Delivery of a novel membrane-anchored Fc chimera enhances NK cell-mediated killing of tumor cells and persistently virus-infected cells.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the most powerful mechanisms for Natural Killer (NK) cells to kill cancer cells or virus-infected cells. A novel chimeric protein (NA-Fc) was created, which when expressed in cells, positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. This NA-Fc chimera was tested with PM21-NK cells, produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Real time viability assays revealed higher PM21-NK killing of both ovarian and lung cancer cells expressing NA-Fc, which correlated with increased release of TNF-α and IFN-γ cytokines from NK cells and was dependent on CD16-Fc interactions. Lentivirus delivery of NA-Fc to target cells increased the rate of PM21-NK cell killing of A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells. This NA-Fc-directed killing was extended to virus infected cells, where delivery of NA-Fc to lung cells that were persistently infected with Parainfluenza virus resulted in increased killing by PM21-NK cells. In contrast to its effect on PM21-NK cells, the NA-Fc molecule did not enhance complement mediated lysis of lung cancer cells. Our study lays the foundation for application of the novel NA-Fc chimera that could be delivered specifically to tumors during oncolytic virotherapy to mark target cells for ADCC by co-treatment with adoptive NK cells. This strategy would potentially eliminate the need to search for unique cancer specific antigens for development of new antibody therapeutics.

Oncolytic parainfluenza virus combines with NK cells to mediate killing of infected and non-infected lung cancer cells within 3D spheroids: role of type I and type III interferon signaling.

BACKGROUND: There is intense interest in developing novel oncolytic viruses, which can be used in cancer therapies along with immune cells such as natural killer (NK) cells. We have previously developed a particle-based method for in vitro expansion of highly cytotoxic human NK cells (PM21-NK cells). Here, we have tested the hypothesis that oncolytic parainfluenza virus 5 (P/V virus) can combine with PM21-NK cells for targeted killing of lung cancer cells. METHODS: PM21-NK cells were assayed for killing of P/V virus-infected A549, H1299 and Calu-1 lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cultures using flow cytometry, luminescence and kinetic imaging-based methods. Blocking antibodies were used to evaluate NK cell activating receptors involved in PM21-NK cell killing of infected target cells. Media transfer experiments tested soluble factors that increase PM21-NK cell killing of both P/V virus-infected and uninfected tumor cells. RESULTS: In 2D cultures, PM21-NK cells efficiently killed P/V virus-infected cancer cells compared with non-infected cells, through involvement of the viral glycoprotein and NK cell receptors NKp30, NKp46 and NKG2D. In 3D spheroid cultures, P/V virus infection was restricted to the outer layer of the spheroid. However, PM21-NK cells were able to more efficiently kill both the outer layer of infected cells in the spheroid and progressing further to kill the uninfected interior cells. Media transfer experiments demonstrated that P/V virus infection produced both type I and type III interferons, which decreased cell growth, which contributed to a reduction in the overall number of uninfected tumor cells in conjunction with PM21-NK cells. Across five cancer cell lines, the contribution of P/V virus infection on PM21-NK cell killing of target cells correlated with interferon induction. CONCLUSION: Our data support the potential of combining oncolytic parainfluenza virus with PM21-NK cell adoptive therapy against lung cancer.

Therapeutic potential of Pnmt+ primer cells for neuro/myocardial regeneration.

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine into epinephrine, and thus serves as a marker of adrenergic cells. In adults, adrenergic cells are present in the adrenal medullae and the central and peripheral (sympathetic) nervous systems where they play key roles in stress responses and a variety of other functions. During early embryonic development, however, Pnmt first appears in the heart where it is associated with specialized myocytes in the pacemaking and conduction system. There is a transient surge in cardiac Pnmt expression beginning when the first myocardial contractions occur, before any nerve-like or neural crest cells appear in the heart. This early expression of Pnmt denotes a mesodermal origin of these "Instrinsic Cardiac Adrenergic" (ICA) cells. Interestingly, Pnmt+ cells are found in all four chambers of the developing heart, but by adult stages, are found primarily concentrated on the left side of the heart. This regionalized expression occurs in the left atrium and in specific regions of the left ventricle roughly corresponding to basal, mid, and apical sections. A second distinct population of Pnmt-expressing (Pnmt+) cells enters the embryonic heart from invading neural crest, and these "Neural Crest-Derived" (NCD) Pnmt+ cells appear to give rise to a subpopulation(s) of cardiac neurons. Pnmt expression thus serves as a marker not only for adrenergic cells, but also for precursor or "primer" cells destined to become specialized myocytes and neurons in the heart. This review discusses the distribution of Pnmt in the heart during development, including the types of cells where it is expressed, and their potential use for regenerative medicine therapies for cardiovascular disease.

Targeting of the enhanced green fluorescent protein reporter to adrenergic cells in mice.

Adrenaline and noradrenaline are important neurotransmitter hormones that mediate physiological stress responses in adult mammals, and are essential for cardiovascular function during a critical period of embryonic/fetal development. In this study, we describe a novel mouse model system for identifying and characterizing adrenergic cells. Specifically, we generated a reporter mouse strain in which a nuclear-localized enhanced green fluorescent protein gene (nEGFP) was inserted into exon 1 of the gene encoding Phenylethanolamine n-methyltransferase (Pnmt), the enzyme responsible for production of adrenaline from noradrenaline. Our analysis demonstrates that this knock-in mutation effectively marks adrenergic cells in embryonic and adult mice. We see expression of nEGFP in Pnmt-expressing cells of the adrenal medulla in adult animals. We also note that nEGFP expression recapitulates the restricted expression of Pnmt in the embryonic heart. Finally, we show that nEGFP and Pnmt expressions are each induced in parallel during the in vitro differentiation of pluripotent mouse embryonic stem cells into beating cardiomyocytes. Thus, this new mouse genetic model should be useful for the identification and functional characterization of adrenergic cells in vitro and in vivo.