In humans, natural killer (NK) cells comprise 5–10% of circulating lymphocytes and constitute a key component of the innate immune system as they have the ability to immediately secrete interferon-γ, as well as to release cytotoxic granules containing perforin and granzymes upon target cell recognition. Unlike B cells and T cells of the adapted immune system, NK cells do not have antigen receptors that are diversified by somatic recombination and mutation events. However, based on their hematopoietic lineage, cell surface receptor repertoire, and killing mechanism, NK cells are more closely related to T cells than to cells of the innate immune system.1
NK cells express two types of cell surface receptors referred to as inhibitory receptors
and activating receptors
. Both types of cell surface receptors consist of several different protein families. Inhibitory receptors recognize MHC class I ligands on target cells, whereas the activating receptors can recognize MHC class I, MHC class I-related, and non-MHC ligands on target cells. The integration of activating and inhibitory signals provided by the target cells ultimately controls NK cell reactivity through processes that remain incompletely understood. Tumor cells and virally infected cells often express activating receptors and downregulate inhibitory receptors, thus rendering them susceptible to NK cell mediated killing. Prominent NK cell surface receptors include CD16 (FcγRIIIA; an activating receptor that binds IgG and mediates antibody-dependent cellular cytotoxicity), CD56 (NCAM; a cell adhesion molecule), the polymorphic KIR protein family which recognizes conventional MHC class I ligands (HLA-A, HLA-B, and HLA-C), the polymorphic CD94/NKG2A/NKG2C/NKG2E/NKG2F protein family which recognizes nonconventional MHC class I ligands (HLA-E), the natural cytotoxicity receptors NKp30/NKp44/NKp46/NKp80 which are activating receptors whose ligands have remained largely elusive, and NKG2D which is an activating receptor that recognizes MHC class I-related ligands.
Despite its name, NKG2D2,3
is functionally and structurally distinct from the CD94/NKG2A/NKG2C/NKG2E/NKG2F protein family. NKG2D is a type II transmembrane glycoprotein with an N-terminal cytoplasmic domain (amino acids 1–51) followed by a transmembrane anchor (amino acids 52–72) and a C-terminal extracellular domain (amino acids 73–216) with three potential N-glycosylation sites. At the cell surface, NKG2D forms a homodimer stabilized by a disulfide bridge and associates with the adaptor protein DAP10, a type I transmembrane protein that exclusively mediates NKG2D signaling. The NKG2D homodimer associates with two DAP10 homodimers, thereby forming a hexameric structure.4
In the absence of DAP10, NKG2D is retained inside the cell.5
NKG2D recognizes transmembrane proteins MICA, MICB, and ULBP4 as well as GPI-anchored membrane proteins ULBP1, ULBP2, and ULBP3. Like MHC class I molecules but in striking contrast to their receptor NKG2D, these MHC I-related ligands are polymorphic. Unlike MHC class I molecules, however, MIC and ULBP proteins do not bind and display peptides and do not associate with β2 microglobulin. Induced in stressed cells by the DNA damage response pathway6
, NKG2D ligands are often expressed on tumor cells and virally infected cells but not on healthy cells. Through recognition of NKG2D ligands, NKG2D is a key molecule in mediating immunosurveillance by NK cells as evidenced by the findings that both tumors and viruses have developed molecular strategies to evade NKG2D immunosurveillance. For example, whereas tumors often secrete soluble MICA and MICB as decoys, cytomegalovirus (CMV) expresses a protein known as UL16 which prevents the cell surface expression of MICB, ULBP1, and ULBP2. In addition, the direct involvement of NKG2D in tumor immunosurveillance was recently shown in NKG2D-deficient mice that were crossed with transgenic mouse models of spontaneous malignancy.7,8
Expression of NKG2D ligands on healthy cells has been detected in several autoimmune diseases. In NOD mice, a mouse model of type I diabetes, mouse NKG2D ligand RAE-1 is expressed on pancreatic islet cells. A rat anti-mouse NKG2D monoclonal antibody (mAb) that did not deplete NKG2D+ NK cells and NKG2D+ T cells was shown to completely prevent type I diabetes in NOD mice.9
In humans, in addition to NK cells, NKG2D is also expressed on T cells, including CD8+ αβ TCR+ T cells, γδ TCR+ T cells, and NKT cells. The participation of NKG2D in fighting cancer and viral infections as well as in promoting autoimmune diseases likely involves both NKG2D+ NK cells and NKG2D+ T cells. On T cells, however, rather than being an activating receptor NKG2D functions as a costimulatory receptor similar to CD28,10
albeit more restricted.11
The central role that NKG2D plays in mediating immune responses in autoimmune diseases, infectious diseases, and cancer, makes NKG2D an attractive target for therapeutic intervention. This perception is further supported by the fact that a single receptor mediates recognition of a diverse and polymorphic group of ligands12
, singling out NKG2D as unique target for a broad range of applications. In particular mAbs that interfere with NKG2D receptor/ligand interactions and thereby, depending on the context, antagonize or agonize immune responses mediated by NKG2D could be of broad and potent therapeutic utility. Nonetheless, mAbs to NKG2D that are suitable for clinical investigations have not been published yet.
To facilitate clinical investigations of the therapeutic potential of NKG2D targeting, we here report the generation, affinity maturation, and characterization of a fully human anti-human NKG2D mAb. Taking into consideration that the immunogenicity of mouse, chimeric mouse/human, or even humanized anti-human NKG2D mAbs would likely interfere with their therapeutic potential, our efforts focused on the generation of a fully human mAb with minimal deviation from natural human antibody sequences. Over the past three decades, a number of different technologies have been developed for the generation of human mAbs. Arguably the most successful and accessible technology has been phage display. Not only does phage display provide a potent platform for the de novo generation of human mAbs to human antigens but also for their affinity maturation in vitro.13,14
Among the most widely applied phage display technologies for affinity maturation of human mAbs in Fab or scFv format are chain shuffling15,16
and CDR walking.17
Although both methods have their merits, chain shuffling provides access to a more diverse structural repertoire without introducing intentional synthetic mutations.