Clinical manufacture of CRISPR/Cas9 knockout tumor infiltrating lymphocytes to treat gastrointestinal cancers

Gastrointestinal (GI) cancer is a highly aggressive malignancy that accounts for 22.5% of cancer deaths globally. Novel therapeutic strategies are urgently required, but while treatments such as adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) have demonstrated efficacy for hematologic cancers, their application to solid tumors has been less successful. The use of CRISPR/Cas9 to knockout (KO) the intracellular immune checkpoint, cytokine-induced SH2 protein (CISH), has been shown to improve the efficacy of mutation reactive (MR) TIL and provide durable regression of established tumors in an animal model. Now, this approach has been optimized for current good manufacturing practices (cGMP) by scientists at the University of Minnesota to support a first-in-human clinical trial to treat patients with metastatic GI cancers. 

Clinical manufacturing for CISH edited TIL 

The manufacturing process for CISH-edited TIL involves three main steps, beginning with tumor resection, fragmentation, and TIL outgrowth. Briefly, metastatic tumor biopsies from patients with various GI cancers are cut into multiple fragments. The fragments are then seeded into 24-well plates for culture in the presence of IL-2 with the aid of an automated liquid handling system. After 6 weeks, TIL are harvested and cryopreserved for further processing. 

During step two, whole exome sequencing and RNA-seq are conducted on tumors and patient-derived peripheral blood mononuclear cells (PBMCs) to identify single nucleotide variants (SNVs) resulting in an amino acid change. Peptides containing these mutations are then synthesized, loaded onto patient-matched antigen-presenting cells, and cocultured with TIL, allowing for neoantigen-reactive TIL to be identified by cytokine production and surface activation marker upregulation. 

In the third and final step, neoantigen-reactive TIL fragments are stimulated with OKT3 antibody and gene edited to knockout CISH. Knockout is accomplished using wild-type spCas9-encoding CleanCap® (Cap 1) capped mRNA and CISH guide RNA, both provided by TriLink. The TIL are then expanded in large-scale containers, prior to being frozen, assessed for lot release conditions including % viability and % editing efficiency, and infused into patients at the clinician’s discretion. 

Observations and future perspectives 

Of the 22 patients enrolled in this trial, the surgery for two individuals did not yield sufficient viable tissue to continue with the study. Six of the remaining patients could not be dosed due to disease progression and one chose to withdraw from the trial, leaving 13 individuals to receive treatment. 

Key findings included the observation that tumor resected from the lung versus the liver allowed for more fragments to pass quality control after TIL outgrowth. It was also established that the greater the cell input for CISH editing and expansion, the more TIL were generated for the final product. Additionally, a comparison of Western blot and Tracking of Indels by DEcomposition (TIDE) to quantify the loss of CISH protein in the final product showed Western blot to consistently report a greater rate of knockout, possibly due to a reduction of CISH protein in the residual non-knockout cells in the population. 

Importantly, this trial demonstrates successful translation of CRISPR/Cas9-based CISH KO MR TIL from a basic research lab to a cGMP facility. However, in common with other TIL-based therapies, the method is limited by an extended manufacturing time that could prevent some patients from receiving treatment. Ways of addressing this challenge include banking tumor material earlier in disease progression and developing novel methods for MR enrichment that avoid the current labor-intensive neoantigen screening protocol. 

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Article reference: Johnson MJ, Sumstad D, Folsom TD, et al. Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers. Cytotherapy. Published online June 21, 2025. doi:10.1016/j.jcyt.2025.06.007