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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  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).

    Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  5. 5.

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

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  10. 10.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  16. 16.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  18. 18.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  21. 21.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  23. 23.

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

    CAS  Article  Google Scholar 

  24. 24.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    PubMed  Google Scholar 

  31. 31.

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

    CAS  Article  Google Scholar 

  32. 32.

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

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  43. 43.

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

    CAS  Article  Google Scholar 

  44. 44.

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

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  46. 46.

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

    CAS  Article  Google Scholar 

  47. 47.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  50. 50.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  55. 55.

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

    CAS  Article  Google Scholar 

  56. 56.

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

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  58. 58.

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

    CAS  Article  Google Scholar 

  59. 59.

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

Download references


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.

Corresponding authors

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

Ethics declarations

Competing interests

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.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Information

Supplementary figures and tables.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kacherovsky, N., Cardle, I.I., Cheng, E.L. et al. Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy. Nat Biomed Eng 3, 783–795 (2019).

Download citation

Further reading


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing