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ma=86400 Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture | Nature Protocols
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Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture

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Abstract

The clinical potential of current chimeric antigen receptor-engineered T (CAR-T) cell therapy is hampered by its autologous nature that poses considerable challenges in manufacturing, costs and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture method to differentiate gene-engineered hematopoietic stem and progenitor (HSP) cells into allogeneic invariant natural killer T (AlloNKT) cells and their CAR-armed derivatives (AlloCAR-NKT cells). We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate AlloCAR-NKT cells, including HSP cell engineering, HSP cell expansion, NKT cell differentiation, NKT cell deep differentiation and NKT cell expansion. In addition, we describe procedures for evaluating the pharmacology, antitumor efficacy and mechanism of action of AlloCAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature AlloCAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.

Key points

  • This protocol describes an ex vivo feeder-free culture method to differentiate genetically engineered hematopoietic stem and progenitor cells into allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy. In addition, procedures are provided for evaluating their pharmacology, antitumor efficacy and mechanism of action.

  • This approach overcomes challenges in manufacturing, costs and patient selection that hamper the clinical potential of current autologous CAR-T cell therapies.

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Fig. 1: Generation of allogeneic CAR-NKT cells with genetically engineered HSP cells and feeder-free differentiation culture.
Fig. 2: Comparison between our allogeneic HSP cell-derived CAR-NKT cell products and the conventional CAR-T cell products.
Fig. 3: Downstream assays to evaluate the allogeneic CAR-NKT cells.
Fig. 4: All lentivectors show successful gene delivery on CD3-overexpressing 293T (293T-CD3) cells.
Fig. 5: Ex vivo development of Allo/15BCAR-NKT cells.
Fig. 6: Phenotype, functionality and antitumor efficacy of allogeneic CAR-NKT cells.

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Data availability

All associated data are presented in the protocol paper or Supplementary Information. The genomics data were reanalyzed from the public repository Gene Expression Omnibus database: GSE241996 (scRNA-seq, related to Fig. 5c–e). Additional information and materials will be made available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the UCLA Technology Centre for Genomics and Bioinformatics facility for providing RNA-seq services, the UCLA Center for AIDS Research (CFAR) Virology Core for providing human cells and the UCLA Broad Stem Cell Research Center (BSCRC) Flow Cytometry Core Facility for cell sorting support. This work was supported by a Partnering Opportunity for Discovery Stage Research Projects Award and a Partnering Opportunity for Translational Research Projects Awards from the California Institute for Regenerative Medicine (grant nos. DISC2-11157 and TRAN1-12250, to L.Y.), a Department of Defense Congressionally Directed Medical Research Program (CDMRP) Peer Reviewed Cancer Research Program (PRCRP) Impact Award (grant no. CA200456 to L.Y.), a UCLA BSCRC Innovation Award (to L.Y.) and an Ablon Scholars Award (to L.Y.). Y.-R.L. is a postdoctoral fellow supported by a UCLA MIMG M. John Pickett Post-Doctoral Fellow Award, a California Institute for Regenerative Medicine (CIRM)-BSCRC Postdoctoral Fellowship and a UCLA Sydney Finegold Postdoctoral Award. K.Z. is a predoctoral fellow supported by a CIRM-BSCRC Predoctoral Fellowship. D.L. is a postdoctoral fellow supported by T32 Tumor Immunology Training Grant postdoctoral fellowships (USHHS Ruth L. Kirschstein Institutional National Research Service Award, T32-CA009120). Figures were created with BioRender.com.

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  1. These authors contributed equally: Yan-Ruide Li, Kuangyi Zhou, Derek Lee.

Authors and Affiliations

  1. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Yan-Ruide Li, Kuangyi Zhou, Derek Lee, Yichen Zhu, Tyler Halladay, Jiaji Yu, Yang Zhou, Zibai Lyu, Ying Fang, Yuning Chen, Sasha Semaan & Lili Yang

  2. Eli and Edythe Broad Centre of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang

  3. Jonsson Comprehensive Cancer Centre, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang

  4. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang

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Contributions

Y.-R.L., K.Z., D.L. and L.Y. designed the experiments, analyzed the data and wrote the manuscript. L.Y. conceived and oversaw the study, with assistance from Y.-R.L., K.Z. and D.L. Y.-R.L., K.Z. and D.L. performed all experiments, with assistance from Y. Zhu, T.H., J.Y., Y. Zhou, Y.F., Z.L., Y.C. and S.S.

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Correspondence to Lili Yang.

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Y.-R.L., D.L., J.Y., Y. Zhou and L.Y. are inventors on patents relating to this study filed by UCLA. J.Y. is currently an employee of Appia Bio. Y. Zhou is currently an employee of Amberstone Biosciences. L.Y. is a scientific advisor to AlzChem and Amberstone Biosciences, and a cofounder, stockholder and advisory board member of Appia Bio. Appia Bio licensed some patents relating to this study from UCLA. None of the declared companies contributed to or directed any of the research reported in this article. The other authors declare no competing interests.

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Nature Protocols thanks Haopeng Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Li, Y.-R. et al. Nat. Biotechnol. (2024): https://doi.org/10.1038/s41587-024-02226-y

Li, Y.-R. et al. Mol. Ther. 32, 1849–1874 (2024): https://doi.org/10.1016/j.ymthe.2024.04.005

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Li, YR., Zhou, K., Lee, D. et al. Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture. Nat Protoc (2025). https://doi.org/10.1038/s41596-024-01077-w

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