Article Text

Download PDFPDF

333 Targeting the apical intracellular checkpoint CISH unleashes T cell neoantigen reactivity and effector program
  1. Douglas Palmer1,
  2. Beau Webber2,
  3. Yogin Patel1,
  4. Matthew Johnson2,
  5. Christine Kariya1,
  6. Walker Lahr2,
  7. Maria Parkhurst1,
  8. Jared Gartner1,
  9. Todd Prickett1,
  10. Frank Lowery1,
  11. Rigel Kishton1,
  12. Devikala Gurusamy3,
  13. Zulmarie Franco1,
  14. Suman Vodnala1,
  15. Miechaleen Diers2,
  16. Natalie Wolf2,
  17. Nicholas Slipek2,
  18. David McKenna2,
  19. Darin Sumstad2,
  20. Lydia Viney4,
  21. Tom Henley4,
  22. Tilmann Bürckstümmer4,
  23. Oliver Baker4,
  24. Ying Hu5,
  25. Chunhua Yan5,
  26. Daoud Meerzaman5,
  27. Kartik Padhan6,
  28. Winnie Lo1,
  29. Parisa Malekzadeh1,
  30. Li Jia1,
  31. Drew Deniger1,
  32. Shashank Patel1,
  33. Paul Robbins1,
  34. R Scott McIvor2,
  35. Modassir Choudhry4,
  36. Steven Rosenberg1,
  37. Branden Moriarity2 and
  38. Nicholas Restifo1
  1. 1Surgery Branch, NCI, Bethesda, MD, USA
  2. 2University of Minnesota, Minneapolis, MN, USA
  3. 3Surgery Branch, NIH, Bethesda, USA
  4. 4Intima Biosciences, London, UK
  5. 5The Center for Biomedical Informatics and Information Technology, NIH, Bethesda, USA
  6. 6National Institute of Allergy and Infectious Disease, NIH, Bethesda, USA


Background Neoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL.

Methods To explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing.

Results CISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy.

Conclusions These pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted.

Trial Registration NCT04426669

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. 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.