Expansion of natural killer cells with optimal killing activity for cancer immunotherapy

Abstract

Abstract Natural killer (NK) cells play a key role in the elimination of microbial pathogens and cancer cells. NK cells rapidly respond to stressed cells that are infected or bear tumorigenic mutations that reduce “self” markers of MHC class 1 and/or display increased presentation of activating ligands. These signals prompt NK cells to form an immunological synapse with an infected or tumor cell and discharge lytic granules containing cytotoxic proteins such as IFNγ, perforin and granzyme B leading to target cell destruction. Because of their natural ability to recognize and kill cancer cells, a plethora of preclinical studies and clinical trials have shown that NK cell therapies offer a potentially powerful weapon to fight both advanced-stage blood cancers and solid tumors. Given the great potential of NK cell therapies, we developed clinically relevant serum- and xeno-free methods for human NK cell expansion. Using optimized media and cytokines, either with anti-NKp46 coated tissue culture dishes or Cloudz™ CD2/NKp46 microspheres we have greatly expanded highly purified NK cells from human peripheral blood mononuclear cells (PBMCs) that are capable of potent cytolytic activity, as indicated with a novel flow cytometry-based NK cytotoxicity assay. Using this NK expansion methodology, we performed high-throughput screening methods to identify novel agents that enhance NK cell killing activity as assessed by IFNγ and Granzyme B quantikine ELISA assays which were then confirmed with NK cytotoxicity assays. The methods established here provide a novel workflow for generating highly purified NK cells that are applicable to clinical translation. Furthermore, the agents identified in this work provide insight for augmenting NK killing potential.