Poster S19 - Targeted granzyme B delivery with conditional nanocarriers induces pyroptosis and synergizes with checkpoint inhibitors in immunological cold tumors

Introduction:

Introduction: Although immune checkpoint inhibitors (ICIs) have emerged as a revolutionary type of cancer immunotherapy, a low response rate to the ICIs is especially prominent in immunologically ‘cold’ tumors as T cells are unable to penetrate tumor parenchyma. Pyroptosis, an inflammatory form of programmed cell death, holds great potential to sensitize ‘cold’ tumors to checkpoint blockade. Proteases—enzymes that break down proteins, play critical roles in tumorigenesis, and are broadly dysregulated and functionally distinct in almost every hallmark of cancer. Therefore, we harnessed catalytic activity of those dysregulated proteases in cancer to engineer novel conditional fusogenic lipid nanocarriers (C-FLIPs) that enable on-target delivery of granzyme B directly to the cytosol of cancer cells, which then efficiently triggers the pyrotosis via a granzyme B-caspase 3-gasdermin E axis. Conditionally targeted delivery relies on the hypothesis that C-FLIPs fuse with plasma membrane of cancer cells only after catalytic reactions performed by cancer-associated proteases remove fusion blockers. This fusion reaction subsequently releases impermeable granzyme B directly into cytoplasm (Fig. 1). In contrast, C-FLIP fusion capability is silenced in normal tissues and circulation due to absent target proteases to mitigate off-target toxicity.

Materials and

Methods: Materials and

Methods: C-FLIPs are comprised of 3 major components – fusion inhibitors, disease-specific protease substrates, and drug-loaded fusogenic liposomes. The C-FLIPs were conjugated with pre-designed fusion blockers via protease-activatable substrates. The C-FLIPs were subsequently optimized for substrate cleavage kinetics, for fusion with various cell types, and for delivering different protein-based biologics. We then leveraged the C-FLIPs to deliver granzyme B (C-FLIP/GzmB) to induce gasdermin E-mediated pyroptosis in immunologically cold lung tumor models and evaluate efficacy synergy of C-FLIP/GzmB with ICIs. Finally, we sought to mechanistically understand the crosstalk between C-FLIP/GzmB and tumor immune microenvironment by performing immune cell profiling.

Results, Conclusions, and Discussions: Our C-FLIPs that can be conditionally activated by disease-specific proteases have demonstrated in vivo functional capacity to fuse with various cells and to efficiently deliver peptide/protein-based biologics into the cytoplasm. We then optimized a C-FLIP formulation for targeted cytosolic granzyme B delivery into tumor cells in a pore-forming motif-free (e.g., perforin) manner. The C-FLIP/GzmB administered via intravenous administration substantially suppressed tumor growth in a genetically modified lung tumors (KrasG12D;Trp53fl/fl) in mouse models by triggering gasdermin E-mediated pyroptosis. Strikingly, the combination of C-FLIP/GzmB with an ICI blocking programmed cell death protein 1 (αPD-1) attains tumor regression in all treated mice and even eradicated lung tumors in some treated animals. This may be attributed to the increased penetration of C-FLIP/GzmB into the immunologically cold lung tumors where infiltrated cytotoxic lymphocytes are always excluded. Immune cell profiling reveals that the pyroptosis triggered by these penetrating C-FLIP/GzmB inflames tumor microenvironment by promoting the infiltration of effector CD8+ T cells, down-regulating regulatory CD4+ T cells and increasing the population of stem-like CD8+ T cells (progenitor of effector T cells). The combination of C-FLIP/GzmB with αPD-1 further augment these immune-inflaming functions.

Concusion: Targeted cytosolic delivery of granzyme B with conditionally activatable nanocarriers inflames cold tumors by triggering pyroptosis and synergizes with immune checkpoint inhibitors. Broadly, the non-viral, cell-free C-FLIPs represent a promising platform that not only addresses imprecise delivery to target sites and inefficiency in the cytosolic delivery of impermeable biomacromolecules, and could open new opportunities for intracellular delivery of a large spectrum of macromolecular therapeutics for immunotherapy and gene editing.

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