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4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors

Nature Medicine volume 21, pages 581–590 (2015)Cite this article

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Abstract

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.

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Figure 1: GD2.28z CAR T cells have discrepant in vitro and in vivo activity.
Figure 2: GD2.28z CAR T cells become exhausted during ex vivo expansion.
Figure 3: Tonic CAR signaling during ex vivo expansion leads to early exhaustion.
Figure 4: Tonic GD2.28z CAR signaling is antigen independent.
Figure 5: 4-1BB endodomain ameliorates exhaustion in CAR T cells.
Figure 6: Ameliorating effect of 4-1BB signaling is associated with a unique transcriptional profile.

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GenBank/EMBL/DDBJ

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Acknowledgements

We kindly thank S. Gottschalk (Texas Children's Hospital) for providing sequences for the 14g2a scFv and IgG1 CH2CH3; J. Kochenderfer (National Cancer Institute, NCI, US National Institutes of Health, NIH) for the H3 MSGV-FMC63-28z retroviral vector producer line; S. Grupp (Children's Hospital of Philadelphia) for the NALM6-GL cell line; R. Morgan (BlueBird Bio, previously NCI, NIH) for the MSGV-139-28z and MSGV-4D5-28BBz vectors; and C. June (University of Pennsylvania) for sequences to the 4-1BB CAR endodomain. We also thank L. Cooper (M.D. Anderson) for providing the 136.20.1, FMC63 anti-idiotype antibody; the Biological Research Branch of NCI for providing the 1A7, 14g2a anti-idiotype antibody; the Clinical Support Laboratory of the Frederick National Laboratory for Cancer Research (FNLCR) for assisting in MesoScale cytokine release assays; and the Laboratory of Molecular Technology (LMT) Microarray Group of FNLCR for assisting in microarray assays. We thank N. Restifo and M. Roederer for careful review of this manuscript. This work was supported by the Intramural Research Program of the NIH, including the NCI and the National Institute of Biomedical Imaging and Bioengineering (NIBIB): ZIA BC 011073 (A.H.L., K.M.W., J.P.S., A.J.W., M.E.K., V.R.V., R.J.O. and C.L.M); ZIA BC 011565 (W.M.H. and T.J.F.); ZIA BC 011332 (M.M. and R.N.K.); and ZIA EB 000071-06 (M.I. and G.H.P.) This research was also supported by a Stand Up To Cancer – St. Baldrick's – NCI Pediatric Dream Team Translational Cancer Research Grant. Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research.

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  1. Rimas J Orentas

    Present address: Present address: Lentigen Technology, Inc., Gaithersburg, Maryland, USA.,

Authors and Affiliations

  1. Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Adrienne H Long, Waleed M Haso, Jack F Shern, Kelsey M Wanhainen, Meera Murgai, Jillian P Smith, Alec J Walker, M Eric Kohler, Vikas R Venkateshwara, Rosandra N Kaplan, Terry J Fry, Rimas J Orentas & Crystal L Mackall

  2. Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Adrienne H Long

  3. Department of Biology, Colgate University, Hamilton, New York, USA

    Kelsey M Wanhainen

  4. Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA

    Maria Ingaramo & George H Patterson

  5. Department of Pediatrics, Johns Hopkins Hospital, Baltimore, Maryland, USA

    M Eric Kohler

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Contributions

A.H.L., W.M.H., J.F.S., M.M., M.I., M.E.K., R.N.K., G.H.P., T.J.F., R.J.O. and C.L.M. designed the research; A.H.L., W.M.H., K.M.W., M.M., M.I., J.P.S., A.J.W., M.E.K., V.R.V. and G.H.P. conducted experiments; A.H.L., W.M.H., J.F.S., M.M., M.I., G.H.P., T.J.F., R.J.O. and C.L.M. analyzed data; and A.H.L. and C.L.M. wrote the paper.

Corresponding author

Correspondence to Crystal L Mackall.

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Competing interests

R.J.O. and C.L.M. are co-inventors of the CD22-CAR described in this report. The authors declare no other competing financial interests.

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Long, A., Haso, W., Shern, J. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21, 581–590 (2015). https://doi.org/10.1038/nm.3838

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