A preclinical study published in Cancer Biology & Medicine (DOI:10.20892/j.issn.2095-3941.2025.0441) describes a novel tumor-infiltrating lymphocyte (TIL) expansion platform that significantly reduces dependence on high-dose interleukin-2 (IL-2), a cytokine associated with severe toxicity and T-cell exhaustion. The feeder-free system, developed by researchers from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics, maintains robust anti-tumor activity while simplifying manufacturing and improving tolerability when combined with low-dose PD-1 blockade.
Conventional TIL therapy, recently validated by the FDA approval of lifileucel for advanced melanoma, relies on high-concentration IL-2 (3,000-6,000 IU/mL) and feeder cells, such as irradiated peripheral blood mononuclear cells, to drive T-cell proliferation. This process complicates manufacturing, promotes T-cell exhaustion, and necessitates systemic high-dose IL-2 administration post-infusion, which carries significant toxicity risks. Additionally, tumor immune evasion mechanisms, including MHC-I down-regulation, limit therapeutic efficacy.
The researchers designed a two-phase expansion protocol eliminating feeder cells entirely. In 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 IL-2 concentration (300 IU/mL) alongside CD3/CD28 co-stimulation. The feeder-free system achieved expansion success rates of at least 90% across multiple tumor types, including melanoma, pancreatic, gastric, cervical, and colorectal cancers. Melanoma-derived TILs expanded approximately 2,500-fold, with high purity (CD45+CD3+ cells >93%) and potent cytotoxic activity. Expanded TILs exhibited a less exhausted phenotype, with minimal PD-1 expression (<0.5%) and a predominantly effector memory T-cell composition.
In a colorectal cancer patient-derived xenograft (PDX) model, combining TIL therapy with low-dose PD-1 blockade (2 mg/kg) 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; HCQ significantly up-regulated 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 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 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. If confirmed in clinical studies, this approach could expand the reach of 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 Science and Technology Commission of Shanghai Municipality (Grant No. 22XD1432200). For more details, see the original publication at https://doi.org/10.20892/j.issn.2095-3941.2025.0441.
