Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers

Matthew J Johnson ¹, Darin Sumstad ², Timothy D Folsom ¹

¹Department of Pediatrics, Division of Hematology and Oncology, University of Minnesota, Minneapolis, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, USA; Center for Genomic Engineering, University of Minnesota, Minneapolis, USA.
²Cell Therapy Laboratory, M Health, Fairview, University of Minnesota Medical Center, Minneapolis, USA.
³Cell Therapy Laboratory, M Health, Fairview, University of Minnesota Medical Center, Minneapolis, USA; Molecular & Cellular Therapeutics, University of Minnesota, Minneapolis, USA.
⁴Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, USA.
⁵Department of Obstetrics, Gynecology, and Women's Health, University of Minnesota, Minneapolis, USA.
⁶Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, USA.
⁷Intima Bioscience Inc., New York, New York, USA.
⁸Department of Pediatrics, Division of Hematology and Oncology, University of Minnesota, Minneapolis, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, USA; Center for Genomic Engineering, University of Minnesota, Minneapolis, USA; Stem Cell Institute, University of Minnesota, Minneapolis, USA.
⁹Cell Therapy Laboratory, M Health, Fairview, University of Minnesota Medical Center, Minneapolis, USA; Molecular & Cellular Therapeutics, University of Minnesota, Minneapolis, USA; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, USA; Stem Cell Institute, University of Minnesota, Minneapolis, USA.

⁰Department of Pediatrics, Division of Hematology and Oncology, University of Minnesota, Minneapolis, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, USA; Center for Genomic Engineering, University of Minnesota, Minneapolis, USA.

Cytotherapy +

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July 17, 2025

View abstract on PubMed

Summary

This study successfully translated CRISPR/Cas9 gene editing of tumor-infiltrating lymphocytes (TIL) to knockout CISH for treating metastatic gastrointestinal cancers. The optimized process supports a first-in-human clinical trial, aiming to improve patient outcomes.

Area Of Science

Oncology
Immunotherapy
Gene Editing

Background

Stage IV gastrointestinal carcinomas have a poor prognosis, with a 15% five-year survival rate for colorectal cancer.
Tumor-infiltrating lymphocytes (TIL) show promise for cancer treatment, but their efficacy can be limited.
CISH (Cotsar, Inhibitor of STAT signaling) is an intracellular checkpoint protein that can be knocked out to enhance T cell function.

Purpose Of The Study

To isolate mutation-reactive TIL and employ CRISPR/Cas9 to knockout CISH, enhancing T cell expansion and function.
To develop an optimized, large-scale manufacturing process for CISH-knockout TIL.
To support a first-in-human clinical trial for metastatic GI cancers.

Main Methods

TIL were initiated from tumor fragments, expanded, and mutation-reactive TIL were identified using neoantigen peptides.
CRISPR/Cas9 was used to knockout CISH in TIL via electroporation of Cas9 mRNA and guide RNA.
Expanded TIL were cryopreserved and underwent rigorous lot release testing, including CISH editing efficiency and protein loss assessment.

Main Results

Nineteen of 22 tumor biopsies proceeded to knockout and expansion, yielding a mean fold expansion of 327.1.
The mean CISH knockout efficiency was 75%, with a mean editing efficiency of 59.9%.
Thirteen patients received TIL therapy; six were not treated due to disease progression.

Conclusions

The translation of CRISPR/Cas9-based CISH knockout TIL from research to current good manufacturing practices (cGMP) was successful.
This process allows for optimized, large-scale expansion of TIL for clinical application.
The developed method supports a first-in-human clinical trial for patients with metastatic GI cancers.

Keywords:

CISH (cytokine-inducible SH2-containing protein) CRISPR/cas9 Tumor-infiltrating lymphocytes (TIL) cGMP manufacturing gastrointestinal cancer