Although conventional TIL therapy has recently been clinically validated with the U.S. Food and Drug Administration’s approval of Rifilucel for advanced melanoma, it still faces significant hurdles. The standard expansion process relies on high concentrations of IL-2 (3,000–6,000 IU/mL) and feeder cells, such as irradiated peripheral blood mononuclear cells, to promote T cell expansion. This approach not only complicates manufacturing, but also promotes T cell depletion and requires large systemic administration of IL-2 after injection, which carries significant toxicity risks. Additionally, tumor immune evasion mechanisms, such as downregulation of major histocompatibility complex class I (MHC-I), 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 Advanced Department of Oncology of the Chinese People’s Liberation Army General Hospital and Shanghai Juncel Therapeutics has developed a feeder-free TIL expansion system using low concentrations of IL-2 and other components. Public (DOI: 10.20892/j.issn.2095-3941.2025.0441) Cancer biology and medicine this study demonstrates that this approach consistently generates functional TILs across multiple solid tumor types, including melanoma, pancreatic cancer, gastric cancer, cervical cancer, and colorectal cancer, and that when combined with a low dose of a PD-1 inhibitor, antitumor activity is significantly enhanced in a colorectal cancer patient-derived xenograft (PDX) model.
The researchers designed a two-step expansion protocol that completely removes feeder cells. In the first rapid pre-expansion protocol (pre-REP), TILs were cultured in low concentrations of IL-2 (2,000 IU/mL) supplemented with IL-7 and IL-15. In the subsequent rapid expansion protocol (REP), an even lower concentration of IL-2 (300 IU/mL) was used in parallel with CD3/CD28 costimulation. This feeder-free system achieved at least 90% growth success across multiple tumor types, with melanoma-derived TILs expanded approximately 2,500-fold. The resulting TIL products exhibited high purity (>93% CD45+CD3+ cells) and potent cytotoxic activity, secreted substantial interferon gamma (IFN-γ), and exhibited effector-to-target (E:T) ratio-dependent killing of tumor cells. Remarkably, 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, adding a low dose of PD-1 inhibitor (2 mg/kg) to TIL therapy significantly reduced tumor volume (P=0.002) compared to the control group and maintained higher body weight while completely preventing tumors. Ulceration - a complication observed in the TIL-only and control groups. Researchers also investigated hydroxychloroquine (HCQ) as an immunomodulator. HCQ significantly upregulated MHC-I expression on tumor cells and enhanced early-stage T cell receptor-modified T cells (TCR-T cells)-mediated tumor killing in vitro without affecting programmed death ligand 1 (PD-L1) levels or impairing TIL proliferation.
”Our goal was to eliminate the dependence of TIL therapy on high doses of IL-2, which is a major barrier to widespread clinical use.” said the authors.We have shown that functional and less wasting TILs can be generated from multiple tumor types by creating a feeder-free system with carefully tailored cytokine support. Addition of a low dose of PD-1 inhibitor not only improved the antitumor efficacy but also improved treatment tolerability, as mice in the combination group maintained better health status and avoided the ulceration observed with TILs alone. Although HCQ showed interesting immunomodulatory effects, the in vivo benefits were limited in this small study. We believe that this feeder-free IL-2 sparing strategy has real potential to make TIL therapy safer and more widely available for patients with solid tumors.. ”
This discovery has significant implications for the future of TIL-based immunotherapy. This protocol may simplify manufacturing and reduce manufacturing costs by eliminating feeder cells and reducing IL-2 doses, potentially making TIL therapy more affordable and available beyond specialized treatment centers. PD-1 inhibitors already have well-characterized safety profiles and are widely used in clinical practice, so the demonstration that low-dose PD-1 inhibitors can serve as an alternative to high-dose post-infusion IL-2 support addresses major safety concerns. This IL-2-independent strategy has already been investigated in clinical trials for advanced gynecological cancers, where early positive safety signals were obtained. Future studies should validate these findings across larger animal models and different tumor types and investigate the mechanisms underlying TIL persistence and tumor microenvironment regulation. If this approach is confirmed in clinical studies, it could expand the reach of TIL therapy to a broader population of patients with solid tumors who currently have limited treatment options.
sauce:
Chinese Academy of Sciences
Reference magazines:
Zhang Yu others. (2026). IL-2-independent tumor-infiltrating lymphocyte proliferation by feeder-free processes: a preclinical study in solid tumors. Cancer biology and medicine. DOI: 10.20892/j.issn.2095-3941.2025.0441. https://www.cancerbiomed.org/content/early/2026/03/05/j.issn.2095-3941.2025.0441
