A fusion protein complex that combines IL12, IL15, and IL18 signaling to induce memory-like NK cells for cancer immunotherapy

NK cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly priming blood NK cells with rhIL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. We developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15 and IL-18 receptor engagement (HCW9201) and the second adds CD16 engagement (HCW9207). HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNAseq and multidimensional mass cytometry revealed strong parallels between HCW9201 and 12/15/18. Moreover, HCW9201 stimulation improved NK cell metabolic fitness, and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201- and 12/15/18-primed similar increases in short-term and memory-like NK cell cytotoxicity and IFN-g production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multi-signal receptor engagement on immune cells, and HCW9201-primed NK cells will be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.erm and memory-like NK cell cytotoxicity and IFN-g production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multi-signal receptor engagement on immune cells, and HCW9201-primed NK cells will be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy. Overall design: NK cells (> 95% CD56+CD3-) were isolated from healthy donor PBMCs using RosetteSep (STEMCELL Technologies). ML and control NK were generated essentially as described previously with NK media (Miltenyi) with 5% (v/v) heat-inactivated human AB pooled sera (Millipore Sigma) (CNKM) or RPMI-1640, supplemented with 2 mM L-glutamine, antibiotics (penicillin, 100 U/mL; streptomycin, 100 µg/mL) (ThermoFisher), and 10% (v/v) fetal bovine serum (Cytiva). NK cells were incubated with individual cytokines; IL-12p70 (10 µg/mL), IL-15 (50 µg/mL), and IL-18 (50 µg/mL) (12/15/18) (7); or HFPCs at 50 to 100 nM. After 12-16 hours of activation, NK cells were washed three times and plated in 1 ng/mL rhIL-15 with every other day media exchanges. RNA was isolated for RNA sequencing as described elsewhere.