Guest Column | May 30, 2025

The Latest On CRISPR Deployment In TIL Therapy Development

By Liang Li, Resilient Collective

DNA engineering, gene editing, genetic modification-GettyImages-1404205939

The first tumor-infiltrating lymphocyte (TIL) therapy was approved by the U.S. FDA in 2024.1 Traditional TIL manufacturing involves extracting T cells from a patient’s tumor, suspending them, expanding the cells ex vivo, and reinfusing them back into the patient. CRISPR/Cas9 gene editing has recently been incorporated into the TIL manufacturing process and clinical studies.2 This article explores the impact of CRISPR-engineered TIL therapy.

Impact Of CRISPR/Cas9 On TIL Manufacturability And Patient Delivery

TIL manufacturing typically takes approximately 22 days.3 Therefore, it's essential to assess whether incorporating CRISPR/Cas9 gene editing significantly affects this timeline. Recent studies demonstrate that non-viral, non-plasmid-based CRISPR/Cas9 gene editing — specifically for PD-1 knockout in TILs — achieves high efficiency.4 In this study, ribonucleoprotein (RNP) delivery systems, composed of single-guide RNA and Cas9 protein, were used.4 The RNPs, combined with an electroporation enhancer (EE), were introduced into TILs via electroporation.4

According to published protocols, the CRISPR/Cas9 editing step takes approximately one hour, which does not significantly extend the overall manufacturing timeline.4 These findings suggest that CRISPR/Cas9 integration — when using optimized RNP-based delivery — can be efficiently incorporated into the TIL workflow without major delays.

Impact Of CRISPR/Cas9 On The Tumor Microenvironment, PK, Efficacy, And Tolerability

Preclinical studies have shown that CRISPR/Cas9-edited TILs, in which the SOCS1 and Regnase-1 genes are inactivated, may overcome the immunosuppressive tumor microenvironment in solid tumors.5 For example, CRISPR/Cas9-mediated knockout of the TGFBR2 gene conferred resistance to TGF-β signaling in ovarian cancer TILs, though it potentially reduced tumor retention in vitro.6 Additionally, HER2-specific TILs with CD39 knockout demonstrated effective tumor clearance in colorectal cancer models, both in vitro and in vivo.7 Knocking out AKT1/2 in cervical cancer TILs enhanced activation, cytokine secretion, and cytotoxicity.8

Clinically, the safety and efficacy of CRISPR/Cas9-edited TILs — targeting SOCS1 and Regnase-1 — are under evaluation in Phase 1/2 trials for patients with various solid tumors, including NSCLC, HNSCC, colorectal cancer, melanoma, pancreatic cancer, and cervical cancer.2,9 Separately, CRISPR/Cas9-mediated knockout of the intracellular checkpoint gene CISH has shown promising antitumor activity in a Phase 1 trial involving metastatic colorectal cancer patients.10

Together, these studies support the potential of CRISPR to enhance TIL efficacy and tolerability, while broadening the range of tumor types responsive to this personalized therapy.

Impact Of CRISPR/Cas9 On Stability, Potency, And Mechanism Of Action

CRISPR/Cas9 modifications may influence both the stability and potency of TIL-based therapies. Early data indicate that gene-edited TILs can maintain functional tumor-killing activity post-editing. However, additional investigation is needed to fully assess long-term functionality, persistence, and safety. Understanding off-target effects and their implications on the mechanism of action remains a critical component of ongoing research and clinical validation.

In summary, CRISPR-engineered TIL therapy has demonstrated promising benefits for solid tumor treatment in vitro, in vivo, and in clinical trials. Ongoing studies continue to evaluate its clinical efficacy and underlying mechanisms of action to ensure safe and effective application of this innovative technology. Advanced CRISPR delivery methods — such as RNPs combined with electroporation — enable efficient gene editing without significantly extending the manufacturing timeline.

References:

  1. Carmen C. First Cancer TIL Therapy Gets FDA Approval for Advanced Melanoma.
  2. https://ksqtx.com/press-releases/ksq-therapeutics-announces-fda-clearance-of-ind-application-for-ksq-001ex-a-crispr-cas9-engineered-tumor-infiltrating-lymphocyte-etil-therapy/
  3. https://www.iovance.com/manufacturing/
  4. Chamberlain C A, Bennett E P, et al. Highly efficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptive T cell therapy. Molecular Therapy: Oncolytics. 2022, Vol. 24 March
  5. https://www.genengnews.com/topics/cancer/tils-tomorrow-bringing-more-cancers-under-immune-control/
  6. Fix S M, Forget M A, et al. CRISPR- mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-β signaling. Journal for ImmunoTherapy of Cancer. 2022, 10(7): e003750.
  7. Potenza A, Balestrieri C, et al. Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells. Gut. 2023, 72(10):1887–903.
  8. Feng H, Qiu L, et al. Modulation of intracellular kinase signaling to improve TIL stemness and function for adoptive cell therapy. Cancer Med. 2023, 12(3):3313–27.
  9. https://firstwordpharma.com/story/5948705
  10. Lou E L, Choudhry M S et al. Targeting the intracellular immune checkpoint CISH with CRISPR-Cas9-edited T cells in patients with metastatic colorectal cancer: a first-in-human, single-centre, phase 1 trial. The Lancet Oncology. 2025, Volume 26, Issue 5p559-570

About The Author:

Liang Li, Ph.D., is an independent consultant with the Resilient Collective group who focuses on cell therapy development. He received his Ph.D. in pharmacy and biomolecular science from the University of Brighton and an MBA from the University of Nottingham. Contact him on LinkedIn.