A functional mammalian display screen identifies rare antibodies that stimulate NK cell–mediated cytotoxicity

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell–mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell–mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell–mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell–mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

Cancer immunotherapies have garnered a great deal of success over the last decade. Much of this success has been driven by the development of antibody-based therapeutics that redirect and enhance the cytotoxic potential of CD8+ T cells via immune checkpoint blockade or CD3/T cell receptor (TCR) complex stimulation. Like CD8+ T cells, natural killer (NK) cells are cytotoxic effector cells that mediate antitumor responses (1–3). They play a key role in tumor immunosurveillance and are able to identify and remove target cells by recognizing stress-induced ligands that are frequently overexpressed on cancer cells. NK cells are also known to perform antibody-dependent cellular cytotoxicity (ADCC), a mechanism that is used by multiple current therapeutic monoclonal antibodies to eradicate tumor cells (4–6). Given the crucial role that NK cells play in tumor immunosurveillance, the identification of novel immunotherapies that can target and redirect NK cell cytotoxicity merits further investigation.Whereas all T cells express the CD3/TCR complex that can be exploited by immunomodulatory molecules to redirect T cell activity, NK cells express multiple activating, costimulatory, and inhibitory receptors that govern NK cell activity (7, 8). Moreover, the NK cell repertoire is highly diverse, and the expression of these activating and inhibitory receptors among different cell subsets varies greatly within and among individuals (9, 10). These factors make it difficult to develop antibodies that can recruit and stimulate NK cells. Here, we report an approach to directly evaluate the ability of a curated set of antibodies to induce NK cell–mediated cytotoxicity.We couple a mammalian display screen to a next generation sequencing (NGS) readout to characterize antibodies that bind to and activate NK cells. Antibodies were selected against six NK cell receptors from an antigen-binding fragment (Fab)-phage library that was based on the trastuzumab scaffold and were displayed on a target cell line to generate a mammalian display library. NK cells have the innate ability to recognize and kill unhealthy cells. We reasoned that an antibody against an NK cell surface protein that was displayed on a target cell could drive the interaction between an NK cell and a target cell. If the antibody was also able to activate NK cells, then the cell displaying the antibody would be killed and deselected. All of our antibodies are constructed on the same scaffold, allowing the use of the same set of primers to amplify and sequence the complementarity determining region (CDR) H3 of each clone. Thus, we rationalized that we should be able to screen these antibodies in a pooled manner and quantify the depletion of specific antibody clones through NGS of CDR H3. Indeed, antibody binders that were depleted in our functional screen were able to stimulate NK cell cytotoxicity and interferon-γ (IFN-γ) secretion. We found that the most potent stimulators of NK cell–mediated cytotoxicity were high-affinity binders to previously identified activating NK receptors, like CD16, NCR1, and NCR3, and that binding to an upregulated NK cell surface protein, TNFSF4, or costimulator NK receptors, TNFRSF9 and CD244, were unable to stimulate NK activity. These activating antibodies were applied to the generation of bispecific antibodies to redirect NK cells toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. We believe this method can facilitate the discovery of rare antibodies that can stimulate immune cell activation and promote the design of immunotherapies.