A new preclinical study published in Cancer Biology & Medicine (DOI: 10.20892/j.issn.2095-3941.2025.0441) presents a feeder-free tumor-infiltrating lymphocyte (TIL) expansion protocol that significantly reduces interleukin-2 (IL-2) dependence while maintaining robust anti-tumor activity. The study, conducted by a joint team from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics, further demonstrates that combining TIL therapy with low-dose programmed cell death protein 1 (PD-1) blockade enhances tumor control and treatment tolerability in a colorectal cancer patient-derived xenograft (PDX) model.
Conventional TIL therapy, clinically validated with the U.S. Food and Drug Administration approval of lifileucel for advanced melanoma, still faces substantial hurdles. The standard expansion process relies on high-concentration IL-2 (3,000–6,000 IU/mL) and feeder cells, which complicate manufacturing, promote T-cell exhaustion, and necessitate post-infusion systemic high-dose IL-2 administration with significant toxicity risks. Tumor immune evasion mechanisms, including major histocompatibility complex class I (MHC-I) down-regulation, further impair therapeutic efficacy.
The researchers designed a two-phase expansion protocol that eliminates feeder cells entirely. During the pre-rapid expansion protocol (pre-REP), TILs were cultured with low-concentration IL-2 (2,000 IU/mL) supplemented with IL-7 and IL-15. The subsequent rapid expansion protocol (REP) used an even lower concentration of IL-2 (300 IU/mL) alongside 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 TIL products demonstrated high purity (CD45+CD3+ cells >93%) and potent cytotoxic activity, with minimal PD-1 expression (<0.5%) and a predominantly effector memory T-cell composition.
In a colorectal cancer PDX model, the addition of low-dose PD-1 blockade (2 mg/kg) to TIL therapy significantly reduced tumor volume compared with 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, finding it up-regulated MHC-I expression on tumor cells in vitro without affecting PD-L1 levels or impairing TIL proliferation.
"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 findings carry significant implications for the future of 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 beyond specialized treatment centers. 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 in clinical practice. 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.
