Feeder-Free TIL Expansion Platform Reduces IL-2 Dependence and Enhances Anti-Tumor Efficacy in Preclinical Study
核心洞察
Researchers developed a feeder-free TIL expansion protocol using low-concentration IL-2 (2,000 IU/mL pre-REP, 300 IU/mL REP) supplemented with IL-7 and IL-15, eliminating reliance on high-dose IL-2 and feeder cells.
The protocol achieved at least 90% expansion success rates across melanoma (搜索), pancreatic, gastric, cervical, and colorectal cancers, with melanoma TILs expanding approximately 2,500-fold and demonstrating high purity (CD45+CD3+ cells ≥93%).
In a colorectal cancer (搜索) PDX model, combining TIL therapy with low-dose PD-1 (搜索) blockade (2 mg/kg) significantly reduced tumor volume (P = 0.002) and completely prevented tumor ulceration compared to TIL-only and control groups.
A joint research team from the Senior Department of Oncology of Chinese PLA General Hospital (搜索) and Shanghai Juncell Therapeutics (搜索) has developed a novel feeder-free tumor-infiltrating lymphocyte (TIL) expansion platform that dramatically reduces dependence on high-dose interleukin-2 (IL-2), addressing one of the most significant barriers to broader clinical adoption of TIL therapy. Published in Cancer Biology & Medicine, the preclinical study demonstrates that combining this IL-2-sparing expansion protocol with low-dose PD-1 (搜索) blockade significantly enhances anti-tumor activity while improving treatment tolerability.
Conventional TIL therapy, which recently gained U.S. Food and Drug Administration approval with lifileucel for advanced melanoma (搜索), 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 complicates manufacturing, promotes T-cell exhaustion, and necessitates post-infusion systemic high-dose IL-2 administration with significant toxicity risks. Additionally, tumor immune evasion mechanisms, including MHC-I (搜索) down-regulation, further impair CD8+ T-cell recognition and limit therapeutic efficacy.
A Feeder-Free, IL-2-Sparing Expansion Protocol
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 (搜索), pancreatic, gastric, cervical, and colorectal cancers. Melanoma-derived TILs expanded approximately 2,500-fold. The resulting TIL products demonstrated high purity, with CD45+CD3+ cells reaching at least 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 (搜索) expression (approximately 0.5%) and a predominantly effector memory T-cell composition.
Combination with PD-1 (搜索) Blockade Improves Outcomes
In a colorectal cancer (搜索) patient-derived xenograft (PDX) model, the addition of low-dose PD-1 (搜索) blockade (2 mg/kg) to TIL therapy significantly reduced tumor volume compared with the control group (P = 0.002). Mice in the combination group maintained higher body weights, and the combination therapy completely prevented tumor ulceration — a complication observed in both the TIL-only and control groups.
The researchers also explored hydroxychloroquine (HCQ) as an immunomodulatory agent. HCQ significantly up-regulated MHC-I (搜索) expression on tumor cells in vitro without affecting PD-L1 (搜索) levels or impairing TIL proliferation, and enhanced early-phase T-cell receptor-engineered T cell (TCR-T cell)-mediated tumor killing. However, its in vivo benefit was limited in this small study.
"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 stated. "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 (搜索) blockade not only boosted anti-tumor efficacy but also improved treatment tolerability, as mice in the combination group maintained better health and avoided the ulceration observed with TILs alone."
Implications for Clinical Translation
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 (搜索) 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.