Abstract B136: Optimized messenger RNA immunolipoplexes for cancer immunotherapy: Balancing immunogenicity and adjuvancy
Authors: Verbeke Rein , Lentacker Ine , Breckpot Karine , Calenbergh Serge Van , Smedt Stefaan C. De , Dewitte Heleen 
Published: February 2019

Abstract
Messenger RNA has garnered a lot of attention as a new therapeutic drug class for vaccination (1). Particularly for cancer immunotherapy, mRNA encoding tumor antigens has the potential to design personalized and effective cancer vaccines (2). However, the major challenge remains to directly deliver the mRNA to (professional) antigen presenting cells (APCs), evoking safe and effective antitumor immunity. The mRNA vaccines that are currently being evaluated in first-in-human clinical trials depend on the self-adjuvant effect of mRNA and subsequent signaling via type I IFN (3-4). We and others have reported that type I IFNs have the downside of inducing anti-mRNA (anti-viral) innate responses, which makes it challenging to strike a balance between evoking innate immunity and obtaining adequate levels of mRNA expression (5-6). In addition, high levels of IFN-α are known to cause adverse effects (e.g., flu-like symptoms and autoimmune sequelae) (7).To overcome these issues, we developed a nanoparticle platform which protects mRNA against degradation while successfully delivering mRNA to APCs in vivo (6). In this platform, we choose to minimize the mRNA-based immune recognition using a nucleoside-modified (“immune-silent”) mRNA construct, but instead to co-package clinically-approved immune adjuvants (e.g., MPLA) to achieve strong and controllable immunogenicity. The vaccine potential of mRNA lipid nanoparticles with different immune adjuvants was evaluated by performing biodistribution and immunogenicity studies after systemic delivery in mice. The preclinical antitumor efficacy was assessed in an EG7-OVA lymphoma and B16-OVA melanoma model. In addition to evaluating overall survival, experiments were performed where tumors were isolated after immunization and screened for effector responses (e.g., antigen-specific CD8+ T-cells and NK cells) and suppressive mechanisms that could impact the therapeutic outcome (e.g., immune checkpoints, myeloid derived suppressor cells [MDSCs] and tumor-associated macrophages [TAMs]). To tackle adaptive resistance to activated T-cells, we evaluated a combinatory therapy of the mRNA vaccine with anti-PDL1 antibodies (8). Upon i.v. injection in mice, these particles are mainly detected within APCs (macrophages and dendritic cells) in lungs and spleen. Importantly, this resulted in high mRNA expression as well as functional activation of the particle-loaded immune cells, marked by cytokine production of IL-12p70 and IFN-γ. We were able to optimize a formulation of adjuvanted-nanoparticles with modified mRNA, which resulted in 6 to 7 times higher numbers of tumor-infiltrating antigen-specific T-cells compared to unmodified mRNA particles. In addition to CD8+ T-cell responses, we also observed a 2- to 3-fold increase in intratumoral NK cells, compared to untreated mice. Furthermore, these mice exhibited reduced immune suppression at the tumor site: low numbers of MDSCs whereas TAMs displayed proinflammatory M1-like changes in phenotype. Moreover, we observed clear synergistic antitumor effects between the mRNA nanoparticles and anti-PDL1 checkpoint blocking antibodies. Taken together, we have developed a flexible and versatile mRNA nanoparticle platform, that presents an attractive way of initiating antitumor immunity by targeting and activating immune cells directly in vivo. Importantly, by combining immune-silent mRNA with immune-adjuvants in a single particle, a safe, effective and controllable immune response can be evoked, which can be strengthened by rational combination with state-of-the-art clinically approved immunotherapies. References: 1. Pardi N, et al. Nature Reviews. Drug Discovery 2018. 2. Sahin U, et al. Nature 2017. 3. Kranz L, et al. Nature 2016. 4. Alberer M, et al. Lancet 2017. 5. De Beuckelaer A, et al. Trends in Molecular Medicine 2017. 6. Verbeke R, et al. Journal of Controlled Release 2017. 7. Jonasch E. Oncologist 2001. 8. Sharma P. Cell 2017.
Citation Format: Rein Verbeke, Ine Lentacker, Karine Breckpot, Serge Van Calenbergh, Stefaan C. De Smedt, Heleen Dewitte. Optimized messenger RNA immunolipoplexes for cancer immunotherapy: Balancing immunogenicity and adjuvancy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B136.