Live cell imaging of lytic granule motility in anti-ErbB2 CAR NK cells and FcR NK cells plus Herceptin towards ErbB2+ breast cancer cells

Background & Aim Upon encountering a susceptible target, NK cells mediate directed cytotoxicity by exocytosis of lytic effector molecules such as perforin and granzymes. The steps leading to NK cell granule exocytosis are highly regulated. Granule exocytosis is preceded by convergence of granules to the microtubule organizing center (MTOC) and subsequent polarization of the MTOC and granules to the immunological synapse (IS). In case of antibody-dependent cell-mediated cytotoxicity (ADCC), it has been shown that signaling through the Fc receptor is critical to polarize MTOC and granules to the IS with otherwise resistant targets. Methods, Results & Conclusion Here we used spinning disk confocal microscopy for live cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR expressing NK-92 cells (NK-92/5.28.z) and research-grade high affinity FcR expressing NK-92 cells plus Herceptin™ towards ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. Interestingly, unmodified NK-92 cells in combination with MDA-MB-453 cells showed granule convergence to the MTOC, but failed to polarize MTOC and granules to the IS. In contrast, retargeting by either CAR or mAb/FcR towards the ErbB2 antigen on MDA-MB-453 enabled granule polarization to the IS resulting in highly effective cytotoxicity. Granule polarization was rapid in both the CAR and high affinity FcR expressing NK-92 cells after cell-cell contact was initiated. These observations suggest that retargeting of NK-92 cells by either transgenic CAR or high affinity FcR expression in combination with tumor-specific antibodies confers tumor cell lysis by enabling the otherwise impaired MTOC and granule polarization to the IS which resembles the physiological exocytosis cascade observed in naturally occurring ADCC.