Targeted Cell-to-Cell Delivery of Protein Payloads via the Granzyme-Perforin Pathway

Abstract

There is great potential for engineering cellular therapeutics by repurposing biological systems. Here, we report utilization of the granzyme-perforin pathway of cytotoxic lymphocytes as a cell-to-cell protein delivery module. We designed and constructed granzyme B-derived chaperone molecules fused to a fluorescent protein payload and expressed these constructs in natural killer (NK) cells. Using confocal microscopy and flow cytometry, we investigated the co-localization of the chaperones with lytic granules and the chaperone-mediated transfer of the fluorescent protein payload from NK to target cells in co-culture experiments. A synthetic chaperone consisting of the granzyme B ER signal peptide and a domain encompassing putative N-linked glycosylation sites in granzyme B is insufficient for payload transfer to target cells, whereas full-length granzyme B is sufficient for payload delivery. Combining our functional data with an analysis of the crystal structure of granzyme B suggests that the necessary motifs for granzyme B loading into lytic granules are dispersed throughout the primary amino acid sequence and are only functional when contiguous in the tertiary structure. These results illustrate that by using granzyme B as a molecular chaperone the granzyme-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

Cytolytic activity correlates with the mutational burden and deregulated expression of immune checkpoints in colorectal cancer

“…The patient’s antitumoral immune cytolytic activity (CYT), calculated as the geometric mean of the expression of the genes granzyme A (GZMA) and perforin 1 (PRF1), is also associated with improved patient survival [6, 7]. Cytotoxic T cells (CTL) and natural killer cells (NK) are able to kill tumor cells by overexpressing GZMA and PRF1 [8]. It is also known that effector T cells at the tumor site can predict a favorable outcome across many cancers [9–14].…”

Cytolytic Activity Score to Assess Anticancer Immunity in Colorectal Cancer

“…The granzyme-perforin pathway is a primary method by which cytotoxic lymphocytes destroy cancer cells 11 . Perforin creates pores within the target cell membrane and mediates the entry of granzymes, which are tryptases that cleave caspases and induce apoptosis 11,12 . Rooney etal .…”

KRAS mutations are negatively correlated with immunity in colon cancer

“…As for the inflammation-promoting signature, the KRAS -mutated group showed lower enrichment and 9 out of 15 genes in this signature had decreased expression (p = 0.002, Figure 5C , Supplementary Table 5 ). Granzyme A (GZMA) and perforin 1 (PRF1) secreted by cytotoxic T-cells and NK cells are able to kill tumor cells [ 27 ]. GZMA is a tryptase that leads to caspase- independent apoptosis, while PRF1 is a pore-forming enzyme that facilitates the entry of granzymes into the target cells.…”