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350 Enhancing directly selected tumor-reactive TIL function through genetic modification
  1. Emily Carron,
  2. Sowbarnika Ratliff,
  3. April Fraley,
  4. Justin Rahman,
  5. Niloufar Khojandi,
  6. Bryant Thompson,
  7. Mathew Thayer,
  8. Christophe Pedros,
  9. Larissa Pikor and
  10. Barbara Sennino
  1. Turnstone Biologics, San Diego, CA, 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 cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) has emerged as a potential treatment of various types of solid tumors. However, responsiveness to TIL therapy can be limited by several mechanisms including tumor mediated-immune suppression, limited persistence, exhaustion, and toxicity associated with high dose IL2 treatment.1 We believe the ability to genetically modify TIL has the potential to overcome these limitations, increase the activity of TIL therapy, and expand ACT to a broad range of solid tumor types.2 Herein, we demonstrate the process of genetically engineering directly selected tumor reactive TIL using CRISPR-Cas9 and show that knocking out specific genes of interest can enhance TIL phenotype and function.

Methods Following selection for tumor reactivity, patient-derived TIL underwent CRISPR-Cas9 mediated editing of the PDCD1 locus or other loci of interest followed by rapid expansion. At the end of production, knockout of gene targets of interest was confirmed by flow cytometry, western blot, and Inference of CRISPR Edits analysis. Gene edited and control TIL were characterized in vitro by phenotyping, serial killing assays, intracellular cytokine staining, and autologous tumor reactivity assays.

Results Using CRISPR-Cas9, we were able to generate directly selected knockout TIL with gene editing efficiencies up to 95% with 70% efficiency at the PDCD1 locus. There was no significant change in rapid expansion or viability between gene edited and unedited samples. There was no improvement in memory phenotype in PD1 knockout TIL; however, ablation of other gene targets (Target X and Y) resulted in increased central memory populations. Polyfunctional cytokine secretion was improved in both PD1 knockout TIL and the other targets tested. In serial killing assays, PD1 KO TIL exhibited similar cytolysis as control and unedited TIL while knockout against Target X exhibited enhanced cytolysis in the absence of cytokine support. Moreover, coculture of tumor cells with autologous selected tumor reactive Target X knockout TIL led to increased tumor cell expression of cleaved-caspase 3 as compared to the selected tumor reactive control TIL.

Conclusions Collectively, these studies demonstrate that directly selected tumor reactive TIL can be genetically engineered and expanded, and that CRISPR-Cas9 mediated-knockout of specific loci is a strategy for enhancing TIL phenotype and function.


  1. Zhao Y, Deng J, Rao S, Guo S, Shen J, Du F, Wu X, Chen Y, Li M, Chen M, Li X, Li W, Gu L, Sun Y, Zhang Z, Wen Q, Xiao Z, Li J. Tumor infiltrating lymphocyte (TIL) therapy for solid tumor treatment: Progressions and challenges. Cancers, 2022;14(17):4160.

  2. Weinstein-Marom H, Gross G, Levi M, Brayer H, Schachter J, Itzhaki O, Besser MJ. Genetic modification of tumor-infiltrating lymphocytes via retroviral transduction. Frontiers in Immunology, 2021;11.

Ethics Approval For human sample procurement, we utilize commercial vendors that are in compliance with applicable regulations and ethical guidelines. This includes 45 CFR 46 (Common Rule) and Good Clinical Practice (GCP) which stipulate project approval by an Institutional Review Board (IRB) or Independent Ethics Committee (IEC), along with informed consent for living donors.

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

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