A joint team from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics has developed a feeder-free tumor-infiltrating lymphocyte (TIL) expansion protocol that significantly reduces dependence on high-dose interleukin-2 (IL-2), a major source of toxicity in TIL therapy. Published in Cancer Biology & Medicine (DOI: 10.20892/j.issn.2095-3941.2025.0441), the study demonstrates that this approach generates functional TILs across multiple solid tumor types and, when combined with low-dose PD-1 blockade, enhances tumor control while improving treatment tolerability in a colorectal cancer patient-derived xenograft (PDX) model.
Conventional TIL therapy, recently validated with the FDA approval of lifileucel for advanced melanoma, relies on high-concentration IL-2 (3,000-6,000 IU/mL) and feeder cells, which complicate manufacturing and promote T-cell exhaustion. The new protocol eliminates feeder cells entirely, using low-concentration IL-2 (2,000 IU/mL) supplemented with IL-7 and IL-15 during the pre-rapid expansion phase, followed by an even lower IL-2 concentration (300 IU/mL) with CD3/CD28 co-stimulation during the rapid expansion phase. This feeder-free system achieved expansion success rates of at least 90% across melanoma, pancreatic, gastric, cervical, and colorectal cancers, with melanoma-derived TILs expanding approximately 2,500-fold. The resulting TIL products exhibited high purity (CD45+CD3+ cells >93%), potent cytotoxic activity, and a less exhausted phenotype with minimal PD-1 expression (<0.5%) and a predominantly 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 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, though its in vivo benefit was limited.
"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 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 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. 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.
Funding for this study was provided by the Science and Technology Innovation Action Plan of the Science and Technology Commission of Shanghai Municipality (STCSM) (Grant No. 22XD1432200). The study was published in Cancer Biology & Medicine, a peer-reviewed open-access journal sponsored by China Anti-cancer Association and Tianjin Medical University Cancer Institute & Hospital.
