Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy

Abstract

Chimeric antigen receptor T-cell therapies using defined product compositions require high-purity T-cell isolation systems that, unlike immunomagnetic positive enrichment, are inexpensive and leave no trace on the final cell product. Here, we show that DNA aptamers (generated with a modified cell−SELEX procedure to display low-nanomolar affinity for the T-cell marker CD8) enable the traceless isolation of pure CD8+ T cells at low cost and high yield. Captured CD8+ T cells are released label-free by complementary oligonucleotides that undergo toehold-mediated strand displacement with the aptamer. We also show that chimeric antigen receptor T cells manufactured from these cells are comparable to antibody-isolated chimeric antigen receptor T cells in proliferation, phenotype, effector function and antitumour activity in a mouse model of B-cell lymphoma. By employing multiple aptamers and the corresponding complementary oligonucleotides, aptamer-mediated cell selection could enable the fully synthetic, sequential and traceless isolation of desired lymphocyte subsets from a single system.

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Fig. 1: Schematic representation of competitive cell−SELEX with magnetic depletion from PBMCs.
Fig. 2: A1, A3 and A8 bind to CD8a glycoprotein.
Fig. 3: Complementary reversal agent designed to occlude binding of A3 aptamer with modified toehold.
Fig. 4: Isolation of label-free CD8+ T cells from PBMCs using a reversible, aptamer-based selection strategy.
Fig. 5: Characterization of CD19 CAR T cells generated from antibody- and aptamer-isolated cells.
Fig. 6: Tumour stress test with antibody- and aptamer-isolated CD8+ CD19 CAR T cells.

Data availability

The data that support the main findings of this study are available in the Article and Supplementary Information. All source data generated for this study and relevant information are available from the corresponding authors on reasonable request. The NanoString nCounter data have been deposited in the NCBI Gene Expression Omnibus, with accession code GSE130185.

References

  1. 1.

    Brentjens, R. J. et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 5, 177ra138 (2013).

  2. 2.

    Davila, M. L. et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci. Transl. Med. 6, 224ra225 (2014).

  3. 3.

    Lee, D. W. et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 385, 517–528 (2015).

  4. 4.

    Mirzaei, H. R., Rodriguez, A., Shepphird, J., Brown, C. E. & Badie, B. Chimeric antigen receptors T cell therapy in solid tumor: challenges and clinical applications. Front. Immunol. 8, 1850 (2017).

  5. 5.

    Hale, M. et al. Engineering HIV-Resistant, anti-HIV chimeric antigen receptor T cells. Mol. Ther. 25, 570–579 (2017).

  6. 6.

    Scholler, J. et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med. 4, 132ra153 (2012).

  7. 7.

    Sommermeyer, D. et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492–500 (2016).

  8. 8.

    Turtle, C. J. et al. CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J. Clin. Invest. 126, 2123–2138 (2016).

  9. 9.

    Gardner, R. A. et al. Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Blood 129, 3322–3331 (2017).

  10. 10.

    Aijaz, A. et al. Biomanufacturing for clinically advanced cell therapies. Nat. Biomed. Eng. 2, 362–376 (2018).

  11. 11.

    Terakura, S. et al. Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells. Blood 119, 72–82 (2012).

  12. 12.

    Wang, X. et al. Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. J. Immunother. 35, 689–701 (2012).

  13. 13.

    Voss, S. & Skerra, A. Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the strep-tag II peptide and improved performance in recombinant protein purification. Protein Eng. 10, 975–982 (1997).

  14. 14.

    Knabel, M. et al. Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nat. Med. 8, 631–637 (2002).

  15. 15.

    Schmitt, A. et al. Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation. Transfusion 51, 591–599 (2011).

  16. 16.

    Stemberger, C. et al. Novel serial positive enrichment technology enables clinical multiparameter cell sorting. PLoS ONE 7, e35798 (2012).

  17. 17.

    Sabatino, M. et al. Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies. Blood 128, 519–528 (2016).

  18. 18.

    Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

  19. 19.

    Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

  20. 20.

    Robertson, D. L. & Joyce, G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344, 467–468 (1990).

  21. 21.

    Bunka, D. H. & Stockley, P. G. Aptamers come of age - at last. Nat. Rev. Microbiol. 4, 588–596 (2006).

  22. 22.

    Hernandez, L. I., Machado, I., Schafer, T. & Hernandez, F. J. Aptamers overview: selection, features and applications. Curr. Top. Med. Chem. 15, 1066–1081 (2015).

  23. 23.

    Zhou, J. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nat. Rev. Drug Discov. 16, 181–202 (2017).

  24. 24.

    Dunn, M. R., Jimenez, R. M. & Chaput, J. C. Analysis of aptamer discovery and technology. Nat. Rev. Chem. 1, 0076 (2017).

  25. 25.

    Daniels, D. A., Chen, H., Hicke, B. J., Swiderek, K. M. & Gold, L. A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment. Proc. Natl Acad. Sci. USA 100, 15416–15421 (2003).

  26. 26.

    Shangguan, D. et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc. Natl Acad. Sci. USA 103, 11838–11843 (2006).

  27. 27.

    Ogasawara, D., Hasegawa, H., Kaneko, K., Sode, K. & Ikebukuro, K. Screening of DNA aptamer against mouse prion protein by competitive selection. Prion 1, 248–254 (2007).

  28. 28.

    Sefah, K., Shangguan, D., Xiong, X., O’Donoghue, M. B. & Tan, W. Development of DNA aptamers using Cell-SELEX. Nat. Protoc. 5, 1169–1185 (2010).

  29. 29.

    Alam, K. K., Chang, J. L. & Burke, D. H. FASTAptamer: a bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections. Mol. Ther. Nucleic Acids 4, e230 (2015).

  30. 30.

    Caroli, J., Taccioli, C., De La Fuente, A., Serafini, P. & Bicciato, S. APTANI: a computational tool to select aptamers through sequence-structure motif analysis of HT-SELEX data. Bioinformatics 32, 161–164 (2015).

  31. 31.

    Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

  32. 32.

    Chen, L. et al. Aptamer-mediated efficient capture and release of T lymphocytes on nanostructured surfaces. Adv. Mater. 23, 4376–4380 (2011).

  33. 33.

    Li, S., Chen, N., Zhang, Z. & Wang, Y. Endonuclease-responsive aptamer-functionalized hydrogel coating for sequential catch and release of cancer cells. Biomaterials 34, 460–469 (2013).

  34. 34.

    Xu, Y. et al. Aptamer-based microfluidic device for enrichment, sorting, and detection of multiple cancer cells. Anal. Chem. 81, 7436–7442 (2009).

  35. 35.

    Yoon, J. W. et al. Isolation of foreign material-free endothelial progenitor cells using CD31 aptamer and therapeutic application for ischemic injury. PLoS ONE 10, e0131785 (2015).

  36. 36.

    Zhu, J., Nguyen, T., Pei, R., Stojanovic, M. & Lin, Q. Specific capture and temperature-mediated release of cells in an aptamer-based microfluidic device. Lab Chip 12, 3504–3513 (2012).

  37. 37.

    Labib, M. et al. Aptamer and antisense-mediated two-dimensional isolation of specific cancer cell subpopulations. J. Am. Chem. Soc. 138, 2476–2479 (2016).

  38. 38.

    Sun, N. et al. Chitosan nanofibers for specific capture and nondestructive release of CTCs assisted by pCBMA brushes. Small 12, 5090–5097 (2016).

  39. 39.

    Wan, Y. et al. Capture, isolation and release of cancer cells with aptamer-functionalized glass bead array. Lab Chip 12, 4693–4701 (2012).

  40. 40.

    Zhang, Z., Chen, N., Li, S., Battig, M. R. & Wang, Y. Programmable hydrogels for controlled cell catch and release using hybridized aptamers and complementary sequences. J. Am. Chem. Soc. 134, 15716–15719 (2012).

  41. 41.

    Nozari, A. & Berezovski, M. V. Aptamers for CD antigens: from cell profiling to activity modulation. Mol. Ther. Nucleic Acids 6, 29–44 (2017).

  42. 42.

    Wang, C.-W. et al. A new nucleic acid−based agent inhibits cytotoxic T lymphocyte−mediated immune disorders. J. Allergy Clin. Immunol. 132, 713–722 (2013).

  43. 43.

    Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 1585–1588 (2006).

  44. 44.

    Yurke, B. & Mills, A. P. Using DNA to power nanostructures. Genet. Program. Evol. Mach. 4, 111–122 (2003).

  45. 45.

    Yurke, B., Turberfield, A. J., Mills, A. P. Jr., Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605–608 (2000).

  46. 46.

    Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 3, 103–113 (2011).

  47. 47.

    Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).

  48. 48.

    Ruella, M. et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat. Med. 24, 1499–1503 (2018).

  49. 49.

    Heczey, A. et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood 124, 2824–2833 (2014).

  50. 50.

    Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

  51. 51.

    Zhao, Z. et al. Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells. Cancer Cell 28, 415–428 (2015).

  52. 52.

    Brentjens, R. J. et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nat. Med. 9, 279–286 (2003).

  53. 53.

    Dahotre, S. N., Chang, Y. M., Wieland, A., Stammen, S. R. & Kwong, G. A. Individually addressable and dynamic DNA gates for multiplexed cell sorting. Proc. Natl Acad. Sci. USA 115, 4357–4362 (2018).

  54. 54.

    Probst, C. E., Zrazhevskiy, P. & Gao, X. Rapid multitarget immunomagnetic separation through programmable DNA linker displacement. J. Am. Chem. Soc. 133, 17126–17129 (2011).

  55. 55.

    Gawande, B. N. et al. Selection of DNA aptamers with two modified bases. Proc. Natl Acad. Sci. USA 114, 2898–2903 (2017).

  56. 56.

    Ni, S. et al. Chemical modifications of nucleic acid aptamers for therapeutic purposes. Int. J. Mol. Sci. 18, 1683 (2017).

  57. 57.

    Pelloquin, F., Lamelin, J. & Lenoir, G. Human blymphocytes immortalization by epstein-barr virus in the presence of cyclosporin a. In Vitro Cell. Dev. Biol. 22, 689–694 (1986).

  58. 58.

    Zadeh, J. N. et al. NUPACK: analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170–173 (2011).

  59. 59.

    Tsai, H. H. et al. Regional astrocyte allocation regulates CNS synaptogenesis and repair. Science 337, 358–362 (2012).

  60. 60.

    Madugula, V. & Lu, L. A ternary complex comprising transportin1, Rab8 and the ciliary targeting signal directs proteins to ciliary membranes. J. Cell Sci. 129, 3922–3934 (2016).

  61. 61.

    Wang, J. et al. Optimizing adoptive polyclonal T cell immunotherapy of lymphomas, using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains. Hum. Gene Ther. 18, 712–725 (2007).

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Acknowledgements

This work was supported by a sponsored research agreement from Juno Therapeutics. We are grateful to C. Ramsborg (Juno Therapeutics), A. Bianchi (Juno Therapeutics), J. Shi (Juno Therapeutics), C. Chan (Juno Therapeutics), B. Olden (University of Washington) and J. Gustafson (Seattle Children’s Research Institute) for their critical discussion and helpful advice and to A. Mills (Juno Therapeutics) for manuscript feedback. We are also grateful to all Pun and Jensen Lab members, especially J. Yokoyama (Seattle Children’s Research Institute) and A. Johnson (Seattle Children’s Research Institute), for experimental support and helpful advice. We also thank the Baker Lab, especially B. Langan, for assistance with Octet BLI studies. We thank C. Saxby (University of Washington) and R. Mukherjee (Seattle Children’s Research Institute) for their valuable input relating to NGS and NanoString nCounter analysis, respectively. We thank M. Meechan (Seattle Children’s Research Institute) for assisting with mouse bioluminescence imaging and cage monitoring. We also thank members of the Statistical Consulting Program in the Departments of Biostatistics and Statistics, especially T. H. Wai (University of Washington), for their valuable input regarding the statistical analysis. We thank H. Y. Lin for preparing the SELEX and cell isolation figures. I. Cardle was supported partly by the National Cancer Institute of the National Institutes of Health under award no. 5T32CA080416-19 for research reported in this publication.

Author information

S.H.P. and M.C.J. conceived the idea and provided experimental advice and funding support. N.K., I.I.C. and S.H.P. designed the project. N.K. and I.I.C. conceived, performed and interpreted the experiments. N.K. designed and performed the SELEX procedure. I.I.C. and E.L.C. evaluated the binding of aptamer libraries and select aptamers and I.I.C., E.L.C., S.J.S. and N.K. analysed the NGS data. I.I.C. performed murine splenocyte and rhesus binding experiments. N.K., J.L.Y., I.I.C. and E.L.C. conducted receptor-binding studies using siRNA knockdown and gene transfection. I.I.C. and N.K. conducted antibody competition and Octet studies. J.L.Y. and E.L.C. performed binding curve studies and E.L.C. and I.I.C. evaluated aptamer binding to human PBMCs. N.K., I.I.C. and J.L.Y. optimized reversal agent and traceless cell isolation conditions. I.I.C. performed CAR T-cell production and characterization studies. M.L.B. conducted in vivo tumour studies and bioluminescence imaging. I.I.C. prepared the figures and performed statistical analyses. I.I.C., N.K., E.L.C. and S.H.P. wrote the manuscript.

Correspondence to Michael C. Jensen or Suzie H. Pun.

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S.H.P., M.C.J., N.K. and I.I.C. are co-inventors on two US provisional patent applications (nos. 62/699,438 and 62/779,946) for the aptamers and complementary reversal agents for traceless isolation described in this manuscript.

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