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CAR-T cells targeting a nucleophosmin neoepitope exhibit potent specific activity in mouse models of acute myeloid leukaemia

Nature Biomedical Engineering volume 5, pages 399–413 (2021)Cite this article

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An Author Correction to this article was published on 16 December 2020

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Abstract

Therapies employing chimeric antigen receptor T cells (CAR-T cells) targeting tumour-associated antigens (TAAs) can lead to on-target–off-tumour toxicity and to resistance, owing to TAA expression in normal tissues and to TAA expression loss in tumour cells. These drawbacks can be circumvented by CAR-T cells targeting tumour-specific driver gene mutations, such as the four-nucleotide duplication in the oncogene nucleophosmin (NPM1c), which creates a neoepitope presented by the human leukocyte antigen with the A2 serotype (HLA-A2) that has been observed in about 35% of patients with acute myeloid leukaemia (AML). Here, we report a human single-chain variable fragment (scFv), identified via yeast surface display, that specifically binds to the NPM1c epitope–HLA-A2 complex but not to HLA-A2 or to HLA-A2 loaded with control peptides. In vitro and in mice, CAR-T cells with the scFv exhibit potent cytotoxicity against NPM1c+HLA-A2+ leukaemia cells and primary AML blasts, but not NPM1c–HLA-A2+ leukaemia cells or HLA-A2– tumour cells. Therapies using NPM1c CAR-T cells for the treatment of NPM1c+HLA-A2+ AML may limit on-target–off-tumour toxicity and tumour resistance.

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Fig. 1: Isolation of human scFv specific for the AIQ–HLA-A2 complex by yeast surface display.
Fig. 2: Specific and high-affinity binding of YG1 scFv–Fc to the AIQ–HLA-A2 complex on AML cells.
Fig. 3: Generation of NPM1c CAR-T cells specific to the AIQ–HLA-A2 complex.
Fig. 4: NPM1c CAR-T cells specifically kill HLA-A2+NPM1c+ AML cells in vitro.
Fig. 5: NPM1c CAR-T cell therapy reduces leukaemia burden and prolongs survival.
Fig. 6: NPM1c CAR-T cells reduce the leukaemia burden in blood, spleen, bone marrow and liver.
Fig. 7: NMP1c CAR-T cells effectively kill primary human AML blasts in vitro and in vivo.

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Data availability

The data that support the main findings of this study are available within the paper and its Supplementary Information. Raw data generated for this study are available in Figshare with the identifier https://doi.org/10.6084/m9.figshare.12922520 (ref. 47).

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Acknowledgements

We are grateful to K. D. Wittrup and A. W. Tisdale for the yeast display library and yeast display/secretion system. We thank B. Turner and S. A. Nordeen for assistance with the Octet biolayer interferometry experiment, V. Spanoudaki for assisting with mouse BLI and the Koch Institute Flow Cytometry Core for assistance. This work was supported in part by National Institutes of Health grant nos. AI69208, CA197605 and NS104315, the Ivan R. Cottrell Professorship and Research Fund, Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute, National Institutes of Health Pre-Doctoral Training Grant T32GM007287 and a gift from C2Y Therapeutics.

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Authors and Affiliations

  1. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Guozhu Xie, Nikola A. Ivica, Bin Jia, Yingzhong Li, Douglas Brown & Jianzhu Chen

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Guozhu Xie, Nikola A. Ivica, Bin Jia, Yingzhong Li, Douglas Brown & Jianzhu Chen

  3. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA

    Han Dong

  4. Department of Microbiology and Immunology, Harvard Medical School, Boston, MA, USA

    Han Dong

  5. Division of Hematologic Malignancies and Transplantation, Dana-Farber Cancer Institute, Boston, MA, USA

    Yong Liang & Rizwan Romee

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Contributions

J.C. conceived of the idea and provided experimental advice and funding support. J.C. and G.X. designed the study. G.X. designed and performed the isolation of AIQ–HLA-A2-specific scFv using the yeast display scFv library. G.X., N.A.I. and Y. Li designed and generated the lentivirus vectors. G.X. and N.A.I. performed scFv–Fc protein production and characterization and determined scFV–Fc affinity by biolayer interferometry. G.X. and D.B. performed the CAR-T-cell production and characterization studies. B.J. and H.D. performed tissue preparation for flow cytometry analysis. G.X., B.J., Y. Li and H.D. performed the flow cytometry analysis. G.X. conducted the in vivo tumour studies and BLI. N.A.I. performed the western blot assay. G.X., H.D., Y. Liang and R.R. prepared and analysed the primary AML samples. G.X. prepared the figures and performed the statistical analyses. G.X. and J.C. wrote the manuscript.

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Correspondence to Jianzhu Chen.

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The authors have filed a provisional patent application on the identified scFv and its applications.

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Supplementary Methods, Figs. 1–8 and references.

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Xie, G., Ivica, N.A., Jia, B. et al. CAR-T cells targeting a nucleophosmin neoepitope exhibit potent specific activity in mouse models of acute myeloid leukaemia. Nat Biomed Eng 5, 399–413 (2021). https://doi.org/10.1038/s41551-020-00625-5

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