Article Text

Download PDFPDF

240 Presentation of DLL4 and VCAM on paramagnetic beads replicates thymic Notch signaling and enables differentiation of functional of iPSC derived TCR T-cells in vitro
  1. Siddarth Chandrasekaran1,
  2. Melanie Kardel1,
  3. Elisa Martinez1,
  4. Nabil Zeidan2,
  5. Avisek Deyati1,
  6. Valerie Wall3,
  7. Dylan Lee2,
  8. Negin Farahani1,
  9. Amanda AuYeung2,
  10. Omar Subedar2,
  11. Michael Cadell3,
  12. Sommer Apelu3,
  13. Laura Prochazka3,
  14. Zahra Sahaf2,
  15. Angela Duong1,
  16. Cole Zmurchok1,
  17. Boyoung Yoon1,
  18. Dan Kirouac3,
  19. Muluken Belew2,
  20. Jasdeep Mann3,
  21. Deepika Rajesh3 and
  22. Chris Bond3
  1. 1Notch Therapeutics, Vancouver, BC, Canada
  2. 2Notch Therapeutics, Toronto, ON, Canada
  3. 3Notch Therapeutics, Seattle, WA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Adoptive TCR T-cell therapies from autologous donor are expensive, time-consuming and depends on quality of T-cells in patients. One of the key challenges for autologous TCR therapy is the requirement of cell numbers that are orders of magnitude higher than CAR-T cells. This has several implications including the inability to manufacture enough cells for a repeat dosing regimen. To this end, we have developed a scalable method by which TCR+ CD8+ lymphocytes can be generated from induced pluripotent stem cells (iPSCs) using 3D engineered thymic niche (ETN) comprised of DLL4- and VCAM-conjugated paramagnetic microbeads.

Methods We have successfully developed a gene-editing workflow for generation and characterization of iPSC clones engineered to express an exogenous TCR. We utilized a defined scalable differentiation process to generate lymphoid competent CD34+ HPCs from multiple iPSC clones. Cryopreserved CD34 cells were differentiated to Pro T cells and CD8+TCR+ cells by leveraging stage specific control of Notch signalling using the 3D ETN. Flow cytometry and single-cell RNA sequencing was used to characterize the phenotypic and transcriptional state of iPSC derived effector cells. Incucyte-based cytotoxic killing assay was used to demonstrate antigen specific effector cell proliferation and cytotoxic function. We have developed a computational tool to quantify the average number of target cells killed by each input effector cell per unit time.

Results Multiple iPSC engineered clones expressing a TCR were successfully differentiated to hematopoietic precursor cells with an average efficiency of ~2.5 lympho-competent HPCs per input iPSC without magnetic sorting. HPCs were further differentiated in the presence of proprietary ETN beads to generate mature single positive T cells expressing TCR. The end stage cells expressed T-cell markers mimicking mature peripheral blood T-cells with a 90% TCR expression detected by an antigen-loaded tetramer. Transcriptional profiling of iPSC-derived and peripheral blood-derived CD8+ T-cells revealed similar gene expression signatures. Cryopreserved end-of-process CD8+ cells displayed target-specific cytotoxic activity against antigen expressing tumor cell lines in vitro across 4 rounds of stimulation in a long-term serial killing assay. iPSC derived CD8 TCR T-cells secreted perforin, granzyme B, IFNg, TNFa and GM-CSF and expanded ~1000-fold in response to target antigen.

Conclusions We have successfully generated CD8+TCR+ T-cells from iPSCs using a defined process that is amenable to scale up. iPSCs derived TCR T-cells, when combined with genetic manipulations to enhance persistence in a suppressive tumor microenvironment and overcome allogeneic rejection, could lead to a new generation of TCR T-cell therapies.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.