Natural Killer Cells: A Promising Cellular Therapy Platform to Conquer Cancer

Background: Natural Killer Cell Biology

In 1973, Ivan Roitt's laboratory identified a new class of lymphocytes dubbed “null” killer cells, since they were neither T-cells nor B cells. The following year, these “null” lymphocytes were found to have spontaneous ability to kill target cells without prior immunization and without antibodies. In 1975, Rolf Keissling and colleagues described a similar natural cytotoxicity and named the effector cell a “natural” killer (NK) cell . We now recognize these cells as key mediators of immunosurveillance.

NK cells represent an important subgroup of peripheral blood lymphocytes and constitute the first line of defense against cancer and virus infected cells. NK cells develop in the bone marrow from CD34+ common lymphoid progenitor cells where they differentiate and mature before traveling to lymphoid and nonlymphoid peripheral organs and tissues. Because of their short half-life as compared to B and T lymphocytes, NK cells are constantly replenished from the bone marrow to ensure a steady pool of circulating cytolytic lymphocytes ready for spontaneous activation when elicited.

Unlike T-cells, NK cells are classically defined as negative for CD3 and T-cell receptor complex, and positive for CD56 and CD16. The two major biologic subsets of NK cells are distinguished based on CD56 and CD16 expression levels: CD56 ^dim CD16 ^bright cells, which represents a more mature and cytotoxic subset or CD56 ^bright CD16 ^dim , which represents a less mature and immunoregulatory subset.

Mechanisms of Natural Killer Cell Cytotoxicity

In contrast to T-cells, NK cells can kill abnormal cells in a major histocompatibility complex (MHC) independent manner and without any prior antigen priming. In fact, NK cell activity is governed by a complex array of germline encoded activating and inhibitory receptors as well as cytokine and chemokine receptors. Hence it is the sophisticated integration of positive and negative signals from these receptors that determines the final disposition of NK cells to kill or not to kill a target cell.

Once NK cells are in a cytotoxic state, they rapidly and effectively kill their target cells through several mechanisms. When NK cells form an immunologic synapse with their targets, they release lytic granules containing perforin (a membrane-disrupting protein) and granzymes (a family of proteolytic enzymes), causing lysis of the target cells. NK cells can also produce interferon gamma, a cytokine that stimulates the adaptive immune response through its effects on other immune effector cells, such as T-cells, macrophages, and dendritic cells. Furthermore, through releasing tumor necrosis factors (TNF), NK cells upregulate surface expression of the death ligands FAS ligand and (TNF)-related apoptosis-inducing ligand that bind to death receptors on cancer cells inducing their apoptosis via the caspase enzymatic cascade. Finally, antibody-dependent cellular cytotoxicity (ADCC) is another important mechanism that NK cells use for killing target cells. ADCC is mediated by CD16, which binds to the Fc portion of immunoglobulin (Ig)G1 antibodies opsonized on the surface of tumor cells leading to activation of NK cells through the CD3ζ pathway.

Role of Natural Killer Cells in Tumor Surveillance

In the 1980s, multiple studies reported that individuals with NK cell dysfunction resulting from genetic disorders, such as X-linked lymphoproliferative syndrome and Chédiak–Higashi syndrome, had a higher propensity to develop cancer. In vitro studies at the time showed that cells from nude mice, which lack functional T-cells, were still able to kill a broad range of tumor cells. During the same period, murine studies revealed that NK cell impairment or depletion was associated with higher tumor burden and metastasis.

In the following years, several studies found that cancer patients had impaired NK cell function and a seminal long-term epidemiologic study reported that subjects with low NK cell activity had a higher risk of developing various types of cancer. Other studies showed that NK cell deficiencies caused by genetic mutations in GATA2 or MCM4 lead to higher rates of malignancy.

These studies pointed to an important role of NK cells in tumor immune surveillance. In fact, NK cells can discriminate between healthy cells and abnormal cells via various mechanisms. One strategy that NK cells use to identify transformed cells is the detection of “stress ligands” recognized by the activating receptor NKG2D, such as human UL-16-binding proteins (ULBP), MHC class I chain-related A (MICA), and MICB molecules, which are commonly overexpressed on tumor cells. Another important mechanism is the “missing self” recognition. NK cells have inhibitory receptors called killer immunoglobulin like receptors (KIRs) that prevent NK cells from killing cells that express MHC class I molecules. Abnormal or transformed cells often downregulate MHC class I molecules to escape T-cell recognition but, as a consequence, lose the ligands for inhibitory KIR signal on NK cells, and hence become susceptible to NK cell-mediated cell lysis.

Sources of Natural Killer Cells for Immunotherapy

Unlike T-cells, NK cells have not been associated with graft-versus-host disease (GVHD) in the allogeneic setting, which opens the potential to produce “off-the-shelf” allogeneic cell therapy products that would be readily available for the treatment of cancer patients at the point of care. For adoptive cellular therapy, NK cells can be derived from multiple sources including NK cell lines, peripheral blood mononuclear cells (PBMCs), umbilical cord blood (CB), CD34+ hematopoietic progenitor cells (HPCs), and induced pluripotent stem cells (iPSCs).