Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has emerged as a powerful immunotherapy for solid tumors, yet its broader application is constrained by reliance on high-dose interleukin-2 (IL-2), a cytokine that activates T cells but also drives T-cell exhaustion and causes severe toxicity. In a new preclinical study, researchers developed a feeder-free TIL expansion protocol that significantly reduces IL-2 dependence while maintaining robust anti-tumor activity. The study further demonstrates that combining TIL therapy with low-dose programmed cell death protein 1 (PD-1) blockade enhances tumor control and treatment tolerability, offering a potential pathway toward safer and more accessible TIL-based immunotherapies for a range of solid malignancies.
Conventional TIL therapy, clinically validated recently 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 such as irradiated peripheral blood mononuclear cells to drive T-cell proliferation. This approach not only complicates manufacturing but also promotes T-cell exhaustion and necessitates post-infusion systemic high-dose IL-2 administration, which carries significant toxicity risks. Moreover, tumor immune evasion mechanisms, including major histocompatibility complex class I (MHC-I) down-regulation, further impair CD8+ T-cell recognition and limit therapeutic efficacy. Based on these challenges, there is an unmet need to develop TIL expansion strategies that reduce IL-2 dependence, preserve T-cell function, and integrate complementary approaches to overcome tumor immune evasion.
A joint team from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics developed a feeder-free TIL expansion system using low-concentration IL-2 and other ingredients. Published in Cancer Biology & Medicine (DOI:10.20892/j.issn.2095-3941.2025.0441), the study demonstrates that this approach consistently generates functional TILs across multiple solid tumor types—including melanoma, pancreatic, gastric, cervical, and colorectal cancers—and that combining it with low-dose PD-1 blockade significantly enhances anti-tumor activity in a colorectal cancer patient-derived xenograft (PDX) model.
The researchers designed a two-phase expansion protocol that eliminates feeder cells entirely. During the initial 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, with melanoma-derived TILs expanding approximately 2,500-fold. The resulting TIL products demonstrated high purity (CD45+CD3+ cells >93%) and potent cytotoxic activity, secreting substantial interferon-gamma (IFN-γ) and exhibiting effector-to-target (E:T) ratio-dependent tumor cell killing. Notably, expanded TILs exhibited features consistent with a less exhausted phenotype, including 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; HCQ significantly up-regulated MHC-I expression on tumor cells in vitro without affecting programmed death-ligand 1 (PD-L1) levels or impairing TIL proliferation, and enhanced early-phase T-cell receptor-engineered T cell (TCR-T cell)-mediated tumor-killing.
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, and investigate the underlying mechanisms of TIL persistence and tumor microenvironment modulation. 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.
