Conditionally activatable fusogenic liposomes for precision cancer therapy

Introduction:: Macromolecular drugs, such as peptides, enzymes, intrabodies, and ribonucleoprotein particles (RNPs), are currently in development. These tools are designed to act directly on intracellular targets for applications ranging from disrupting metabolic abnormalities to regulating signaling pathways, to immune response modulation, as well as gene editing. However, on-target drug delivery to pathological environments and subsequent internalization by abnormal cells – either via translocation across the plasma membrane, or via endosomal escape – remain long-standing physiological and subcellular challenges that limit therapeutic efficacy. To address these issues, we harnessed the catalytic activity of proteases present at lesion sites to engineer a class of conditional fusogenic liposomes (C-FLIPs) to enable efficient cytosolic delivery of amnio acid-based biomacromolecules (e.g., peptides, proteins). Our hypothesis is that C-FLIPs fuse with target cells only after catalytic reactions performed by disease-dysregulated proteases successfully remove fusion inhibitors. This fusion reaction at the cell membrane subsequently releases impermeable payloads directly into the cytoplasm, thus bypassing endocytic pathways in which peptide bonds are susceptible to enzymatic degradation (Fig. 1). In contrast, C-FLIP fusogenicity is silenced in normal tissues and cells due to the absence of the target protease activity.


Materials and Methods:: C-FLIPs are comprised of 3 major components – fusion inhibitors, disease-specific protease substrates, and drug-loaded fusogenic liposomes (FLIPs). The C-FLIPs were conjugated with pre-designed fusion inhibitors via protease-activatable substrates. The C-FLIPs were subsequently optimized for substrate cleavage kinetics, for fusion with various cell types, and for delivering different peptide/protein-based biologics. We then leveraged the C-FLIPs to deliver protein drugs, termed synthetic T cells to assess their antitumor efficacy in two immunologically cold tumor models – lung and colorectal cancer. Synergistic effects of synthetic T cells with immune checkpoint blockade (ICB) and/or RNAi therapeutics were also evaluated in the same cancer models. Finally, we sought to understand the crosstalk between synthetic T cells and the immune microenvironment in tumors.

Results, Conclusions, and Discussions:: Our C-FLIPs have demonstrated in vivo functional capacity to fuse with various cells and to deliver cytotoxic proteins that induce cell death and peptide-based proteolysis-targeting chimera (PROTAC) to promote targeted protein degradation. Building on the optimal C-FLIP formulation, we developed targeted synthetic T cells that release granzyme family proteins into malignant cells in a protease-activatable, pore-forming motif-free (e.g. perforin) manner. Synthetic T cells via intravenous administration significantly suppressed tumor growth in mouse lung and colorectal tumor models by triggering gasdermin E-mediated pyroptosis, and increased infiltration of CD8+ effector T cells. Furthermore, ICB and/or silencing of intracellular serine protease inhibitors strongly synergized with the therapeutic impact of synthetic T cells, and even eradicated tumors in some of the treated animals. The combination of synthetic T cells and ICB also promotes the expansion of resident CD8+ memory T cells in the lungs, which prolongs improved protection against tumor recurrence and metastasis. Therefore, the non-viral, cell-free C-FLIPs represent a promising platform that addresses both imprecise delivery to target sites and inefficiencies in the cytosolic delivery of impermeable biomacromolecules, and could enable new opportunities in tackling intracellular delivery of a large palette of macromolecular therapeutics for immunotherapy and gene editing.


Acknowledgements (Optional): :

References (Optional): : B. Kim et al. Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus. Nat Comms, 2018.