Manipulating Tumor-Infiltrating Lymphocytes for Cancer Therapy

Cancer immunotherapy continues to evolve beyond first-generation approaches, as researchers seek to overcome the limitations of existing treatments in solid tumors. While therapies such as CAR T cells have demonstrated remarkable success in hematological malignancies, their translation into solid tumor settings remains challenging due to antigen heterogeneity and immunosuppressive microenvironments.

Tumor-infiltrating lymphocytes (TILs)—and more recently, CRISPR-edited TILs—are emerging as a promising alternative. 

Dr. Beau Webber, an associate professor at the University of Minnesota, is working to develop gene-edited TIL therapies to enhance anti-tumor immunity. His research explores how genome engineering can improve T-cell function, persistence, and therapeutic efficacy. Technology Networks spoke with Webber to discuss how TILs differ from other cell therapies, how CRISPR editing is reshaping their capabilities, and what recent clinical findings reveal about their future potential.

Broad antigen recognition vs single-target engineering in cell therapies 

How do TILs and gene-edited TILs differ from other immune cell-based therapies, like CAR T-cell therapy? 

CAR T-cell therapy is built around a highly targeted strategy: patient T cells are genetically engineered to express a synthetic receptor directed at a single tumor-associated antigen. This precision has driven successes in cancers where the target is uniformly expressed, such as CD19-positive B cell malignancies. However, this same specificity limits their application in solid tumors, where antigen expression is often heterogeneous. 

By contrast, TILs are derived directly from a patient’s tumor, providing an inherently diverse and polyclonal population of T cells. These cells recognize multiple tumor-specific neoantigens via their native T-cell receptors (TCRs), allowing them to respond to a broader array of cancer targets. This characteristic is particularly important in solid tumors, where antigen variability can undermine single-target approaches. 

“Gene-edited TILs represent the next evolution,” explained Webber. “While traditional TIL therapy relies on the cells' natural fitness, we use CRISPR to ‘upgrade’ them.” Rather than introducing new receptors, genome editing is used to enhance the intrinsic capabilities of these cells.  

“By knocking out inhibitory genes like CISH, we remove the molecular brakes that tumors use to shut down or evade immune responses,” said Webber. “This creates a therapy that combines the broad recognition capabilities of natural TILs with the enhanced persistence and potency afforded by genome editing.” 

Key differences between TILs and CAR T-cell therapies: 

• TILs recognize multiple neoantigens, while CAR T-cells typically target a single surface antigen 

• Polyclonal TIL populations are better suited to heterogeneous solid tumors 

• CRISPR editing enhances natural TIL function rather than adding synthetic receptors 

Engineering TIL fitness through CRISPR-mediated checkpoint removal 

How do you approach engineering TILs to reshape their activation, persistence, or metabolic fitness in the tumor microenvironment? 

The tumor microenvironment imposes a range of suppressive signals that dampen T-cell activity. One strategy to counteract this involves targeting intracellular checkpoint pathways that regulate TCR signaling. Webber’s group focuses on the CISH gene, which encodes a protein that acts as a negative regulator of T-cell activation. 

Using CRISPR/Cas9, the team deletes CISH from TILs, effectively removing an internal “brake” that limits their responsiveness. This intervention amplifies TCR signaling, enabling T cells to respond more robustly to tumor antigens. The result is a marked increase in functional avidity—the ability of T cells to detect and respond to even low levels of antigen. 

This heightened sensitivity translates into improved anti-tumor activity. Edited TILs demonstrate stronger cytokine production and enhanced cytotoxicity, key features for effective tumor clearance. “Essentially, CISH-deleted TILs become ‘hypersensitive’ to the tumor in the best way possible,” said Webber. 

 CRISPR strategies enhancing TIL function:  

• Knockout of CISH removes intracellular inhibitory signaling 

• Increased functional avidity improves detection of low antigen levels 

• Enhanced cytokine production and cytolytic activity boost tumor killing 

First-in-human CRISPR-edited TIL trial demonstrates safety and early efficacy 

Can you tell us more about the CRISPR-edited TILs trialed in humans for advanced gastrointestinal cancer? What were the key takeaways from this trial? 

Translating CRISPR-edited TILs into the clinic represents a significant milestone in cell therapy. In a trial involving patients with advanced gastrointestinal cancers who had exhausted standard treatments, Webber’s team evaluated the safety and feasibility of CISH-edited TILs. 

Scaling Genome Editing From Discovery to Manufacturing

In this webinar, our expert speaker will discuss how non-viral electroporation-based genome editing supports applications ranging from CRISPR screening to complex cell engineering.
View Webinar

Advertisement

“This was a trial of multiple firsts,” he said. “This was the first time that CRISPR had been deployed in a TIL therapy, and the first time that CISH knockout T cells had been tested in humans.” 

One achievement from the trial was demonstrating that CRISPR editing could be successfully integrated into TIL manufacturing without compromising cell viability. This was a critical step, as the production process is already complex and highly individualized.  

Encouraging signs of clinical activity were also observed. Half of the patients experienced disease stabilization, and notably, one patient achieved a durable complete response that has persisted for three years. While early-stage, these results underscore the therapeutic potential of engineered TILs in difficult-to-treat cancers.  

Clinical outcomes and implications: 

• CRISPR editing integrated into TIL manufacturing successfully 

• First-in-human demonstration of CISH knockout T cells 

• Evidence of disease stabilization and durable response in advanced cancer 

Overcoming manufacturing and scalability barriers in TIL therapy 

What challenges are associated with using TILs for cancer therapy, and what innovations do you see as key for overcoming these hurdles? 

Despite their potential, TIL therapies face significant logistical and scalability challenges. The process requires tumor resection, ex vivo expansion, and, in some cases, neoantigen selection before large-scale expansion and reinfusion. “The long 'vein-to-vein' time can be critical for patients with rapidly progressing disease,” noted Webber. 

Another limitation lies in the variability of tumor samples. Not all biopsies yield sufficient numbers of high-quality, tumor-reactive TILs, making consistent manufacturing difficult. These challenges have historically constrained the scalability of TIL-based therapies. 

Synthetic Biology: Engineering Life

This infographic provides a clear overview of the workflow, technologies, and applications shaping synthetic biology.
View Infographic

Advertisement

To address these issues, researchers are developing faster and more efficient manufacturing methods, such as accelerated expansion protocols and techniques to enrich for neoantigen-reactive T cells without labor-intensive screening.  

“We are also implementing multiplex, non-viral engineering methods to further enhance the TIL to increase their persistence and resilience in the tumor microenvironment,” said Webber. 

Innovations improving TIL scalability: 

• Accelerated manufacturing protocols reduce production timelines 

• Enrichment strategies improve yield of tumor-reactive TILs 

• Multiplex and non-viral engineering enhance cell durability 

Combining CRISPR-edited TILs with checkpoint inhibitors to target “cold” tumors 

Looking ahead, how might CRISPR‑engineered TILs integrate with existing therapies, and are there combinations that you think have potential to unlock responses in historically “cold” tumors? 

Combination therapies are likely to play a central role in maximizing the efficacy of CRISPR-engineered TILs. One particularly promising strategy involves pairing these cells with immune checkpoint inhibitors (ICIs), such as PD-1 blockade therapies. 

“While ICIs like pembrolizumab release the ‘external’ brakes, our gene-edited TILs have their ‘internal’ brakes removed. Together, this creates a dual-layered assault that is much harder for the tumor to suppress.”— Dr. Beau Webber. 

Preclinical studies from the group have already demonstrated synergy between CISH-edited TILs and PD-1 inhibitors. Such combinations could be especially valuable in “cold” tumors, which typically lack sufficient immune infiltration and are resistant to immunotherapy alone.  

Combination strategies advancing immunotherapy: 

• CRISPR edits remove internal T cell inhibition 

• Checkpoint inhibitors release external immune suppression 

• Synergistic effects may improve responses in immunologically “cold” tumors  

Advancing multiplex genome editing in next-generation TIL therapies 

At the American Society of Gene & Cell Therapy meeting 2026, Webber presented insights from translating CRISPR-edited TILs into clinical practice. His talk reflected both the successes and the challenges encountered during the development of first-in-human therapies. 

Advancing AAV Characterization Through Mass Photometry

This infographic shows how single-particle mass photometry delivers AAV purity analytics in less than five minutes while supporting development and GMP manufacturing workflows.
View Infographic

Advertisement

Beyond single-gene editing, his group has now advanced into multiplex genome engineering, enabling simultaneous modification of multiple targets within T cells. This approach opens new possibilities for refining TIL function, improving persistence and overcoming multiple suppressive pathways at once. 

These developments signal a shift towards increasingly sophisticated cell therapies that integrate multiple layers of engineering to enhance efficacy.  

Next-generation TIL engineering trends:

• Transition from single-gene to multiplex CRISPR editing 

• Focus on improving multiple aspects of T-cell performance simultaneously 

• Ongoing clinical translation informing next-stage innovation 

This content includes text that has been created with the assistance of generative AI and has undergone editorial review before publishing. Technology Networks' AI policy can be found here.