Immune checkpoint therapies (ICT) have revolutionized cancer treatment, but resistance remains a significant hurdle. Primary resistance, inherent to the tumor's characteristics, and adaptive resistance, which develops during treatment, limit the effectiveness of ICT. This necessitates the exploration of novel therapeutic strategies to enhance anti-tumor immunity and overcome resistance. Innate immune activators, such as Toll-like receptor (TLR) agonists, are promising candidates for combination therapies due to their ability to stimulate the immune system. TLR5, a receptor for bacterial flagellin, is a key component of the innate immune response. This study focuses on evaluating the efficacy of TLR5 agonists, flagellin and its derivative CBLB502, in combination with ICT against established, highly refractory tumor models in mice. The study aims to determine if TLR5 activation can enhance anti-tumor immunity, overcome ICT resistance, and establish long-term survival and immunological memory. The importance of this research lies in its potential to identify a new and effective combination therapy for patients with ICT-resistant cancers, addressing a significant unmet medical need. The highly refractory nature of triple-negative breast cancer (TNBC) and the poor immunogenicity of melanoma present significant challenges to current cancer therapies, making these models ideally suited to evaluate the potential benefits of TLR5 agonists in conjunction with ICT. Understanding the mechanisms behind the observed antitumor effects and identifying potential predictive biomarkers will be crucial for translating these findings into clinical practice. The overall goal is to provide a foundation for the development of a novel therapeutic strategy that improves outcomes for patients with ICT-resistant cancers.

Numerous studies have explored the use of immune checkpoint inhibitors (ICT) in cancer treatment, such as anti-PD-1 and anti-CTLA-4 antibodies. While these therapies have shown remarkable success in some patients, resistance remains a major challenge. Primary resistance, inherent to the tumor's properties, and acquired resistance, which arises during treatment, often lead to treatment failure. Researchers have explored various strategies to overcome ICT resistance, including the combination of ICT with other immunotherapies or conventional therapies. The role of innate immunity in shaping the adaptive immune response and influencing cancer treatment outcomes has gained increasing attention. Toll-like receptors (TLRs), particularly TLR5, which recognizes bacterial flagellin, have been studied for their potential to enhance anti-tumor immunity. Previous research has shown promising results in preclinical models using TLR5 agonists in combination with other therapies. However, comprehensive investigations into the use of TLR5 agonists to specifically overcome ICT resistance in established, highly refractory tumor models are limited. This gap in knowledge highlights the need for further research in this area.

This study utilized syngeneic mouse models of highly refractory triple-negative breast cancer (4T1) and poorly immunogenic melanoma (B16-F10). The 4T1 cell line was stably transfected with a bioluminescent reporter to monitor tumor growth in vivo. In vitro studies assessed NF-κB activation in 4T1 cells following treatment with flagellin and CBLB502, a flagellin derivative. Cytokine profiles were analyzed using protein arrays. In vivo studies evaluated the therapeutic efficacy of intratumoral and intraperitoneal administrations of flagellin and CBLB502, alone and in combination with anti-CTLA-4 and anti-PD-1 antibodies. Treatment regimens and doses are detailed in Table 1. Bioluminescence imaging (BLI) was used to monitor tumor growth. Kaplan-Meier survival curves assessed the impact of treatment on overall survival. Immunological memory was assessed by tumor re-challenge. Peripheral blood cytokine profiles were analyzed in long-term survivors and non-survivors. Tumor immune infiltrates were analyzed by flow cytometry. Statistical analyses included Kaplan-Meier analysis, Log-rank test, Gehan-Breslow-Wilcoxon test, Pearson correlation, and receiver operating characteristic (ROC) curve analysis.

The study's key findings demonstrate that combining TLR5 agonists with ICT significantly enhanced survival in both 4T1 and B16-F10 tumor models. Intratumoral administration of flagellin or CBLB502 (low dose) combined with ICT resulted in a substantial increase in long-term survival compared to ICT alone or TLR5 agonist alone. Long-term survivors showed immunological memory upon tumor re-challenge. A distinctive cytokine profile was observed in long-term survivors, characterized by low serum levels of G-CSF and CXCL5 and high levels of IL-15. These cytokines emerged as potential predictive biomarkers for treatment response. TLR5 was found to be essential for the observed anti-tumor response as evidenced by the lack of efficacy in TLR5 knockout mice. Analysis of tumor immune infiltrates revealed that long-term survivors had a shift in the tumor microenvironment from immune suppression to immune activation, characterized by a decrease in myeloid-derived suppressor cells (MDSCs) and an increase in CD3+ and CD4+ T cells. Bioluminescence imaging (BLI) at week 3 showed strong predictive power (AUC = 0.75) for survival at week 12 or later. This provides a non-invasive method to assess treatment response and predict patient outcomes.

These findings support the development of a novel therapeutic strategy for treating ICT-refractory solid tumors by combining TLR5 agonists with ICT. The synergistic effect observed likely stems from the ability of TLR5 agonists to activate innate immunity, thereby enhancing the efficacy of ICT-mediated adaptive immune responses. The identification of G-CSF, CXCL5, and IL-15 as potential biomarkers is crucial for clinical translation. Low G-CSF and CXCL5 levels and high IL-15 levels may serve as predictive indicators of a positive treatment response. The dependence of this therapeutic effect on TLR5 suggests that the presence of functional TLR5 is necessary for optimal combination therapy efficacy. Future studies should investigate the specific mechanisms of action involved, explore the optimal dosing strategies for TLR5 agonists and ICT, and evaluate the combination therapy's efficacy in other cancer types and in larger clinical trials.

This study demonstrates the potential of combining TLR5 agonists with immune checkpoint therapy to overcome resistance and improve outcomes in patients with ICT-refractory solid tumors. The identification of potential biomarkers and the observed immunological memory suggest a promising therapeutic avenue for future clinical investigation. Further research should focus on optimizing treatment regimens, investigating the specific mechanisms of action, and conducting larger clinical trials to confirm these findings in humans. This approach offers a new strategy for enhancing anti-tumor immunity and improving survival rates in patients with currently difficult-to-treat cancers.

The study was conducted using preclinical mouse models, and the results may not fully translate to human patients. The sample size in some analyses could have been larger to enhance statistical power, particularly in the biomarker analysis. The study primarily focused on intratumoral administration; further research is needed to determine the efficacy of systemic delivery methods. While potential biomarkers were identified, more research is required to validate their clinical utility and predictive accuracy in humans.