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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.

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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.


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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.

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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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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).

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