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Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells

Nature Biomedical Engineering (2023)Cite this article

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Abstract

On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent ‘universal’ CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.

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Fig. 1: Design and characterization of a covalent CAR T cell and folate receptor adapter molecules.
Fig. 2: Design and characterization of a covalent CAR T cell and PSMA adapter molecules.
Fig. 3: In vivo activity of CovCAR T cells and DUPA-3-diketone, DUPA-4-diketone and DUPA-5-diketone conjugates in a prostate cancer xenograft model.
Fig. 4: Pharmacological control of cytokine release and CovCAR-T-cell propagation following DUPA-3-diketone adapter administration in vivo.
Fig. 5: Selection and characterization of PSMA-specific adapter molecules from DEL for covalent CAR T-cell therapy.
Fig. 6: Selection and characterization of HER2-specific adapter molecules from DEL for covalent CAR T-cell therapy.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. All data generated during the study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank N. Lerner and I. A. Wilson for help with the preparation of the manuscript. This work was supported in part by a grant from the JPB Foundation to R.A.L. A.G.G. was personally supported by grant no. 17-74-30019. Open-access funding was provided by Scripps Research.

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  1. These authors contributed equally: Alexey V. Stepanov, Jia Xie.

Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Alexey V. Stepanov, Jia Xie, Geramie Shaver, Lacey Douthit, Ding Zhang, Xiang Fu, Yingying Zhao, Phil S. Baran & Richard A. Lerner

  2. WuXi AppTec Co., Ltd, Shanghai, China

    Qiaoqiao Zhu, Zuyuan Shen, Wenji Su, Letian Kuai & Richard Soll

  3. Department of Immunology and Microbiology, UF Scripps Biomedical Research, University of Florida, Jupiter, FL, USA

    Christoph Rader

  4. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation

    Roman Kalinin & Alexander G. Gabibov

  5. The University of Texas Southwestern Medical Center, Dallas, TX, USA

    Tian Qin

  6. Structural Biology, School of Medicine, Stanford University, Stanford, CA, USA

    David Bushnell & Roger D. Kornberg

  7. Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Zurich, Switzerland

    Dario Neri

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Contributions

A.V.S, J.X., Q.Z., Z.S., W.S., G.S., L.D., D.Z., R.K., X.F., Y.Z. and T.Q. performed experiments, analysed and interpreted data; L.K., R.S., P.B., A.G.G., D.B. and D.N. analysed and interpreted data and revised the manuscript; and A.V.S., J.X., R.D.K. and R.A.L. designed the research, analysed and interpreted data and wrote the paper. C.R. analysed and interpreted data, and revised the manuscript.

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Correspondence to Alexey V. Stepanov or Roger D. Kornberg.

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Extended data

Extended Data Fig. 1 Effect of folate-diketone concentration on cell lysis and cytokine release of acute monocytic leukemia and T cell leukemia by the CovCAR T cells in vitro.

a, Comparison of FOLR1 and FOLR2 expression by THP-1, THP-1 FOLR1, and THP-1 FOLR2 cells. Open histogram: unstained cells; gray histogram: isotype control; red: folate receptor. b, THP-1 and THP-1 FOLR2 cells were incubated with conventional FOLR2-specific CAR-T cells (m909h-CAR) or in the presence of CovCAR T cells plus different concentrations of folate-diketone prior to analysis of tumor cell lysis (effector:tumour cell ratio 2:1). c, THP-1, THP-1 FOLR1 and THP-1 FOLR2 cells were incubated in the presence of CovCAR T cells plus different concentrations of folate-diketone prior to analysis of tumor cell lysis (effector:tumour cell ratio 5:1). d, CovCAR T cells release IL-2 cytokine in the presence of acute monocytic leukemia cells expressing the folate receptor and folate-diketone in a dose-dependent manner (effector:tumour cell ratio 5:1). Data represent mean ± s.d., n = 4.

Extended Data Fig. 2 In vivo activity of CovCAR T cells and constant dosage of DUPA-3-diketone injections in a prostate cancer xenograft model.

a NSG mice were implanted s.c. with 1 × 106 PC3-PSMA ffLuc cells. On day 3 after tumor inoculation 10 × 106 of CovCAR-T cells with transduction efficacy of 68% were infused i.v. into animals from all experimental groups. Mice were then injected i.v. with either PBS or DUPA-3-diketone (500 nmol/kg) on day 3, 5, 7, 9, 11, 13, 15, 17. b Representative IVIS images of the PC3-PSMA ffLuc tumors implanted mice treated with CovCAR T cells and PBS or DUPA-3-diketone. c Survival plots for animals from experimental and control groups. d Quantified tumor burden (as average radiance from luciferase activity per mouse) from (B) for the 5–44 day period. e Individual animal tumor growth kinetics of mice from control and treated groups. Overall survival curves were plotted using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. Statistical significance: *p < 0.05.

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Stepanov, A.V., Xie, J., Zhu, Q. et al. Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01102-5

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