Abstract LB028: Reprogramming of tumor-infiltrating immune cells using a tumor-associated macrophage-targeted TLR7 agonist
Authors: Napoleon John Victor , Zhang Fenghua , Alfar Rami A. , Wolt Taylor A. , Low Philip S. 
Published: April 2023

Abstract
Myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) are two of the more immunosuppressive cell types in a tumor microenvironment (TME). Although TAMs and MDSCs can be converted from tumor-supporting to tumor-suppressing phenotypes by treatment with proinflammatory immune stimulants (e.g. TLR7 agonists) in vitro, such strategies have proven to be too toxic in vivo because of their systemic activation of immune cells. Therefore, for such immune stimulants to be used to reprogram the immune system in solid tumors, they must be targeted specifically to the immune cells in the TME.
One strategy for targeting TAMs/MDSCs is to exploit a receptor that is expressed solely on these cell types. Because folate receptor beta (FRβ) is expressed exclusively on myeloid cells and since FRβ acquires its ability to bind folic acid (FA) only in inflamed tissues, we have previously exploited FA to target attached TLR7 agonists (TLR7a) to FRβ+ TAMs/MDSCs in solid tumors. While significant repolarization of TAMs and MDSCs was observed in this previous study, premature systemic release of the attached TLR7a caused unwanted toxicity. However, since FRβ was later found to reside in the same endosome as TLR7, we hypothesized that constructing a more stable FA-TLR7a conjugate with a non-cleavable linker should not only prevent premature TLR7a release, but also deposit the FA-TLR7a into an endosome containing a TLR7 receptor, thereby enabling selective activation of TAMs and MDSCs in the TME. The data summarized here confirm this hypothesis. Thus, free TLR7-1A was found to stimulate massive production of IL-6, TNF-α and IFNγ upon addition to human peripheral blood in vitro or injected into live mice, suggesting cells in both species respond aggressively to TLR7-1A. However, performance of the same studies with FA-targeted TLR7-1A stimulated barely or nondetectable levels of the inflammatory cytokines, confirming that FA-TLR7-1A is unable to enter and activate cells that lack a folate receptor (FRβ). More importantly, intravenous administration of FA-TLR7-1A into mice bearing an orthotopic 4T1 breast tumor totally blocked metastases and induced tumor regression without causing detectable toxicity. Flow cytometric analyses of the residual tumor tissue further demonstrated that FA-TLR7-1A increased M1/M2 macrophage ratios and elevated infiltration of CD8+ T cells into the tumor masses. Taken together, these data demonstrate that a targeted TLR7 agonist can rejuvenate immune cells in a solid tumor without systemically activating the immune system.
Citation Format: John Victor Napoleon, Fenghua Zhang, Rami A. Alfar, Taylor A. Wolt, Philip S. Low. Reprogramming of tumor-infiltrating immune cells using a tumor-associated macrophage-targeted TLR7 agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB028.