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 platform that significantly reduces reliance on high-dose interleukin-2 (IL-2), a major source of toxicity in TIL therapy. The approach, developed by a joint team from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics, consistently generates functional TILs across multiple solid tumor types and demonstrates that combining TIL therapy 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, clinically validated with FDA approval of lifileucel for advanced melanoma, relies on high-concentration IL-2 (3,000-6,000 IU/mL) and feeder cells to drive T-cell proliferation. This process complicates manufacturing, promotes T-cell exhaustion, and necessitates post-infusion systemic high-dose IL-2 administration, which carries significant toxicity risks. Additionally, tumor immune evasion mechanisms such as MHC-I down-regulation further limit efficacy. The new protocol addresses these challenges by eliminating feeder cells and using lower IL-2 concentrations: 2,000 IU/mL during the pre-rapid expansion phase (supplemented with IL-7 and IL-15) and 300 IU/mL during the rapid expansion phase (with CD3/CD28 co-stimulation).
The 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 with substantial interferon-gamma secretion, and a less exhausted phenotype characterized by minimal PD-1 expression (<0.5%) and a predominance of effector memory T cells. In the PDX model, adding low-dose PD-1 blockade (2 mg/kg) to TIL therapy significantly reduced tumor volume compared to controls (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 that it 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. However, its in vivo benefit was limited in this small study.
By simplifying manufacturing and reducing IL-2 doses, the protocol may lower production costs and expand access to TIL therapy beyond specialized centers. The use of low-dose PD-1 blockade as an alternative to post-infusion high-dose IL-2 addresses a major safety concern, as PD-1 inhibitors are 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 clinically, this approach could broaden the reach of TIL therapy to more patients with solid tumors who currently have limited options.
The study was funded by the Science and Technology Commission of Shanghai Municipality (Grant No. 22XD1432200). More information about the journal can be found at Chuanlink Innovations.
