Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown promise for solid tumors, but its broader use is limited by reliance on high-dose interleukin-2 (IL-2), which drives T-cell exhaustion and causes severe toxicity. In a new preclinical study published in Cancer Biology & Medicine, researchers from the Senior Department of Oncology of Chinese PLA General Hospital and Shanghai Juncell Therapeutics developed a feeder-free TIL expansion system that significantly reduces IL-2 dependence while maintaining robust anti-tumor activity. The study also demonstrates that combining this approach 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.
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 such as irradiated peripheral blood mononuclear cells. This approach complicates manufacturing, 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.
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—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, 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 patient-derived xenograft (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.
"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. We believe this feeder-free, IL-2-sparing strategy has real potential to make TIL therapy safer and more widely available for patients with solid tumors."
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.
