A preclinical study published in Cancer Biology & Medicine (DOI: 10.20892/j.issn.2095-3941.2025.0441) presents a novel feeder-free tumor-infiltrating lymphocyte (TIL) expansion platform that significantly reduces reliance on high-dose interleukin-2 (IL-2), a major barrier to broader clinical use of TIL therapy. The study, conducted by researchers from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics, demonstrates that combining this approach with low-dose PD-1 blockade enhances anti-tumor activity while improving treatment tolerability in a colorectal cancer patient-derived xenograft (PDX) model.
Conventional TIL therapy, despite FDA approval of lifileucel for advanced melanoma, faces challenges including reliance on high-dose IL-2 (3,000-6,000 IU/mL) and feeder cells, which complicate manufacturing, promote T-cell exhaustion, and cause severe toxicity. The new protocol eliminates feeder cells entirely. During the pre-rapid expansion protocol (pre-REP), TILs are cultured with low-concentration IL-2 (2,000 IU/mL) plus IL-7 and IL-15. The subsequent rapid expansion protocol (REP) uses even lower IL-2 (300 IU/mL) with CD3/CD28 co-stimulation. This feeder-free system achieved expansion success rates of at least 90% across multiple tumor types, including melanoma, pancreatic, gastric, cervical, and colorectal cancers, with melanoma-derived TILs expanding approximately 2,500-fold. The resulting TILs showed high purity (CD45+CD3+ cells >93%), potent cytotoxic activity, and a less exhausted phenotype with minimal PD-1 expression (<0.5%) and a predominant effector memory T-cell composition.
In a colorectal cancer PDX model, adding low-dose PD-1 blockade (2 mg/kg) to TIL therapy significantly reduced tumor volume compared to the control group (P = 0.002) and maintained higher body weights, while completely preventing tumor ulceration—a complication observed in TIL-only and control groups. The researchers also explored hydroxychloroquine (HCQ) as an immunomodulatory agent; HCQ upregulated MHC-I expression on tumor cells in vitro without affecting PD-L1 levels or impairing TIL proliferation, and enhanced early-phase TCR-T cell-mediated tumor-killing.
"Our goal was to eliminate TIL therapy's dependency on high-dose IL-2, which has been a major barrier to broader clinical use," the authors said. "By creating a feeder-free system with carefully calibrated cytokine support, we've shown that we can generate functional, less exhausted TILs from multiple tumor types. The addition of low-dose PD-1 blockade not only boosted anti-tumor efficacy but also improved treatment tolerability."
The findings carry significant implications for TIL-based immunotherapy. By eliminating feeder cells and reducing IL-2 doses, the protocol simplifies manufacturing and may lower production costs, potentially making TIL therapy more affordable and accessible. The demonstration that low-dose PD-1 blockade may serve as an alternative to post-infusion high-dose IL-2 support addresses a major safety concern, as PD-1 inhibitors are already widely used with well-characterized safety profiles. This IL-2-independent strategy has already been explored in a clinical trial for advanced gynecologic cancers with early favorable safety signals. Future research will need to validate these findings in larger animal models and across diverse tumor types, and investigate mechanisms of TIL persistence and tumor microenvironment modulation. If confirmed, this approach could expand TIL therapy to a broader population of patients with solid tumors who currently have limited treatment options.
The study was supported by the Science and Technology Innovation Action Plan of the Shanghai Municipality (Grant No. 22XD1432200). The original source article can be accessed at https://doi.org/10.20892/j.issn.2095-3941.2025.0441.
