Conventional TIL therapy, recently clinically validated with the US Food and Drug Administration’s approval of lifileucel for advanced melanoma, faces significant hurdles. The standard expansion procedure relies on high concentration IL-2 (3,000-6,000 IU/mL) and feeder cells such as irradiated peripheral blood mononuclear cells to promote T-cell proliferation. This approach not only complicates production, but also promotes T-cell exhaustion and requires systemic administration of a high dose of IL-2 after infusion, which carries significant risks of toxicity. In addition, tumor immune evasion mechanisms, including major histocompatibility complex class I (MHC-I) downregulation, further damage CD8+ T-cell recognition and limitation of therapeutic efficacy. Based on these challenges, there is an outstanding need to develop TIL expansion strategies that reduce IL-2 dependence, preserve T-cell function, and incorporate 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 components. Published (DOI: 10.20892/j.issn.2095-3941.2025.0441) in Cancer Biology & Medicine study shows that this approach stably generates functional TILs in multiple types of solid tumors—including melanoma, pancreatic, gastric, cervical, and colon—and that its combination with low-dose PD-1 blockade significantly enhances antitumor activity in a colon cancer patient-derived xenograft (PDX) model.
The researchers designed a two-phase expansion protocol that completely eliminates feeder cells. 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% in multiple tumor types, with melanoma-derived TILs expanding approximately 2,500-fold. The resulting TIL products showed high purity (CD45+CD3+ cells >93%) and potent cytotoxic activity, secreting significant interferon-gamma (IFN-γ) and demonstrating effector-to-target (E:T) ratio-dependent killing of tumor cells. Specifically, expanded TILs displayed features consistent with a less exhausted phenotype, including minimal PD-1 expression (<0.5%) and a predominantly effector memory T-cell composition. In a PDX model of colon cancer, adding low-dose PD-1 blockade (2 mg/kg) to treatment with TILs with higher body volume (compared to 0, higher volume) weights while completely preventing tumor ulceration—a complication seen only in TIL and control groups. Researchers have also investigated hydroxychloroquine (HCQ) as an immunomodulatory agent. HCQ significantly upregulated 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-mediated tumor killing.
“We aimed to eliminate the dependence of TIL therapy on high-dose IL-2, which has been a major barrier to wider clinical use,” the authors said.By creating a feeder-free system with carefully calibrated cytokine support, we have shown that we can generate functional, less depleted TILs from multiple tumor types. The addition of low-dose PD-1 blockade not only enhanced antitumor efficacy but also improved tolerability to the treatment, as mice in the combination group maintained better health and avoided ulceration seen with TILs alone. While HCQ showed interesting immunomodulatory effects, its in vivo benefit was limited in this small study. We believe that this non-feedback IL-2 sparing strategy has real potential to make TIL therapy safer and more widely available for patients with solid tumors.”
The findings have important implications for the future of TIL-based immunotherapy. By eliminating feeder cells and reducing IL-2 doses, the protocol simplifies manufacturing and may reduce production costs, potentially making TIL therapy more affordable and accessible beyond specialized treatment centers. The demonstration that low-dose PD-1 blockade can serve as an alternative to high-dose IL-2 support after infusion 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 investigated in a clinical trial for advanced gynecological cancers with early favorable safety signals. Future research will need to validate these findings in larger animal models and in various tumor types and investigate the underlying mechanisms of TIL persistence and shaping the tumor microenvironment. If confirmed in clinical studies, this approach could extend the reach of TIL therapy to a wider population of patients with solid tumors who currently have limited treatment options.
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