Lymph node macrophages drive innate immune responses to enhance the anti-tumor efficacy of mRNA vaccines.

mRNA vaccines are promising for cancer treatment. Efficient delivery of mRNAs encoding tumor antigens to antigen-presenting cells (APCs) is critical to elicit anti-tumor immunity. Herein, we identified a novel lipid nanoparticle (LNP) formulation, L17-F05, for mRNA vaccines by screening 34 ionizable lipids and 28 LNP formulations using human primary APCs. Subcutaneous delivery of L17-F05 mRNA vaccine encoding Gp100 and Trp2 inhibited tumor growth and prolonged the survival of mice bearing B16F10 melanoma. L17-F05 efficiently delivered mRNAs to conventional dendritic cells (cDCs) and macrophages in draining lymph nodes (dLNs). cDCs functioned as the main APCs by presenting antigens along with enhanced expression of co-stimulatory molecules. Macrophages triggered innate immune responses centered on type-I interferon (IFN-I) in dLNs. Lymph node (LN) macrophage depletion attenuated APC maturation and anti-tumor activity of L17-F05 mRNA vaccines. Loss-of-function studies revealed that L17-F05 works as a self-adjuvant by activating the stimulator of interferon genes (STING) pathway in macrophages. Collectively, the self-adjuvanticity of L17-F05 triggered innate immune responses in LN macrophages via the STING-IFN-I pathway, contributing to APC maturation and potent anti-tumor activity of L17-F05 mRNA vaccines. Our findings provide strategies for further optimization of mRNA vaccines based on the innate immune response driven by LN macrophages.

Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency.

We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

Activation of adenosine A2A receptor by lipids from docosahexaenoic acid revealed by NMR.

The lipid composition of the plasma membrane is a key parameter in controlling signal transduction through G protein-coupled receptors (GPCRs). Adenosine A2A receptor (A2AAR) is located in the lipid bilayers of cells, containing acyl chains derived from docosahexaenoic acid (DHA). For the NMR studies, we prepared A2AAR in lipid bilayers of nanodiscs, containing DHA chains and other acyl chains. The DHA chains in nanodiscs enhanced the activation of G proteins by A2AAR. Our NMR studies revealed that the DHA chains redistribute the multiple conformations of A2AAR toward those preferable for G protein binding. In these conformations, the rotational angle of transmembrane helix 6 is similar to that in the A2AAR-G protein complex, suggesting that the population shift of the equilibrium causes the enhanced activation of G protein by A2AAR. These findings provide insights into the control of neurotransmissions by A2AAR and the effects of lipids on various GPCR functions.

Novel contamination-free wet milling technique using ice beads for poorly water-soluble compounds.

The aim of this study is to establish a contamination-free milling method using ice beads instead of conventional hard beads such as metal or ceramics. Ice beads, which melt after the milling process to form water, would solve the contamination issue attributed to bead breakage or abrasion. The technique/method for preparing spherical ice beads of mono-dispersed size ranging from 150 to 3000 µm was newly developed. An oscillation beads milling apparatus was used for pulverization. In the initial stages of ice beads milling, the process is dry, but as time passes, the surface of the ice beads begins to melt, resulting in a transition to wet beads milling. It was found that ice beads are an effective milling media for beads milling, and that milling efficiency is strongly affected by the temperature of the coolant, with the peak efficiency occurring when the temperature was set to -2 °C and ice beads around 1500 µm in diameter were used. The spray-dried powder obtained from suspension after ice beads milling had dissolution improvement equivalent to that obtained after zirconia beads milling, resulting from its spontaneous rapid dispersion into nanosuspension.

Evaluation of disintegration properties of orally rapidly disintegrating tablets using a novel disintegration tester.

This report describes a new disintegration tester that can determine not only the disintegration time of orally rapidly disintegrating tablets (ODT), but also the disintegration behavior and mechanism. Using the tester, the disintegration properties of the tablets prepared in a previous study were examined. The purpose of this study is to confirm the utility of the tester as an instrument for evaluating the disintegration properties of ODT and determine relations among time, behavior and mechanism of the disintegration. Results demonstrated that in vitro disintegration time in the tester is similar to that in the commercial disintegration tester for ODT and is highly correlated with oral disintegration time. Observations of disintegration process revealed that a difference in disintegration behavior between tablets compressed at 50-75 MPa and 100 MPa; the disintegration behavior of the tablets were designated immediate disintegrating type and gradual disintegrating type, respectively. The dynamic swelling profile and water absorption profile indicated that the disintegration mechanism of the tablets involved wicking action induced by swelling of the disintegrant; the disintegration time was closely related to the initial rates of swelling and water absorption. Furthermore, the mechanism of water absorption of tablets compressed at 50-75 MPa and 100 MPa shows anomalous diffusion and case-II transport, respectively. The shift in this mechanism is consistent with differences in disintegration time and behavior between the tablets. These findings suggest that information on disintegration properties obtained by our tester is useful for understanding of disintegration phenomena of ODT.