Abstract 715: Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment
Authors: Andon Fernando Torres , Pensado-López Alba , Anfray Clement , Ummarino Aldo , Fernández-Mariño Iago , Sanjurjo Lucia , Crecente-Campo Jose , Fernández-Megía Eduardo , Castro Flavia , Oliveira Maria Jose , Calvo Alfonso , Campelo Rosario García , Mantovani Alberto , Alonso Maria Jose , Allavena Paola 
Published: March 2024

Abstract
The stroma of solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Increasing evidence demonstrates their ability to hamper cancer patients' response to most treatments currently applied in the clinic, including immunotherapy. Therefore, strategies to counteract negative effects of TAMs are nowadays gaining momentum at preclinical, translational, and clinical levels.
Here, we present the development and evaluation of nanoparticles (NPs) loaded with TLR agonists and/or inhibitors of immunosuppressive pathways to reprogram TAMs and the tumor microenvironment (TME) to unleash an effective immune response to fight against the tumor. Firstly, we have evaluated in vitro the ability of poly(I:C) and/or R848, agonists of TLR3 and TLR7/8 respectively, to reprogram TAMs into antitumor effector cells, and also the efficacy of Stattic and/or Galunisertib, inhibitors of STAT3 and TGF-β pathways respectively, to inhibit immunosuppression by cancer cells and/or TAMs. The best combinations of these drugs were encapsulated in nanoemulsions or polymeric nanocapsules for improved TAM-targeting and pharmacokinetics in vivo. These NPs were characterized for their physicochemical properties and also tested in vitro using primary human macrophages. For in vivo evaluation, subcutaneous and orthotopic murine models of lung cancer (CMT167) were used, showing antitumoral efficacy and TME reprogramming as evaluated by FACS, RNA analysis and multiplex immunofluorescence. Protamine-NCs-loaded with poly(I:C)+R848 and coated with an additional layer of hyaluronic acid functionalized with mannose were used to target the CD206 receptors, showing antitumoral efficacy mediated by TAM reprogramming, evaluated as higher CD86 while lower CD206 and Arg1 expression. Nanoemulsions with a PEGylated surfactant, encapsulating Stattic+Galunisertib+R848 showed faster antitumoral activity versus the free drugs. Experiments performed in IFN-γ KO mice and immune deficient mice (NSG and Balb/c nude) revealed that a fully functional immune system is crucial for the response to the treatment.
In conclusion, our work demonstrates the antitumoral efficacy and reprogramming of the tumor microenvironment by combinations of TLR agonists and/or inhibitors of immunosuppression, which can be improved by nanotechnological approaches. Further investigations are ongoing to assess their antitumoral efficacy in other tumor models (i.e. breast and pancreatic cancer), with the final aim of clinical translation.
Citation Format: Fernando Torres Andon, Alba Pensado-López, Clement Anfray, Aldo Ummarino, Iago Fernández-Mariño, Lucia Sanjurjo, Jose Crecente-Campo, Eduardo Fernández-Megía, Flavia Castro, Maria Jose Oliveira, Alfonso Calvo, Rosario García Campelo, Alberto Mantovani, Maria Jose Alonso, Paola Allavena. Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 715.