

  7. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Lamia Lamrani, E. John Wherry & Cecile Alanio

  8. Agora Cancer Research Center, Lausanne, Switzerland

    Eliana Marinari & Denis Migliorini

  9. Center for Translational Research in Onco-Hematology, University of Geneva, Geneva, Switzerland

    Eliana Marinari & Denis Migliorini

  10. Swiss Cancer Center Léman, Lausanne and Geneva, Geneva, Switzerland

    Eliana Marinari & Denis Migliorini

  11. Department of Oncology, University Hospital of Geneva, Geneva, Switzerland

    Eliana Marinari & Denis Migliorini

  12. Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    MacLean P. Nasrallah

  13. Department of Radiation Oncology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Robert A. Lustig, Goldie Kurtz & Michelle Alonso-Basanta

  14. Department of Translational Research, PSL Research University, Institut Curie Research Center, Paris, France

    Wilfrid Richer & Joshua J. Waterfall

  15. Department of Hematology and Medical Oncology, Emory University, Atlanta, GA, USA

    Shawn Kothari

  16. Cytometry Platform, CurieCoreTech, Institut Curie, Paris, France

    Lea Guyonnet & Coralie L. Guerin

  17. INSERM U830, PSL University, Institut Curie Research Cente, Paris, France

    Joshua J. Waterfall

  18. Department of Radiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Suyash Mohan

  19. Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Wei-Ting Hwang

  20. Warren Alpert Medical School of Brown University, Providence, RI, USA

    Oliver Y. Tang

  21. Cooper Medical School of Rowan University, Camden, NJ, USA

    Meghna Bhattacharyya

  22. Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    E. John Wherry

  23. Institute for Immunology and Immune Health, Cambridge, MA, USA

    E. John Wherry

  24. Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    Jennifer L. Brogdon

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Contributions

S.J.B., A.S.D., E.M., A.M., J.L.B., E.H. and D.M.O. designed the study. S.J.B., A.S.D., E.M., S.B., R.L., G.K., M.A.B., S.M., E.H. and D.M.O. recruited and treated patients in the study. L.L., C.A., Z.A.B., M.P.N., S.K., W.T.H., O.Y.T., M.L., M.B., K.M. and D.D. generated, curated and analyzed data. C.A.,P.E.B., C.G., W.R., E.M., D.M., E.P. and J.J.W. performed bioinformatic analyses. S.J.B., Z.A.B.,L.L. and C.A. wrote the original paper, and S.J.B., Z.A.B., L.L., M.P.N., S.B., S.M., S.K., E.J.W, W.T.H., O.Y.T.,E.P,.J.J.W., S.A.,D.M., G.L.B., J.L.B., C.A. and D.M.O. reviewed and edited the paper. S.J.B., Z.A.B., G.L.B., E.J.W., D.M., C.A. and D.M.O. supervised the study. Funding support was provided by C.A. and J.L.B. C.A. and D.M.O. contributed equally to this study.

Corresponding authors

Correspondence to Stephen J. Bagley or Cecile Alanio.

Ethics declarations

Competing interests

S.J.B. has received consulting fees from Kiyatec, Novocure and Bayer and has received research funding from Incyte, Tmunity, Novocure, GSK and Eli Lilly, all outside of the submitted work. S.J.B., A.S.D. and D.M.O. have inventorship interest in intellectual property owned by Novartis and the University of Pennsylvania: U.S. patent 62/809,245: “Combination therapies of EGFRvIII CARs and PD-1 inhibitors”. Z.A.B. has inventorship interest in intellectual property owned by the University of Pennsylvania and has received royalties related to CAR T-cell therapy in solid tumors. S.M. has received consulting fees from AI Integrated Radiological Solutions Medial and Qynapse SAS and has received research funding from Novocure and Galileo CDS, all outside of the submitted work. G.L.B. has received consulting fees from Seattle Genetics (now Seagen), Adicet Bio, Aduro Biotech, AstraZeneca, BiolineRx, BioMarin Pharmaceuticals, Bristol-Myers Squibb, Cantargia, Cour Pharmaceuticals, Boehinger Ingelheim, Genmab, Hibercell, HotSpot Therapeutics, Incyte Corporation, Janssen, Merck, Molecular Partners, NanoGhost, Pancreatic Cancer Action Network, Shattuck Labs and Verastem and reports receiving research funding from Incyte Corporation, Bristol-Myers Squibb, Verastem, Halozyme, Biothera, Newlink, Novartis, Arcus Biosciences and Janssen, all outside of the submitted work. E.J.W. receives honoraria and/or research support from BMS, Merck, MedImmune, Surface Oncology, Takeda and KyMab. E.J.W. has a patent licensing agreement for the PD-1 pathway. J.L.B. reports employment with, is a current holder of stock options and holds patents with Novartis Institutes for Biomedical Research. D.M. is inventor of patents in the field of cell and gene therapy filed by the University of Pennsylvania and the University of Geneva, is scientific co-founder of Cellula Therapeutics and is a member of the scientific advisory board of MPC, Limula and Cellula Therapeutics. D.M.O. has received consulting fees from Celldex Therapeutics, Prescient Therapeutics and Century Therapeutics; has received research funding from Celldex Therapeutics, Novartis and Tmunity Therapeutics; and is an inventor of intellectual property (U.S. patent numbers 7,625,558 and 6,417,168 and related families) and has received royalties related to targeted ErbB therapy in solid cancers previously licensed by the University of Pennsylvania. C.A. is a consultant for Biotherapy Partners. All other authors declare no competing interests.

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Nature Cancer thanks Jon Amund Kyte, Wendy London, Julie Park and Mario Suva for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 CAR T cells in the infusion products are activated and exhausted effector T cells, with comparable features across patients.

(a) Representative example of EGFRvIII CAR T cells staining in the infusion product. (b) Scatterplot shows proportion of EGFRvIII CAR T cells staining in the infusion products of the patients. (c) Representative example of CD4/CD8 staining in EGFRvIII CAR T cells staining in the infusion product. (d) Scatterplot shows proportion of CD4 T cells within EGFRvIII CAR T cells in the infusion products of the patients. (e) Representative example of CD127/CD25 regulatory T cells staining in the infusion product. (f) Representative example of Foxp3 expression in the different CD4 T-cell subsets in the infusion product. (g) Expression of CD4, CD40LG, Foxp3 in the infusion product as detected by RNA in scRNAseq data projected on a UMAP. (h) Representative example of CD45RA/CD127 staining in CD4 CAR T cells in the infusion product (P1, left; P4, right). (i) Representative example of CD45RA/CD127 staining in CD8 CAR T cells in the infusion product (P1, left; P4, right). (j) Representative example of PD1, CD39, Ki67 staining in CD8 T cells in the infusion product (P1, top; P4, bottom). (k) Heatmap shows expression in CD8 T cells in the infusion product of P1 and P4 as compared to a normal donor.

Source data

Extended Data Fig. 2 Bioinformatic analysis.

(a) Example of our strategy for detecting CAR T cells by RNA on scRNAseq data, here applied to one healthy donor where we spiked small amount of CAR T cells. (b) Clonal space homeostasis indicating percentage of clones in distinct proportions in the six paired samples. (c) Clonal Proportion where clonotypes are ranked by copy or frequency of occurrence in the six paired samples. (d) UMAP shows the overall cell space for the TME of three paired patients as in Fig. 4c, here split by 3 Pre (left) and 3 Post (right). (e) UMAP shows the overall cell space for the TME of three paired patients as in Fig. 4c, here split by sample.

Extended Data Fig. 3 Increased myeloid inflammation with therapy.

(a) UMAP shows the overall cell space for the TME of three paired patients (3 Pre and Post, total six samples) after filtering myeloid cells from the overall UMAP as in Fig. 4c. (b) Top 10 genes for each cluster as in A. (c) Gene expression on top of each cluster as in A. GSEA of cluster 0 (d), 2 (e), and 3 (f) as defined in A. GSEA employs a permutation-based test usng Kolmogorov-Smirnov; Benjamini-Hochberg procedure was used to adjust for multiple comparisons. (g) UMAP shows the overall cell space for the myeloid TME of three paired patients as in A, here split by 3 Pre (left) and 3 Post (right).

Extended Data Fig. 4 Flow cytometry analysis of PD-1 expression.

(a) Flow cytometry gating schema for quantification of CD4+/CAR+ PD-1 expression in infusion product. (b) Exemplary flow cytometry plots taken from 6 total samples.

Supplementary information

Supplementary Information

Study protocol, statistical analysis plan, and CONSORT diagram.

Reporting Summary

Supplementary Tables

Supplementary Table 1: Markers for Cytek panel. Supplementary Table 2: Antibodies and staining reagents used for flow cytometry.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

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Source Data Extended Data Fig./Table 1

Statistical source data.

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Bagley, S.J., Binder, Z.A., Lamrani, L. et al. Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial. Nat Cancer 5, 517–531 (2024). https://doi.org/10.1038/s43018-023-00709-6

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