From endotoxin to engineered agonists: A translational strategy to enable TLR4-based immunomodulation in pancreatic cancer.

Abstract

e14605 Background: Pancreatic ductal adenocarcinoma (PDAC), a lethal malignancy with a dismal 5-year overall survival is highly resistant to immunotherapy. Previously, we established that TLR4 agonism using lipopolysaccharide (LPS) remarkably reduces tumor burden in PDAC by inducing an early inflammatory cytokine surge involving TNF⍺ and IFN𝛾, that promotes cytotoxic CD4⁺ T-cell-mediated antitumor immunity. However, the systemic toxicity of LPS represents a major barrier to clinical translation. Here, we sought to define whether TLR4-agonism mediated anti-tumor effects can be preserved while progressively mitigating toxicity through structural modification of LPS-derived molecules. Methods: KPC pancreatic cancer cells were implanted subcutaneously into syngeneic C57BL/6 mice. Tumor-bearing mice were treated with LPS, Lipid A (TLR4 binding moiety), and structurally modified TLR4 agonists that were generated through bacterial enzymatic combinatorial chemistry, called BECC compounds. Tumor growth was assessed and systemic toxicity was evaluated using murine sepsis scoring and physiologic monitoring. Immune activation was characterized by serum cytokine profiling, flow cytometric analysis of splenic and intratumoral immune populations, and ex vivo CD4⁺ T-cell cytotoxicity assays. Dose-response relationships were assessed where toxicity profiles permitted escalation. Results: As expected, LPS treatment resulted in significant tumor reduction but was associated with marked systemic toxicity. Lipid A failed to reproduce consistent antitumor efficacy in this PDAC model. This is consistent with a skewed Type I interferon (IFN) response, a typical characteristic of vaccine adjuvants, rather than a pro-inflammatory anti-tumor response. Guided by these structure-function observations, BECC compounds were engineered to selectively retain antitumor-relevant immune signaling while eliminating toxicity-associated moieties. Preliminary analyses demonstrate that select BECC compounds (including BECC44 and BECC470) exhibit minimal systemic toxicity and are associated with promising reductions in tumor burden based on endpoint tumor weight measurements. Mechanistic studies are ongoing to determine whether they engage the same immune pathways as defined for LPS. Conclusions: These findings support a structure-guided, iterative approach to reducing the risks associated with TLR4 agonism for cancer immunotherapy. By selectively modifying molecular features, antitumor immune activity may be preserved while progressively reducing systemic toxicity. Next-generation TLR4 agonists such as BECC compounds represent a promising therapeutic strategy for PDAC and warrant further optimization and mechanistic validation.