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Engineering primary T cells with chimeric antigen receptors for rewired responses to soluble ligands

Nature Protocols volume 15, pages 1507–1524 (2020)Cite this article

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Abstract

The expression of synthetic receptors in primary T cells enables the programming of user-defined responses when designing T-cell therapies. Chimeric antigen receptors (CARs) are synthetic receptors that have demonstrated efficacy in cancer therapy by targeting immobilized antigens on the surface of malignant cells. Recently, we showed they can also rewire T-cell responses to soluble ligands. In contrast to other synthetic receptors, CARs are not only readily engineered by rational design, but also clinically translatable, with robust function in primary human T cells. This protocol discusses design principles for CARs responsive to soluble ligands and delineates steps for producing T cells expressing synthetic receptors. Functional assays for quantifying the ability of CAR T cells to sense and respond to soluble ligands are also presented. This protocol provides a framework for proficient immune-cell researchers to test novel T-cell therapies targeting soluble ligands in <2 weeks.

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Fig. 1: CAR structure and mechanisms for response to soluble ligands.
Fig. 2: Troubleshooting CAR expression and effector activity.
Fig. 3: Steps in the generation of CAR+ primary human T cells from human whole blood.
Fig. 4: Assessing functionality of the TGF-β CAR.
Fig. 5: Degranulation in response to soluble and immobilized target ligand.

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Data availability

The data analyzed to generate what is shown in this manuscript are compiled in the Supplementary Spreadsheet file. Individual examples of gating strategy are shown when appropriate. Data from Fig. 4 and Supplementary Fig. 3 have been used in a prior publication6.

References

  1. Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).

    Article  Google Scholar 

  2. Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018).

    Article  CAS  Google Scholar 

  3. Porter, D. L. et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci. Transl. Med. 7, 303ra139 (2015).

    Article  Google Scholar 

  4. Brentjens, R. J. et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118, 4817–4828 (2011).

    Article  CAS  Google Scholar 

  5. Kochenderfer, J. N. et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J. Clin. Oncol. 33, 540–549 (2015).

    Article  CAS  Google Scholar 

  6. Chang, Z. L. et al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat. Chem. Biol. 14, 317–324 (2018).

    Article  CAS  Google Scholar 

  7. Hou, A. J., Chang, Z. L., Lorenzini, M. H., Zah, E. & Chen, Y. Y. TGF‐β–responsive CAR‐T cells promote anti‐tumor immune function. Bioeng. Transl. Med 3, 75–86 (2018).

    Article  CAS  Google Scholar 

  8. Chen, L. C. & Chen, Y. Y. Outsmarting and outmuscling cancer cells with synthetic and systems immunology. Curr. Opin. Biotechnol. 60, 111–118 (2019).

    Article  CAS  Google Scholar 

  9. Leen, A. M. et al. Reversal of tumor immune inhibition using a chimeric cytokine receptor. Mol. Ther. 22, 1211–1220 (2014).

    Article  CAS  Google Scholar 

  10. Mohammed, S. et al. Improving chimeric antigen receptor-modified T cell function by reversing the immunosuppressive tumor microenvironment of pancreatic cancer. Mol. Ther. 25, 249–258 (2017).

    Article  CAS  Google Scholar 

  11. Wilkie, S. et al. Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4. J. Biol. Chem. 285, 25538–25544 (2010).

    Article  CAS  Google Scholar 

  12. Chang, Z. L. & Chen, Y. Y. CARs: synthetic immunoreceptors for cancer therapy and beyond. Trends Mol. Med. 23, 430–450 (2017).

    Article  CAS  Google Scholar 

  13. Schwarz, K. A., Daringer, N. M., Dolberg, T. B. & Leonard, J. N. Rewiring human cellular input-output using modular extracellular sensors. Nat. Chem. Biol. 13, 202–209 (2017).

    Article  CAS  Google Scholar 

  14. Baeumler, T. A., Ahmed, A. A. & Fulga, T. A. Engineering synthetic signaling pathways with programmable dcas9-based chimeric receptors. Cell Rep. 20, 2639–2653 (2017).

    Article  CAS  Google Scholar 

  15. Scheller, L., Strittmatter, T., Fuchs, D., Bojar, D. & Fussenegger, M. Generalized extracellular molecule sensor platform for programming cellular behavior. Nat. Chem. Biol. 14, 723 (2018).

    Article  CAS  Google Scholar 

  16. Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2012).

  17. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  18. Tiscornia, G., Singer, O. & Verma, I. M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 (2006).

    Article  CAS  Google Scholar 

  19. Liu, L. et al. Inclusion of Strep-tag II in design of antigen receptors for T-cell immunotherapy. Nat. Biotechnol. 34, 430–434 (2016).

    Article  CAS  Google Scholar 

  20. Ghassemi, S. et al. Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol. Res. 6, 1100–1109 (2018).

    Article  CAS  Google Scholar 

  21. Hollyman, D. et al. Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy. J. Immunother. 32, 169–180 (2009).

    Article  CAS  Google Scholar 

  22. Vormittag, P., Gunn, R., Ghorashian, S. & Veraitch, F. S. A guide to manufacturing CAR T cell therapies. Curr. Opin. Biotechnol. 53, 164–181 (2018).

    Article  CAS  Google Scholar 

  23. Chang, Z. L. et al. Rewiring T-cell signaling responses to extracellular soluble cues with chimeric antigen receptors. Mol. Ther. 27, abstr. 832 (2019).

    Google Scholar 

  24. Zah, E., Lin, M.-Y., Silva-Benedict, A., Jensen, M. C. & Chen, Y. Y. T cells expressing CD19/CD20 bispecific chimeric antigen receptors prevent antigen escape by malignant B cells. Cancer Immunol. Res. 4, 498–508 (2016).

    Article  CAS  Google Scholar 

  25. Caruso, H. G. et al. Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res. 75, 3505–3518 (2015).

    Article  CAS  Google Scholar 

  26. Liu, X. et al. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res. 75, 3596–3607 (2015).

    Article  CAS  Google Scholar 

  27. Wang, X. et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 118, 1255–1263 (2011).

    Article  CAS  Google Scholar 

  28. Wei, P. et al. Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells. Nature 488, 384–388 (2012).

    Article  CAS  Google Scholar 

  29. Wang, D. et al. A requirement for CARMA1 in TCR-induced NF-κB activation. Nat. Immunol. 3, 830–835 (2002).

    Article  CAS  Google Scholar 

  30. Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581–590 (2015).

    Article  CAS  Google Scholar 

  31. Frigault, M. J. et al. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol. Res. 3, 356–367 (2015).

    Article  CAS  Google Scholar 

  32. Aivazian, D. & Stern, L. J. Phosphorylation of T cell receptor ζ is regulated by a lipid dependent folding transition. Nat. Struct. Biol. 7, 1023–1026 (2000).

    Article  CAS  Google Scholar 

  33. Zhang, H., Cordoba, S.-P., Dushek, O. & van der Merwe, P. A. Basic residues in the T-cell receptor ζ cytoplasmic domain mediate membrane association and modulate signaling. Proc. Natl Acad. Sci. USA 108, 19323–19328 (2011).

    Article  CAS  Google Scholar 

  34. Dobbins, J. et al. Binding of the cytoplasmic domain of CD28 to the plasma membrane inhibits Lck recruitment and signaling. Sci. Signal. 9, ra75–ra75 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

Several protocols presented reflect discussion and contributions from present and past members of the Chen lab, notably E. Zah, P. Ho, M.-y. Lin, and M. Lorenzini, as well as members of the M. Jensen lab (Seattle Children’s Research Institute). The NFAT reporter Jurkat T-cell line was a gift from A. Weiss (University of California, San Francisco). The NFκB reporter Jurkat T-cell line was a gift from X. Lin (University of Texas MD Anderson Cancer Center). This work was supported by the National Institutes of Health (grant DP5OD012133, UC CAI grant U54HL119893, and UCLA CTSI grant UL1TR001881 to Y.Y.C.). Z.L.C. was supported by an NIH F30 Fellowship (F30CA183528). A.J.H. was supported by the NIH Biotechnology Training in Biomedical Sciences and Engineering Program (T32 GM067555).

Author information

Author notes

  1. ZeNan L. Chang

    Present address: Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA

  2. These authors contributed equally: ZeNan L. Chang, Andrew J. Hou.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Los Angeles (UCLA), Los Angeles, CA, USA

    ZeNan L. Chang, Andrew J. Hou & Yvonne Y. Chen

  2. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA

    Yvonne Y. Chen

  3. Parker Institute for Cancer Immunotherapy Center at UCLA, Los Angeles, CA, USA

    Yvonne Y. Chen

Authors
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Contributions

Z.L.C., A.J.H., and Y.Y.C. conceptualized and wrote the manuscript. Z.L.C. and Y.Y.C. developed the protocol, with additional contributions as reflected in the Acknowledgements section.

Corresponding author

Correspondence to Yvonne Y. Chen.

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Competing interests

Y.Y.C. and Z.L.C. declare competing interests in the form of a patent application whose value may be affected by the publication of this work.

Additional information

Peer review information Nature Protocols thanks Carl June, Atsushi Okuma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Chang, Z. L. et al. Nat. Chem. Biol. 14, 317–324 (2018): https://doi.org/10.1038/nchembio.2565

Hou, A. J., Chang, Z. L., Lorenzini, M. H., Zah, E. & Chen, Y. Y. Bioeng. Transl. Med. 3, 75–86 (2018): https://doi.org/10.1002/btm2.10097

Integrated supplementary information

Supplementary Fig. 1 Comparison of quantifying cytokine secreted into supernatant and intracellular cytokine staining.

CD4 TGF-β CAR-T cells were stimulated with 5 ng/mL TGF-β for 24 hours. Side-by-side samples were prepared for analysis of supernatant or intracellular cytokine staining. a, Quantification of IFN-γ and TNF-α levels secreted into supernatant using a cytometric bead array showed comparable IFN-γ and TNF-α production. Data points from n = 3 (without TGF-β) or n = 2 (with TGF-β) samples are shown with means ± 1 standard deviation or total ranges, respectively. b, In contrast, intracellular cytokine staining detected more robust TNF-α production than IFN-γ production, indicating lower sensitivity for IFN-γ staining using the protocol described.

Supplementary Fig. 2 Cell sorting results are impacted by transduction efficiency of starting material.

Cells lines A and B were generated by transduction with a synthetic construct linked via a T2A “self-cleaving” peptide to truncated EGFR (EGFRt). The poorly transduced cell line (Cell Line A) and well-transduced cell line (Cell Line B) were then sorted according to steps 34-41. The poorly transduced starting material yielded an enriched product that was only 68.5% pure, while the well-transduced cell line yielded a 97.1% pure population.

Supplementary Fig. 3 Gating strategy for flow cytometry experiments.

a, An example gating scheme for all flow cytometry experiments is shown. Initial FSC-Area/SSC-Area gates are drawn to remove debris and dead cells. A subsequent FSC-Area/FSC-Height gate demarcates singlet events. b-d, Histograms are shown for flow cytometry of TGF-β CAR-expressing cell lines, gated for viable singlets, demonstrating upregulation of (b) NFAT-driven EGFP, (c) NFκB-driven EGFP, and (d) CD69. Plots in b-d are representative of data used to generate the bar graphs in Fig. 4a–c. b–d adapted from ref. 6, Springer Nature.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Data 1

Supplementary Spreadsheet

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Chang, Z.L., Hou, A.J. & Chen, Y.Y. Engineering primary T cells with chimeric antigen receptors for rewired responses to soluble ligands. Nat Protoc 15, 1507–1524 (2020). https://doi.org/10.1038/s41596-020-0294-8

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