Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)

Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1β release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1β release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.